ivision - biovis.com

iVisionFor Macintosh OS X

Version 4.0.0

Extensions ManualIncluding Camera and Device Control

rev December 20, 2006

BioVision Technologies, Inc.80 West Welsh Pool RoadExton, PA 19341Phone: 610-524-9740Email: [email protected]

© 1989 - 2006 BioVision Technologies, Inc. All rights reserved.

End User License Agreement for BioVision Technologies, Inc. Software

PLEASE READ THIS SOFTWARE LICENSE AGREEMENT (“LICENSE”) CAREFULLY BEFOREPRESSING THE “ACCEPT” BUTTON BELOW. BY PRESSING “ACCEPT”, YOU ARE AGREEING TO BEBOUND BY THE TERMS OF THIS LICENSE. IF YOU DO NOT AGREE TO THE TERMS OF THISLICENSE, PRESS “DECLINE” AND (IF APPLICABLE) RETURN THE SOFTWARE TO THE PLACEWHERE YOU OBTAINED IT FOR A REFUND.

THIS SOFTWARE END USER LICENSE AGREEMENT (EULA) IS A LEGAL AGREEMENT BETWEEN YOU(EITHER AS AN INDIVIDUAL OR AN ENTITY, WHO WILL BE REFERRED TO IN THIS EULA AS “YOU”)AND BIOVISION BIOSCIENCE, INC. (“BIOVISION”) FOR THE iVision SOFTWARE PRODUCT THATACCOMPANIES THIS EULA, INCLUDING ANY ASSOCIATED MEDIA, PRINTED MATERIALS ANDELECTRONIC DOCUMENTATION (“THE SOFTWARE PRODUCT”). READ IT CAREFULLY BEFORECOMPLETING ANY INSTALLATION PROCESS OR OPERATING THE SOFTWARE. BY INSTALLING ORUSING THE SOFTWARE, YOU CONFIRM YOUR ACCEPTANCE OF THE SOFTWARE AND AGREE TO BEBOUND BY THE EULA. IF YOU DO NOT AGREE TO THE TERMS OF THIS EULA, DO NOT INSTALL,ACCESS OR USE THE SOFTWARE PRODUCT, INSTEAD YOU SHOULD RETURN IT TO BIOVISION FOR AFULL REFUND.

I. Grant of License.

• General License Grant. This Software Product is licensed, not sold. Subject to the terms and conditions of this EULA,YOU are granted a limited, non-transferable, non- exclusive license to use a copy of the Software Product on one (1)computer CPU. YOUR right to use the Software Product includes the right to reproduce the documentation as necessaryfor use of the Software Product. YOU may make one (1) backup copy of the Software Product for your own use.

• Not for Resale. The Software Product is not for resale and is labeled “Not For Resale” or “NFR”. Notwithstanding anyother provision of this EULA, YOU may not resell, or otherwise transfer for value, the Software Product.

• Limitation on Reverse Engineering, Decompilation and Disassembly. The Software Product in its entirety is protectedby copyright laws. YOU may not reverse engineer, decompile or disassemble the Software Product, except and only tothe extent that such activity is expressly permitted by applicable law notwithstanding this limitation.

• Trademarks. This EULA does not grant YOU any rights in connection with any trademarks of BIOVISION.

• Reservation of Rights. All rights not expressly granted are reserved by BIOVISION.

II. Termination. An event of default occurs when (i) either party fails to comply with any material obligation under thisAgreement, and (ii) the noncompliance remains uncured for than thirty (30) days after receipt of written notice thereof.

In the event a default occurs, the non-defaulting party, in addition to any other rights available to it under law or equity,may terminate this EULA and all licenses granted hereunder by giving written notice to the defaulting party. Except asotherwise specifically stated herein, remedies will be cumulative and there will be no obligation to exercise a particularremedy.

Within fifteen (15) days after termination of this EULA, YOU will certify in writing to BIOVISION that all copies of theSoftware Product and documentation in any form, including backup copies, have been destroyed or returned toBIOVISION.

III. Confidentiality Provision. YOU agree and acknowledge that the Software Product contains the proprietary andconfidential information and assets of BIOVISION, and furthermore that the access codes issued by BIOVISION areconfidential. YOU shall hold the BIOVISION confidential information in confidence in perpetuity and shall not use ordisclose any BIOVISION confidential information except as authorized hereunder. YOU shall take all reasonable stepsto ensure that such BIOVISION confidential information is not disclosed or distributed in violation of the terms of thisAgreement. If YOU discover or suspect that the confidentiality of BIOVISION’s confidential information has beencompromised, YOU shall promptly report the same to BIOVISION.

4 Device Independent Camera Controls - Overview

The parties agree that the BIOVISION confidential information constitutes valuable assets and trade secrets ofBIOVISION. The parties acknowledge and agree that irreparable injury will result to BIOVISION as a result of a breachof confidentiality. Because it would be difficult to measure the damages to the respective parties from any breach, thatinjury to such party from any such breach would be impossible to calculate, and that money damages would therefore bean inadequate remedy for any such breach, the parties agree that in the event of any such breach, the non-breaching partymay seek in addition to all other remedies it may have at law or in equity, injunctions or other appropriate orders torestrain any such breach without showing or proving any actual damage to such party .

IV. Intellectual Property Rights. This Software Product, and any copies, modifications, updates or derivative works arethe intellectual property of BIOVISION. The structure, organization and code of the software are the confidentialinformation of BIOVISION, and may not be disclosed without prior written permission from BIOVISION. All rights notspecifically granted in this EULA, including federal and international copyrights are reserved by BIOVISION .

V. Limited Warranty. BIOVISION warrants that the Software Product shall substantially comply with thespecifications set forth in the Software Product documentation for a period of ninety (90) days from the date of receipt .

VI. Disclaimer of Warranties. EXCEPT FOR THE LIMITED WARRANTIES SET FORTH HETEINABOVE,BIOVISION HEREBY DISCLAIMS, TO THE MAXIMUM EXTENT PERMITTTED BY LAW, ALL OTHERWARRANTIES, EXPRESS, IMPLIED OR STATUTORY, INCLUDING, BUT NOT LIMITED TO, IMPLIEDWARRANTIES OF MERCHANTABLITY OR FITNESS FOR A PARTICULAR PURPOSE, OR LACK OF VIRUSESOR NON-INFRINGEMENT WITH RESPECT TO THE SOFTWARE PRODUCT .

VII. Exclusion of Incidental, Consequential and Certain Other Damages. BIOVISION WILL NOT BE LIABLEFOR ANY INDIRECT, INCIDENTAL, PUNITIVE, SPECIAL, OR CONSEQUENTIAL DAMAGES, INCLUDINGBUT NOT LIMITED TO LOST DATA OR LOST PROFITS, HOWEVER ARISING, EVEN IF IT HAS BEENADVISED OF THE POSSIBILITY OF SUCH DAMAGES. BIOVISION’s liability for damages under this EULA,whether in contact or tort, will in no event exceed the amount paid by YOU to BIOVISION for Software Product in theone-year period preceding the claim. The parties agree to the allocation of liability risk set forth in this provision .

VIII. Indemnification. YOU hereby agrees to indemnify, defend and hold harmless BIOVISION, its shareholders,officers, directors, employees and agents against any and all losses, damages, liabilities, costs, expenses, claims, actions,lawsuits and judgments thereon (including attorneys’ fees through final resolution) that may be incurred by or broughtagainst BIOVISION, as a result of and to the extent of any claim or allegation based upon (i) YOUR use of SoftwareProduct in manner not permitted by this Agreement, including misuse that infringes upon the intellectual property orother rights of a third party or (ii) related to the use or misuse of servers on which the Software Product resides .

IX. Third Party Software. This Software Product may contain third party software requiring notice or additional termsand conditions. Such required third party notice or additional terms are available at BIOVISION, and made a part of, andincorporated by reference, into this EULA .

X. Software Product Delivered to U.S. Government. This Software Product and Documentation were developed atprivate expense, and are provided with “RESTRICTED RIGHTS.” Use, duplication or disclosure by the U.S.Government is subject to restrictions as set forth in this EULA and as provided in DFARS 227.7202-1(a) and 227.7202-3(a) (1995), DFARS 252.227- 7013(c)(1)(ii)(Oct. 1998), FAR 12.212(a) 1995, FAR 52.227-19 or FAR 52.227-14, asapplicable .

XI. Assignment. YOU may not sign this EULA, without the prior written consent of BIOVISION. Any such action inviolation of the foregoing shall be void .

XII. Entire Agreement. This EULA is the entire agreement between YOU and BIOVISION relating to the SoftwareProduct and it supersedes all prior or contemporaneous oral or written communications, proposals and representationswith respect to the Software Product or any other subject matter covered by this EULA .

XIII. Governing Law. All matters affecting the interpretation, validity, and performance of this Agreement shall begoverned by the internal laws of the State of New Jersey without regard to its conflict of law principles, except asotherwise expressly provided herein.

iVision is a trademark of BioVision Technologies, Inc. Macintosh is a trademark of Apple Computer, Inc.

vii

Table Of ContentsPart I Acquisition 11 Overview of the Acquisition Process ..................................................1

1.1 Identifying Your Camera the First Time..............................................................................11.2 Switching Cameras .............................................................................................................11.3 Setting Acquisition Parameters............................................................................................21.4 Previewing the Acquisition .................................................................................................21.5 Grabbing the Image.............................................................................................................3

2 Camera Menu Commands..................................................................52.1 Camera Toolbar ..................................................................................................................52.2 Select Camera .....................................................................................................................5

2.2.1 Troubleshooting ..................................................................................................52.3 Preview...............................................................................................................................6

2.3.1 Scripting the Preview Command .........................................................................62.4 Snapshot .............................................................................................................................72.5 Autofocus ...........................................................................................................................72.6 Single Exposure ................................................................................................................10

2.6.1 Scripting the Single Exposure Command ..........................................................112.7 Full Acquire......................................................................................................................11

2.7.1 Preview (Full Acquire)......................................................................................122.7.2 Trigger (Full Acquire).......................................................................................152.7.3 Processing (Full Acquire)..................................................................................152.7.4 Exposure (Full Acquire)....................................................................................152.7.5 Image Size (Full Acquire) .................................................................................172.7.6 Destination (Full Acquire).................................................................................182.7.7 Do Timelapse (Full Acquire).............................................................................192.7.8 Timelapse (Full Acquire) ..................................................................................192.7.9 Use External Shutter (Full Acquire) ..................................................................202.7.10 Shutter (Full Acquire) .....................................................................................202.7.11 Create Timing Window (Full Acquire)............................................................212.7.12 Scripting the Full Acquire Command ..............................................................21

2.8 RGB Color Acquire ..........................................................................................................212.8.1 Do Preview (RGB Color Acquire).....................................................................232.8.2 Filters (RGB Color Acquire) .............................................................................232.8.3 Exposure (RGB Color Acquire) ........................................................................232.8.4 Image Size (RGB Color Acquire)......................................................................242.8.5 Destination (RGB Color Acquire) .....................................................................252.8.6 Timelapse (RGB Color Acquire).......................................................................272.8.7 Use External Shutter (RGB Color Acquire).......................................................272.8.8 Shutter (RGB Color Acquire)............................................................................272.8.9 Create Timing Window (RGB Color Acquire) ..................................................282.8.10 Scripting the RGB Color Acquire Command...................................................28

2.9 Multi Dimensional Acquire...............................................................................................292.9.1 Multi Dimensional Acquire: General Tab..........................................................302.9.2 Multi Dimensional Acquire: Size Tab ...............................................................322.9.3 Multi Dimensional Acquire: Shutter & Filters Tab ............................................332.9.4 Multi Dimensional Acquire: Z-Steps Tab..........................................................362.9.5 Multi Dimensional Acquire: Time Lapse Tab....................................................372.9.6 Multi Dimensional Acquire Preview: Preview Dialog .......................................39

2.10 Set Color Balance .............................................................................................................452.11 Set Gain and Offset ...........................................................................................................46

viii

2.12 Camera Settings ................................................................................................................473 Acquisition Hardware Specifics .......................................................49

3.1 Cooke-PCO Cameras ........................................................................................................503.1.1 Supported Cameras ...........................................................................................503.1.2 Hardware Installation........................................................................................503.1.3 Possible Settings for Cooke Cameras ................................................................503.1.4 Camera Settings Command ...............................................................................50

3.2 Diagnostic Instruments Cameras .......................................................................................523.2.1 The Different Cameras and How to Use Them ..................................................523.2.2 Possible Settings for Spot Cameras ...................................................................533.2.3 Camera-Specific Data Files...............................................................................533.2.4 Camera Settings Command ...............................................................................543.2.5 Spot-Specific Commands..................................................................................55

3.3 Hamamatsu Cameras.........................................................................................................583.3.1 Supported Cameras ...........................................................................................583.3.2 Conflicts ...........................................................................................................583.3.3 Camera Setup....................................................................................................583.3.4 Camera Settings Command ...............................................................................59

3.4 Roper Photometrics Cameras ............................................................................................613.4.1 Supported Cameras ...........................................................................................613.4.2 Possible Settings for the Photometrics Cameras ................................................613.4.3 Camera Settings Command ...............................................................................61

3.5 QImaging Cameras ...........................................................................................................633.5.1 Supported Cameras ...........................................................................................633.5.2 Conflicts ...........................................................................................................633.5.3 Possible Settings for QImaging Cameras...........................................................633.5.4 Camera Settings Command ...............................................................................63

3.6 Virtual Camera .................................................................................................................653.6.1 Purpose.............................................................................................................653.6.2 Camera Settings Command ...............................................................................65

Part II Motion Control 674 Control Menu Commands ................................................................67

4.1 Introduction ......................................................................................................................674.2 About Device Modules .....................................................................................................674.3 Device Setup.....................................................................................................................68

4.3.1 The Setup Button ..............................................................................................694.3.2 The Connect and Connect All Buttons ..............................................................694.3.3 Serial Status ......................................................................................................704.3.4 Done .................................................................................................................704.3.5 Determining Step Size for Stages ......................................................................70

4.4 Device Select ....................................................................................................................714.5 Device Toolbar .................................................................................................................724.6 Device Specific.................................................................................................................734.7 Microscope Control ..........................................................................................................734.8 Microscope Variable .........................................................................................................754.9 Microscope Lamp .............................................................................................................764.10 Microscope Speed.............................................................................................................764.11 Stage Control ....................................................................................................................774.12 Stage Set Zero Position .....................................................................................................784.13 Stage Get Position.............................................................................................................784.14 Stage Record Position .......................................................................................................794.15 Stage Move to Recorded Position......................................................................................804.16 Stage Z-Sections Setup .....................................................................................................81

ix

4.17 Stage Speed ......................................................................................................................834.18 StageScan Array Calibrate ................................................................................................84

4.18.1 Calibration Menu ............................................................................................854.18.2 Single Array Tab.............................................................................................854.18.3 Multiple Arrays Tab........................................................................................864.18.4 Calculate All Positions ....................................................................................884.18.5 Duplicate, Save, Delete ...................................................................................884.18.6 Scripted Behavior............................................................................................88

4.19 StageScan Array Setup......................................................................................................884.19.1 Setup Menu.....................................................................................................894.19.2 Calibration Data ..............................................................................................904.19.3 Path From Start ...............................................................................................904.19.4 Single Array Tab.............................................................................................914.19.5 Multiple Arrays Tab........................................................................................914.19.6 Create Locations Window ...............................................................................924.19.7 Scripted Behavior............................................................................................92

4.20 StageScan Mosaic Calibrate ..............................................................................................934.20.1 Capture Dimensions ........................................................................................934.20.2 Initial Camera Position....................................................................................944.20.3 X/Y Movement ...............................................................................................944.20.4 Create Locations Window ...............................................................................944.20.5 Scripted Behavior............................................................................................95

4.21 StageScan Move/Iterate.....................................................................................................954.21.1 Position Window.............................................................................................954.21.2 Move Tab........................................................................................................964.21.3 Iterate Tab.......................................................................................................974.21.4 Scripted Behavior............................................................................................97

4.22 Define User Devices .........................................................................................................984.22.1 Using Device Definitions ................................................................................984.22.2 Managing Device Definition ...........................................................................984.22.3 Device Type Tab.............................................................................................994.22.4 Port Setup Tab (Serial) ....................................................................................994.22.5 Commands Tab (Serial).................................................................................1004.22.6 Positions Tab (Linked) ..................................................................................101

5 Motion Control Hardware Specifics ..............................................1035.1 Common Device Setup ...................................................................................................1035.2 Common Device Information..........................................................................................103

5.2.1 Filter Information............................................................................................1045.2.2 Microscope Information..................................................................................1045.2.3 Simple Positioner Information.........................................................................104

5.3 Applied Scientific Instruments Filter Wheel and Shutter .................................................1055.3.1 ASI S&F Setup ...............................................................................................105

5.4 ASI Stage........................................................................................................................1065.4.1 ASI Stage Setup ..............................................................................................1065.4.2 ASI Stage Lock...............................................................................................106

5.5 BD Biosciences CARV II................................................................................................1085.5.1 BD CARV II Setup .........................................................................................1085.5.2 CARV II Options ............................................................................................109

5.6 Cambridge Research Inc. RGB Liquid Crystal Tunable Filter .........................................1105.6.1 CRI Filter Setup ..............................................................................................110

5.7 Generic S&F Device Module ..........................................................................................1115.7.1 Generic S&F Setup .........................................................................................111

5.8 Leica DM6000 Microscope (Upright and Inverted) .........................................................1125.8.1 Device Setup Dialog .......................................................................................1125.8.2 Leica DM6000 Setup ......................................................................................112

x

5.9 Leica Two Microscope (DM IRE2, DM RXA2)..............................................................1135.9.1 Device Setup Dialog .......................................................................................1135.9.2 Leica Two Setup .............................................................................................113

5.10 Ludl Shutter and Filter Wheel .........................................................................................1155.10.1 Ludl S&F Setup ............................................................................................115

5.11 Ludl Stage.......................................................................................................................1175.11.1 Ludl Stage Setup...........................................................................................1175.11.2 Ludl Stage Lock............................................................................................117

5.12 Nikon Eclipse E1000 Microscope Control.......................................................................1195.12.1 Nikon E1000 Setup .......................................................................................119

5.13 Nikon Remote Focus.......................................................................................................1215.13.1 Nikon Remote Focus Setup ...........................................................................121

5.14 Nikon Eclipse TE2000 Microscope Control ....................................................................1225.14.1 Device Setup Dialog .....................................................................................122

5.15 Olympus BX & IX Microscope Control ..........................................................................1235.15.1 Device Setup Dialog .....................................................................................1235.15.2 Olympus BX / IX Setup ................................................................................1235.15.3 Olympus BX / IX Mode ................................................................................124

5.16 The Physik Instrumente Piezo-Electric Focus Device (Models 662 and 665)...................1265.16.1 Setup Dialog .................................................................................................126

5.17 Prior Shutter & Filter ......................................................................................................1275.17.1 Prior S&F Setup............................................................................................127

5.18 Prior Stage ......................................................................................................................1285.18.1 Prior Stage Setup ..........................................................................................128

5.19 Sutter Lambda DG-4 High Speed Filter Changer.............................................................1295.19.1 Sutter DG-4 Setup.........................................................................................1295.19.2 DG-4 Commands ..........................................................................................130

5.20 Sutter Lambda Filter Wheel Controller (Models 10-2 and 10-3)......................................1315.20.1 Sutter Lambda Setup.....................................................................................1315.20.2 Smart Shutters™...........................................................................................132

5.21 Vincent Associates UniBlitz Shutter Control...................................................................1335.21.1 UniBlitz Setup ..............................................................................................133

5.22 Virtual Shutter & Filter ...................................................................................................1345.22.1 Virtual S&F Setup.........................................................................................134

5.23 Virtual Stage...................................................................................................................1355.23.1 Virtual Stage Setup .......................................................................................135

5.24 Yokogawa CSU-22 Confocal Scanning Unit ...................................................................1365.24.1 Yokogawa CSU-22 Setup .............................................................................136

5.25 Zeiss AxioImager Microscope Control............................................................................1375.25.1 Zeiss AxioImager Setup ................................................................................137

5.26 Zeiss AxioPlan Microscope Control................................................................................1395.26.1 Device Setup Dialog .....................................................................................1395.26.2 AxioPlan Setup .............................................................................................139

Part III Processing Extensions 1416 Fluorescence CV..............................................................................141

6.1 Color Join .......................................................................................................................1416.1.1 Color Join Controls Palette..............................................................................1416.1.2 Color Join Dialog Box ....................................................................................145

6.2 Colocalization.................................................................................................................1506.2.1 Important Details ............................................................................................1506.2.2 I/O Palette.......................................................................................................1516.2.3 Colocalization Dialog Box ..............................................................................153

6.3 Line Measure ..................................................................................................................1636.3.1 Draw Lines .....................................................................................................164

xi

6.3.2 Pick the Window and Frame ...........................................................................1646.3.3 Set Up the Measurement .................................................................................1656.3.4 Set Up the Output............................................................................................1656.3.5 See Results......................................................................................................165

7 MicroTome™ ..................................................................................1677.1 About MicroTome ..........................................................................................................1677.2 Overview of Digital Deconvolution.................................................................................168

7.2.1 PSFs (Point Spread Functions) ........................................................................1687.2.2 Number of Iterations .......................................................................................1697.2.3 Background Attenuation, or Scaling the Data ..................................................169

7.3 Images MicroTome Can Deconvolve ..............................................................................1707.3.1 Frames and Sequences ....................................................................................1707.3.2 Size.................................................................................................................170

7.4 Using MicroTome...........................................................................................................1707.4.1 Microscope (Mic) Data ...................................................................................1707.4.2 Rapid Deconvolution ......................................................................................1737.4.3 Constrained Iterative Frame ............................................................................1757.4.4 Constrained Iterative Volume..........................................................................176

7.5 Troubleshooting: Image Acquisition Issues .....................................................................1787.5.1 Image Capture Tips.........................................................................................1797.5.2 Thickness of the Specimen..............................................................................1797.5.3 Sources of Data...............................................................................................1797.5.4 Slice Spacing ..................................................................................................179

7.6 Acknowledgements.........................................................................................................1807.7 Bibliography ...................................................................................................................180

8 Index ................................................................................................183

Acquisition - Overview of the Acquisition Process 1

Part IAcquisition

1 Overview of the Acquisition ProcessiVision offers a device-independent system for acquiring images. The benefit of this system is that iVision, its scripts,and its acquisition commands will function the same regardless of the frame grabber or camera you are using.

This chapter gives you an overview of the basic procedure for collecting data. Please see chapter 2 (page 5) for detailedexplanations of how to use the Camera menu’s commands.

1.1 Identifying Your Camera the First TimeBefore you can use a camera for the first time, you must identify the camera to iVision. Please do the following:

1. Turn on the camera and start iVision.

2. When iVision starts, it will open the Device Setup dialog box. If necessary, you can select Device Setup from theControl menu.

In the Device Setup dialog box:

a. Select your camera and click the Setup button.

b. Change any settings and click OK.

c. Click Done (or Continue) in the Device Setup dialog box. Your camera will now be connected to(communicating with) iVision.

3. Choose Select Camera from the Camera menu.

Pick the camera you wish to use.

4. Choose Camera Settings from the Camera menu.

Configure the camera as you like.

You only have to do this once. After you have set up your camera the very first time, you can start iVision and godirectly to the acquisition commands.

1.2 Switching CamerasYou can easily switch from one camera to another if you have already set them up. Simply choose Select Camera(Camera menu) and pick the camera you wish to use.

When you switch cameras, all of the parameters for the Camera menu commands will switch to those last used for thiscamera. (This does not alter scripted commands.)

2 Acquisition - Overview of the Acquisition Process

1.3 Setting Acquisition ParametersTo acquire an image, choose one of the acquisition commands:

• Preview• Single Exposure• Full Acquire• RGB Color Acquire• Multi Filter Acquire

You can then set the exposure time, image size, and other parameters. Once you have the options set the way you wantthem, you can use the command repeatedly without changing anything.

iVision’s simple scripting mechanism and its usage of variables gives you an even greater control of the acquisitioncommands.

• You can record acquisition commands in scripts, letting you accurately repeat the same data acquisitionprocedure.

• You can also store acquisition parameters and other numbers in variables, which lets you manipulate theparameters to give you detailed control of the data collection.

Please see the iVision User’s Guide for a full description of scripting and using variables within iVision.

1.4 Previewing the AcquisitionAll of the acquisition commands give you the option to see a constantly updated ("live") image from the camera: apreview of your captured picture. While iVision is in preview mode, you will see a Preview palette next to the liveimage. Use the Preview palette to adjust the exposure time, normalization, and other settings while looking at the effectsupon the image. This is a good way to determine the correct exposure time to use in your experiment.

Acquisition - Overview of the Acquisition Process 3

The Preview Palette used bymost of the commands

The Multi -D Preview Palette used bythe Multi -D Acquire command

Described on pages 12-14. Described on page 39

1.5 Grabbing the ImageOnce you are satisfied with the preview, click the Grab button. The acquisition will stop if you click the Cancel button.

After you become comfortable acquiring images, you can uncheck the Preview option and grab the data immediatelyafter clicking OK in the command’s dialog box.

Please see the detailed descriptions of the acquisition commands in the next chapter.

Preview: page 6Single Exposure: page 7Full Acquire: page 11RGB Color Acquire: page 21Multi -D Acquire: page 29

Acquisition - Camera Menu Commands 5

2 Camera Menu CommandsThe Camera menu contains the commands that will enable you to grab images for later analysis.

2.1 Camera ToolbarThe camera toolbar provides an alternative way to run some of the most common commands in the Camera menu.

The Camera Toolbar

The commands are (left to right) Preview, Snapshot, Full Acquire, and Multi-D Acquire. The name of each commandwill be shown in the area below the icons when you place the mouse over each icon.

With the exception of Snapshot, clicking the icon opens the corresponding command’s dialog, as if you had selected itfrom the Camera menu. The Snapshot command will simply take a snapshot, using the settings last set by choosing thecommand from the menu.

The Camera Toolbar icons are scriptable.

2.2 Select CameraAs described on page 1, you must pick the camera using this command. After you choose the camera once, you do notneed to use Select Camera again unless you want to switch to a different camera.

To select a camera, you must first connect it using the Device Setup command (Control menu).

Select Camera Dialog Box

After selecting the camera, you can configure it by using the Camera Settings command (Camera menu). Page 47describes the Camera Settings command.

2.2.1 TroubleshootingIf your camera is grayed out in the Select a Camera pop-up list, then your iVision camera control is installed but youneed to finish setting up and connecting the camera. Please do the following:

6 Acquisition - Camera Menu Commands

1. Make certain the camera is turned on. Also make certain that all cables are solidly connected.

2. Make certain you installed the manufacturer's camera drivers, if any. We put these in the "Camera Drivers"folder inside the "iVision 3.9 Folder".

3. Follow the directions on page 1 to connect the camera to iVision using the Device Setup command,

2.3 PreviewThe Preview command repetitively acquires images, allowing you to see a “live” image while you focus the microscopeor camera. You can also use Preview to interactively find the best acquisition settings. The Preview command does notcreate an image. It does not collect data because its only purpose is to help you focus on the sample.

Preview Dialog Box

Exposure: Click on the Exposure button to set the exposure time in seconds. You can also set up anauto-exposure to make iVision calculate the optimum exposure time.

Please read page 15 for a detailed description of the Exposure dialog box.

Image Size: Click on the Image Size button to set the dimensions of the image you will acquire.

Acquire a smaller image size for faster image updates. Bin the image to increase the imageintensity, reduce its size, and speed up acquisition.

Please read page 17 for a detailed description of the Image Size dialog box.

OK: When you click OK, you will see a Preview palette along with a live preview of your image.Adjust the exposure time, contrast, size, and other parameters of your acquisition.

Please read page 12 for a description of the Preview palette.

When you have finished focusing, click the Done button on the Preview palette or press either the Enter or Return key.The preview image will disappear along with the Preview palette.

Because Preview is a separate command, you may look at a fast preview without affecting the settings for the otheracquisition commands. For example, Preview can give you a fast preview using high bin sizes and low exposure timeswithout affecting the bin sizes and exposure time settings of the Single Exposure command.

2.3.1 Scripting the Preview CommandYou can record the Preview command in scripts. This command is very useful for letting the script's user check thefocusing of the microscope or camera and the placement of the sample.


If you run Preview from a script and close the Preview palette by clicking Cancel or pressing the Escape key, then youwill halt the script.

2.4 SnapshotThe Snapshot command grabs an image at a specific exposure time and binning. It assumes that you have alreadydetermined the proper settings for other camera controls (gain, offset, etc.) with a command like Preview. The name ofthe image will always be “Snapshot”, without any numbers appended to it.

Snapshot Dialog Box

You can only choose the exposure time and the binning to use.

If you check the “Use Exposure from Preview” checkbox, then the exposure time will be automatically taken from thelast exposure used by the Preview command. The time will be modified to take the selected binning into account. So ifthe Preview was at 1x1 binning with an exposure time of 200 ms, then a snapshot at 2x2 binning with the “Use Exposurefrom Preview” box checked will be taken at 50 ms.

If “Use Exposure from Preview” is not checked then the number entered in the Exposure time edit field will be used. It isnot adjusted for different binnings.

2.5 AutofocusiVision supports both software and hardware based auto focus methods, including a combination of both. Both methodsrequire the use of an automated z-stepper device. Hardware-based auto focus requires additional devices specificallysupported by iVision.

Software-based auto focus is implemented by scanning though a series of images acquired through a predeterminedrange of travel along the z axis. iVision uses sophisticated image processing to determine the best focused z positionautomatically.

Hardware-based auto focus methods typically depend on one of two methods. The first method uses a laser to find theinterface between the aqueous phase and the glass or plastic of a tissue culture plate or multi-well plate. The secondmethod uses a video camera, which feeds the live video signal into an auto focus video card. Based on the contrastmethod employed by the device the best focus position is determined and the device is positioned at this location. Witheither method, once the hardware auto focus is completed, iVision will start acquiring images.

The following example is based on using image (software) based auto focus. To use Auto Focus, please follow thesesteps:


1. Before setting the Auto Focus parameters, focus on your sample through the microscope eye pieces or with thecamera.

2. Set the radio button to Image.

3. Select the appropriate filter wheel and position to use during auto focus.

4. Select the correct shutter and shutter positions if a shutter is used during auto focus.

5. Choose the camera binning. The higher the camera binning, the faster the auto focus routine will be performed.

6. Set the Maximum Distance from Starting Point.

7. Enter the number of image planes on each side of the current focal point.

8. Select Gaussian Contrast for the Sharpness and set the Neighborhood to Large.

9. Click the press the Test button and the auto focus routine will start.

10. Confirm by view the location through the microscope eye pieces or camera. The auto focus may also be monitoredby polling the current z-position through the Z-Device located on the Device Toolbar.

11. To further test the current settings, move the sample with the z-stepper away from a location currently in focus.Rerun the test procedure and monitor the z-position. If the routine is working correctly, the current settings may besaved.

Autofocus Dialog Box

Type: Based on your camera and auto focus hardware, select the appropriate auto focus method.


Image: Software only auto focus using the camera to snap a series of images along a definedtravel distance in Z.

Hardware AF: Hardware based auto focus using either a laser- or video-based auto focusing device.

Hardware + Image: A combination of hardware and software auto focus methods. If this option isselected, hardware auto focus will be performed first, followed immediately bysoftware auto focus. This method is employed to reach the correct area first and thenuse software auto focus to do a fine focus adjustment.

Filter Wheel: This option is selected when using image (software) auto focus. Select the correct filter wheelor other positioning device used for auto focus. The filter wheel and filter position used inauto focus are not required to be one of the wavelengths acquired during the experiment. Forexample, your sample is labeled with DAPI and Cy5 and you only want to acquire z-stacks ofthe Cy5 channel while using the DAPI for auto focus.

Filter Position: Select the correct filter position used for auto focus. For example, select the DAPI filterposition if you want to use the DAPI channel for your sample to auto focus on.

Shutter: Select the correct shutter used during auto focus. If you are using fluorescence for auto focus,you will want to automatically control the shutter during this procedure to minimizephotobleaching.

Open/Close Position: Pick the motorized shutter that protects the sample from the light. Also choose the appropriateopen and closed positions.

Hardware AF: These options are available when using hardware-based auto focusing.

Device: Select the auto focus device used for auto focus.

Position: Select the appropriate position to invoke auto focus.

Z Offset After Autofocus: An offset in microns will be added or subtracted after the auto focus is complete.

Exposure Time: Set the camera exposure time for auto focus.

Bin Size: Set the camera binning for the auto focus.

Max Distance from Starting Point: This option sets the travel distance for image based auto focus. The total traveldistance is reported on the right side of the dialog box next to this option. During the autofocus routine, this is the distance over which images will be acquired to determine the optimalfocus location. It is important to set the distance based on the objective, objective’s workingdistance and the objective’s depth of focus. For example, if you are using a 60x oil objective,do not set the maximum distance for 200 microns and risk crashing the objective into theslide.

Number of Planes on Each Side: This option sets the number of images that will be taken on each side of the current z-position when the auto focus routine starts.

Total Number of Planes: Reports the total number of image planes used during the image based auto focus, based onthe number of planes entered into the above setting.

Maximum Resolution: The reported number is based on the maximum distance of travel and the number of planestaken on each side of the current z-position when the image based auto focus routine starts.

Sharpnes: Pick from two methods of calculating contrast pull-down menu: Local Contrast andGaussian Contrast. Local Contrast is best for images of reflective, solid type objects or


samples. The Gaussian Contrast choice is typically used for brightfield, transmitted light andfluorescence microscopy. The Gaussian algorithm is less sensitive to noisy images and thepreferred, but slightly slower, method.

Neighborhood: The relative size of the local neighborhood the contrast map is processing during the autofocus routine. For larger images over 1K x 1K in pixels, the Larger neighborhood should beselected from the pull-down menu.

Contrast Ratio: With a range of 1 to 10, this option calculates the ratio of image signal over the dynamicrange of the camera. This option is used to set a ratio limit for the auto contrast routine. If theratio does not pass the limit set, it is assumed no sample or objects were found during autofocus. If no sample or objects are found during auto focus, iVision will return to the previousz-position before auto focus was started.

Test: Clicking on the Test button will start the auto focus procedure based on the current settings.During the test process, a series of live images will be displayed in order to monitor the autofocus process. The series of images acquired during auto focus will only be displayed duringthe testing process. During a real image acquisition, these images will not be displayed.

2.6 Single ExposureUse the Single Exposure command to acquire a single image for analysis. It is meant to provide a simple way to grab animage when you do not need the numerous features of the Full Acquire command.

Single Exposure Dialog Box

The buttons on the Single Exposure dialog box work as described below. You can read complete descriptions for thesebuttons in the Full Acquire command’s section, starting on page 11.

Do Preview: When this option is checked, the Single Exposure command will give you a “live” previewimage. The Preview palette will let you change the acquisition parameters and see the effectsof your changes. When you click Grab on the palette, your image will be acquired anddisplayed on the screen.

Uncheck this option to grab an image immediately after clicking OK.

Please read page 12 for a description of the Preview palette.

Exposure: Click on the Exposure button to set the exposure time in seconds. You can also set up anauto-exposure to make iVision calculate the optimum exposure time.

Please read page 15 for a detailed description of the Exposure dialog box.


Image Size: Click on the Image Size button to set the dimensions of the image you will acquire.

Acquire a smaller image size for faster image updates. Bin the image to increase the imageintensity, reduce its size, and speed up acquisition.

Please read page 17 for a detailed description of the Image Size dialog box.

Destination: You may choose to put the new image into a new window or into the front window, if it is theright size.

Please read page 18 for a detailed description of the Destination dialog box.

2.6.1 Scripting the Single Exposure CommandYou can record the Single Exposure command in a script. Script the Single Exposure command any time you want tocollect a single frame of data.

2.7 Full AcquireThe Full Acquire command gives you the full range of acquisition options. You can:

• Grab single images or sequences of images.• Use noise-reducing processing .• Trigger the camera from keyboard or from external hardware (with most cameras).• Use external shutters.• Grab time-lapse sequences of images.

Full Acquire Dialog Box

Full Acquire's options are described below on these pages:

Option Page


Preview, Preview palette:Processing:Trigger:

Exposure:Image Size:Do Timelapse, Time Lapse button:

Destination:

Use External Shutter, Shutter button:Create Timing Window:

121515

151719

18

2021

This same information describes the options of the other acquisition commands, as well.

2.7.1 Preview (Full Acquire)The preview is a “live,” constantly updated image. You can focus the microscope or adjust the exposure time and otherparameters while viewing the image on the screen. The Preview pop-up menu offers four options:

Before First Frame: Choose this option to preview before any images are grabbed. When you press the Grabbutton in the Preview palette, the preview will stop, and the image or image sequence will begrabbed without interruption.

Before Each Frame: Choose this option to preview before grabbing each image in a sequence. This allows you tomake changes between frames (for example, you could change the exposure time or focusbetween each frame).

None, Wait for Key: With this option, there will be no preview, but images will not be grabbed until a key on thecomputer keyboard is pressed.

None, No Wait: Choose this option to begin acquiring immediately after the OK button is pressed in the FullAcquire dialog. iVision will neither preview nor wait for a key-press.

When you use a preview, the Preview palette will open and let you adjust your acquisition.

2.7.1.1 Preview Palette

The Camera menu's acquisition commands give you the option to see a constantly updated, or "live", display from thecamera: a preview of your captured image. While iVision is in preview mode, you will see this Preview palette next tothe live image.


Preview Palette

These are the palette's features:

Exposure Time: Adjust the exposure time in milliseconds. Read more about this on page 15.

If you click the arrow keys, iVision will automatically use the new time. If you type the newtime, click the Apply button. Click the Auto button to automatically calculate the optimumexposure time. (You may wish to read about auto-exposures on page 15).

Link to Binning: This feature scales the exposure time to match the Bin Size. When you uncheck thisoption, the exposure time will remain the same when you change the bin size.

For example, changing an image's bin size from 1x1 to 2x2 results in four pixelsbeing counted as one. The Link to Binning option would divide the exposure timeby four to keep the intensity nearly the same.

Data Range: When capturing grayscale data, the Min and Max fields display the minimum and maximumintensity values present in the entire image. Use this information to detect saturation and thenavoid it by adjusting the exposure time.

This section of the Preview palette also tells you what data type iVision is acquiring.

Gain, e Gain, Offset: Gain magnifies the camera's sensitivity. The signal-to-noise ratio will drop with increase ingain. If the camera is not receiving enough light, you can increase the gain to acquire animage. Offset raises the level of the signal. These settings may not be available for all


cameras, and their meanings can be different for some cameras, so please check your camera'sdocumentation for more detail.

Normalization: These controls improve the appearance of the image without altering the data.

Move the sliders to adjust the black and white points, which are the pixel values that will beshown as black and white. The small button to the left of the sliders will return the blackand white points to the extremes that the data type can contain.

Check the Auto-enhance Contrast box to automatically set the black and white points to theminimum and maximum pixel values present. Keeping this option checked will make thepreview intensity appear constant even when the amount of light being captured is changing.

Magnification: You can zoom in and out on the image at your convenience.

Bin Size: Use binning to brighten your image, reduce the exposure time, and/or reduce the size of yourimage. Please read more about binning on page 18.

Resize To: These buttons can change the size of the image you are acquiring. When you select a portionof the live image and click Resize to: ROI, iVision will only acquire that portion of theimage. When you click Resize to: Full Size, iVision will again acquire the full frame of theCCD. Acquiring only the ROI can be faster. You can read more on page 17.

Shutter: This option appears if you chose to use a shutter in the acquisition command (as described onpage 20).

Check the Keep Open While Live box to keep the shutter open during the entire preview.Otherwise, the shutter will open and close repeatedly, taking time. However, leave this boxunchecked if you must minimize photo-bleaching.

You can control the shutter by clicking Open and Close when the preview is paused.

: Click the Pause and Play buttons to temporarily turn the preview on and off.

Color Balance: This option appears when capturing color data from single-pass cameras (with a built-inmosaic filter). Clicking this button improves the image's color by adjusting the data valuescaptured by the camera.

You should first select an appropriate area for the color balance calculation. Select a feature-less (color-less) area for this. The values calculated will remain active for all subsequentimages captures.

Specifically, Color Balance calculates the mean intensity of each channel (red, green, andblue). It then scales the channels so that they all have the same mean intensity. To see thescaling values used, choose Set Color Balance (described on page 45) from the Cameramenu.

Snapshot: Click this button to quickly grab an image from the constantly updating preview. Your imagewill open in the background and the preview will continue.

Cancel: Click Cancel to stop the preview without collecting the data. (Any images grabbed with theSnapshot button will remain, of course.)

Grab: Click the Grab button to stop the preview and begin collecting your data. The acquisition willuse any changes you made to the settings during the preview.


2.7.2 Trigger (Full Acquire)When a Trigger option is selected, iVision will not acquire the image until the triggering signal is sent. The triggeringsignal could be a keyboard key pressed or an electrical pulse sent by hardware attached to the camera. This lets youcontrol exactly when the image is acquired. For example, you may not want the image acquisition to begin until yourdrug delivery hardware injects the drug into the cell. The drug delivery device could send a signal to the externalsynchronization port on your camera to start the image acquisition.

When using Trigger modes, any previews will be displayed first. Image acquisition will then stop, and the messageAwaiting Keypress or Awaiting External Trigger will appear on the image’s title bar. Pressing the Escape key willcancel the acquisition; pressing any other key will acquire the image. The Trigger options are:

No Trigger: With No Trigger selected, image preview or acquisition will begin immediately after Okay isclicked in the Full Acquire dialog.

Keyboard ... First: When Keyboard Trigger First is selected, iVision will not begin acquiring images until aftera keyboard key is pressed.

Keyboard ... Each: If you are grabbing a sequence of images with this option selected, you will have to press akeyboard key before each image acquisition.

External ... First: When External Trigger First is selected, iVision will not acquire the image until the camerareceives a triggering signal.

External ... Each: When External Trigger Each is selected, the camera must receive an external signal beforeacquiring each frame of a sequence.

2.7.3 Processing (Full Acquire)You can sum or average multiple frames into a single frame in order to improve the signal-to-noise ratio. The Processpop-up box becomes unavailable when the Do Timelapse checkbox is checked.

No Processing: When this default option is selected, none of the processing methods will be used.

Average, Sum: Averaging and summing images improves the signal-to-noise ratio for the final image. Wheneither Average or Sum is chosen, a Num Frames text box appears in which you should enterthe number of frames to be averaged or summed. That number of images will be acquired andadded together pixel by pixel. If you are averaging, the intensity value of each pixel will thenbe divided by the number of frames acquired.

2.7.4 Exposure (Full Acquire)When you click the Exposure button, a new dialog box appears to let you set the length of time during which the camerawill collect light from the sample. Please give the exposure time in seconds.


Exposure Dialog Box

Exposure Time: Enter the exposure time in seconds. This is the length of time for which the camera willcollect light before reading out the data to the computer.

You can use an exposure time stored in a variable by typing the variable's number andclicking the # button so it changes to Var.

Auto-Exposure: Auto-Exposure is a function that automatically determines the appropriate exposure time.

You would use this function if you do not know exactly what exposure time to use and youwant the computer to calculate it.

To use auto-exposure, please do the following:

1. Enter a first guess at the exposure time in the Exposure Time field.

2. Next, enter the Desired Maximum Pixel Value.

3. Enter the Maximum Number of Retries.

4. Click Calculate Now, check the Use Auto-Exposure box, or both.

Exposure Time: Enter your initial guess at the exposure time. The Auto-Exposure function willbegin its calculations using this first guess.

Use Auto-Exposure: Check this box if you want iVision to automatically calculate the exposure timebefore grabbing the preview or image.

Calculate Now: Click this button to immediately calculate the exposure time. The Exposure Timebox will display the new exposure time.

If you click Calculate Now and also check the Use Auto-Exposure box, theniVision will calculate the exposure time again before grabbing the preview or image.The second calculation will be better because it will use the newly calculated time asits first guess.

Desired Max. Value: The Desired Maximum Pixel Value is the highest pixel intensity you want in yourfinal image.

You may want to enter a value equal to half or two-thirds of the highest intensityyour camera can deliver. If your desired target value is too high, you will risk


overexposing the image. (The highest value that a 12-bit camera can deliver is 4095,so you may wish to use 2048. The highest given by an 8-bit camera is 255, so youmay wish to aim for 128.)

Maximum… Retries: The Maximum Number of Retries is the highest number of images you want theauto-exposure function to grab.

The higher the number, the more accurate the auto-exposure calculation. However,the light from too many exposures could damage some samples.

You can store the Desired Maximum Pixel Value and/or the Maximum Number of Retriesin variables by typing the variable's numbers and clicking the # buttons so they change to Var.

OK: Click OK to accept these settings and return to the Full Acquire dialog box.

To acquire the image using the settings you just entered, click OK and then click OK again inthe main dialog box.

Cancel: Click Cancel to discard these settings but still return to the Full Acquire dialog box. Theexposure time will remain unchanged since you clicked on the Exposure button.

After you acquire the image, the exposure time produced by Auto-Exposure will be displayed in the Exposure Timetext field and stored in variable #255.

2.7.5 Image Size (Full Acquire)Clicking on the Image Size button opens a dialog box that lets you set the dimensions of the image you will acquire.

Image Size Dialog Box

Image Size: In the section titled Image Size, you can specify the region of the camera’s CCD from whichto acquire the data. You may elect to capture the full size image or a sub-array. Smallerregions are usually read out faster than larger regions.


Full Frame: Click on this radio button if you want the new image to be the full size that the camera canproduce.

ROI on: This radio button lets you acquire only part of the full frame (that is, a sub-region of theCCD). You specify the sub-region by selecting a region within a window and then selectingthat window's name from the ROI on box.

This technique is very handy when you are acquiring a lot of images and want to keep theimage sizes small. Grab one full-size image first and draw a region of interest around theimportant details. Next, acquire images with the size set to ROI on. Your acquisitions willproceed much faster and won’t take up as much hard drive space.

Custom Rectangle: This feature lets you specify the exact borders of the sub-region to acquire from the CCD. Fillin the Left, Top, Right, and Bottom boxes to specify the edges of the image boundary.

You can store boundary locations in variables by typing the variables' numbers and clickingthe # buttons so they change to Vars.

Bin Size: Use binning to brighten your image, reduce the exposure time, and/or reduce the size of yourimage.

Binning sums the charge in adjacent CCD wells (pixels) within the CCD before sending theresults to the computer. When you use this option, you increase the intensity and the signal-to-noise ratio (SNR) of the image, while decreasing the spatial resolution. For example, whenBin Width and Height are set to 2, the image will be a quarter of the full size and about fourtimes as bright. In low light situations, binning may be used to make the image brighter.

If you want to keep the image intensity constant, reduce the exposure time by the same factorthat you are reducing the image size. (That is, when binning 2x2, you are quartering the imagesize and you may want to quarter the exposure time.)

2.7.6 Destination (Full Acquire)Clicking the Destination button raises a dialog box for specifying where to put the acquired image.

Destination Dialog Box


SelectImage Destination: You can put your new data in a new window or in the window of a pre-existing image.

New Window: When the New Window option is selected, the acquired image will be put into a newwindow with the name you give here.

Front Window: This option will place the image into the most recently selected window (the onewith the highlighted title bar). However, the front window must be the same datatype and size as the new image!

Grab to Disk: When this option is checked, iVision will save each image to a hard drive or other disk rightafter it acquires the image. This prevents a large number of images from cluttering yourcomputer’s RAM.

This option becomes available only when you have checked the Do Timelapse option in theFull Acquire dialog box.

The following options become available:

File Type: This is the format of the file you will be saving. Please see the descriptions of the fileformats in the Operation chapter of the iVision User's Guide.

Base Name: Enter the name that you want given to all of the time lapse images. Sequential, zero-filled numbers will be appended to this name. For example, “TimeLapse ” wouldbecome “TimeLapse 0000,” “TimeLapse 0001,” “TimeLapse 0002,” etc.

If you check the Use Window Name checkbox, then the name set in the New textfield will be used as the base name.

Number of Digits: Set the number of digits that will be appended to the base name.

Folder: This is the name of the destination folder where the images will be saved. To selectthis folder, click on the Choose button. A dialog box will appear to help you find thecorrect folder.

2.7.7 Do Timelapse (Full Acquire)Check this box to acquire a time lapse image sequence. Then click the Timelapse button to enter the time lapseparameters (the number of frames and the interval time).

2.7.8 Timelapse (Full Acquire)In a time-lapse acquisition, iVision will acquire a set number of images at set intervals of time. Time lapse is very usefulfor looking at developing, changing, or moving subjects. To gather time lapse data, check the Do Timelapse checkbox,and then click this button to enter the parameters:


Timelapse Dialog Box

Number of Frames: Enter the number of frames you want to acquire (that is, the number of iterations).

Do Timelapse: This allows you to specify a delay between each frame.

If you do not check this box, then the frames will be taken as quickly as possible. For mostcameras, this will be faster than if you had checked the Do Timelapse box and entered a delayof 0. For some cameras it will be measurably faster.

Delay: Enter the length of time (in seconds) between the start of each image acquisition.

Display After Each… This will update the display with the latest image after it is captured. Since this can slow theacquisition speed, it is not recommended for small delay times (less than 1 second) or if youdid not check the Do Timelapse box. It should be checked for long timelapse delays (5seconds and greater), since it lets you check the quality of each image.

When Time Lapse is selected, the Destination dialog changes to let you save each image to disk right after it isacquired. This lets you acquire a lot of data without filling up your RAM. Also, the following options become disabledbecause they are unnecessary: Preview: Before Each Frame, Keyboard Trigger: Trigger Each, and the Number ofFrames field in the Image Size dialog. Also, the Process options become unavailable.

When you begin the time lapse acquisition, iVision will display a progress bar to tell you what is happening.

2.7.9 Use External Shutter (Full Acquire)Check this box if you have a shutter protecting your sample from the light source and you want it to close betweenacquisitions. Then use the Shutter button to pick the hardware.

2.7.10 Shutter (Full Acquire)When Use External Shutter is checked, this button lets you set up the shutter that will work in tandem with the camera.You must have already used the Device Setup command (Control menu) to set up the shutter.


Shutter Dialog Box

Use the features from in order from the top to the bottom:

Select Device: First, select the correct shutter hardware from the Device pop-up menu. This menu only listsshutters unless you check the Show All Devices in List checkbox.

Select Position: Next, choose the correct positions to use for the Open and Closed positions. For mosthardware, this will be obvious, but it is also possible to assign an empty filter wheel positionto Open and an opaquely blocked filter wheel position to Close.

Try Now: You can click the Try Now buttons to test the shutter (or other hardware).

Extra Open Time: In the rare case that the camera has a time lag before it starts to capture an image, increase theamount in the Extra Open Time field. This will keep the shutter open longer to account forthe lag time and not close the shutter too early. If you think you need to change the ExtraOpen Time, add time to it until the sum intensity of the image does not increase.

Keep Open… Clicking Keep Open... saves time by keeping the shutter open during capture of multipleframes. Otherwise, the shutter would have to close and open again between each imagecapture. However, this exposes your sample to more light, which may damage some samples.

2.7.11 Create Timing Window (Full Acquire)This option will create a text window to document the relative time at which each image was grabbed, starting from thefirst capture. The text window will be named Timing Window.

2.7.12 Scripting the Full Acquire CommandYou can record the Full Acquire command in a script.

2.8 RGB Color AcquireThis command grabs a three-pass RGB color image from a monochrome camera with an external filter. RGB ColorAcquire lets your monochrome camera mimic a color camera; you use only one exposure time and then you use Color


Balance to adjust the colors. This is unlike the Multi-Filter Acquire command, which adjusts the colors by usingmultiple exposure times.

Use RGB Color Acquire for bright field images; use Multi-Filter Acquire (described on page 29) for fluorescenceimages.

To use RGB Color Acquire, please do the following:

1. First, you must set up your filter device and any external shutter. If you have already done this, you can skip thisstep.

a. Use the Device Setup command (Control menu) to set up the hardware.

b. Use the Device Select command (Control menu) to identify and name the hardware.

2. After you do that, choose the RGB Color Acquire command and set up its parameters.

3. Click the OK button.

RGB Color Acquire Dialog Box

This command’s options are described briefly below. Please read the referenced sub-sections for more detail.

Do Preview: Check this box to adjust the acquisition settings while watching the effects in a "live" colorimage. Please see page 23.

Filters: Click the Filters button to choose the filter device and the filters. Please see page 23.

Exposure: Enter the exposure time for the image. Please see page 23.

Image Size: You can acquire the whole image or a sub-region, and you can bin the data. Please see page24.

Timelapse: Check this box and click the Timelapse button to grab a sequence of color images over time.Please see page 27.

Destination: Specify where to put the acquired image. Please see page 25.


Use External Shutter: Check this box and click the Shutter button to protect your sample from the light source byusing a motorized shutter. Please see page 27.

Create Timing Win: Record the relative time at which each image was grabbed. Please see page 28.

2.8.1 Do Preview (RGB Color Acquire)Check this box to see a constantly updated, or "live", image before you begin grabbing data. While iVision is in previewmode, you will see a Preview palette next to the live image. The Preview palette lets you adjust the focus and theacquisition settings while viewing the live image on the screen.

Please read the description of the Preview palette that starts on page 12 (within the description of the Full Acquirecommand).

2.8.2 Filters (RGB Color Acquire)When you click on this Filters button, another dialog box appears to let you pick the filter-switching device and thecorrect filter positions. Filter devices include liquid crystal filters and filter wheels. These can be anywhere in the lightpath in front of the camera.

You must have already set up the hardware in the Device Setup and Device Select commands (Control menu) beforechoosing the Multi-Filter Acquire command.

Filters Dialog Box

1. First, select the correct filter-switching hardware from the Select Filter Device box.

2. Next, choose the correct filter positions to use for the Red, Green, and Blue channels.

3. Clicking the Try Now buttons will test the filter device by switching it to the corresponding position.

4. Click OK in order to use this filter setup.

2.8.3 Exposure (RGB Color Acquire)When you click the Exposure button, a new dialog box appears to let you set the length of time during which the camerawill collect light from the sample. Please give the exposure time in seconds.


Exposure Dialog Box

Exposure Time: Enter the exposure time in seconds. This is the length of time for which the camera willcollect light before reading out the data to the computer.

You can use an exposure time stored in a variable by typing the variable's number andclicking the # button so it changes to Var.

Auto-Exposure: Auto-Exposure is a function that automatically determines the appropriate exposure time.

You would use this function if you do not know exactly what exposure time to use and youwant the computer to calculate it.

Please read page 16 for detailed instructions on how to use the Auto-Exposure function.

Exposure Time: Enter your initial guess at the exposure time.

Use Auto-Exposure: Check this box to do an auto-exposure before grabbing the preview or image.

Calculate Now: Click this button to immediately calculate the exposure time.

Desired Max. Value: Enter the highest pixel intensity you want in your final image.

Maximum… Retries: Enter the highest number of images you want the auto-exposure function to grab.

2.8.4 Image Size (RGB Color Acquire)When you click on the Image Size button, a new dialog box appears to help you set the dimensions of the image(s) youwill acquire.


Image Size Dialog Box

Image Size: These options let you specify the region of the camera’s CCD from which to acquire the data.Smaller regions are usually read out faster than larger regions.

Full Frame: Choose this to capture the full size image.

ROI On: You can capture a sub-array by referring to an ROI that has already been drawn onan existing image. The new image will only include the region of interest.

1. Acquire a full frame image.

2. Next, define an ROI on that image around the portion of interest.

3. Finally, acquire another image, but select the ROI On option and choose thename of the already-existing image from the pop-up box.

Custom Rectangle: You can capture a sub-array by specifying the left, top, right, and bottom edges ofthe image boundary.

Bin Size: Binning sums the charge in adjacent CCD wells (pixels) within the CCD before sending theresults to the computer. When you use this option, you increase the intensity and the signal-to-noise ratio (SNR) of the image, while decreasing the spatial resolution.

For example, when Bin Width and Height are set to 2, the image will be a quarter of the fullsize and about four times as bright. In low light situations, binning may be used to make theimage brighter.

2.8.5 Destination (RGB Color Acquire)Clicking the Destination button raises a dialog box for specifying where to put the acquired image.


Destination Dialog Box

SelectImage Destination: You can put your new data in a new window or in the window of a pre-existing image.

New Window: When the New Window option is selected, the acquired image will be put into a newwindow with the name you give here.

Front Window: This option will place the image into the most recently selected window (the onewith the highlighted title bar). However, the front window must be the same datatype and size as the new image!

Grab to Disk: When this option is checked, iVision will save each image to a hard drive or other disk rightafter it acquires the image. This prevents a large number of images from cluttering yourcomputer’s RAM.

This option becomes available only when you have checked the Do Timelapse option in theRGB Color Acquire dialog box.

The following options become available:

File Type: This is the format of the file you will be saving. Please see the descriptions of the fileformats in the Operation chapter of the iVision User's Guide.

Base Name: Enter the name that you want given to all of the time lapse images. Sequential, zero-filled numbers will be appended to this name. For example, “Color_01_” wouldbecome “Color_01_0000,” “Color_01_0001,” “Color_01_0002,” etc.

If you check the Use Window Name checkbox, then the name set in the New textfield will be used as the base name.

Number of Digits: Set the number of digits that will be appended to the base name.

Folder: This is the name of the destination folder where the images will be saved. To selectthis folder, click on the Choose button. A dialog box will appear to help you find thecorrect folder.


2.8.6 Timelapse (RGB Color Acquire)In a time-lapse acquisition, iVision will acquire a set number of images at set intervals of time. Time lapse is very usefulfor looking at developing, changing, or moving subjects. To gather time lapse data, check the Do Timelapse checkbox,and then click this button to enter the parameters:

Timelapse Dialog Box

Do Timelapse: This allows you to specify a delay between each frame.

If you do not check this box, then the frames will be taken as quickly as possible. For mostcameras, this will be faster than if you had checked the Do Timelapse box and entered a delayof 0. For some cameras it will be measurably faster.

Delay: Enter the length of time (in seconds) between the start of each image acquisition.

Display After Each… This will update the display with the latest image after it is captured. Since this can slow theacquisition speed, it is not recommended for small delay times (less than 1 second) or if youdid not check the Do Timelapse box. It should be checked for long timelapse delays (5seconds and greater), since it lets you check the quality of each image.

When Time Lapse is selected, the Destination dialog changes to let you save each image to disk right after it isacquired. This lets you acquire a lot of data without filling up your RAM.

When you begin the time lapse acquisition, iVision will display a progress bar to tell you what is happening.

2.8.7 Use External Shutter (RGB Color Acquire)Check this box if you have a shutter protecting your sample from the light source and you want it to close betweenacquisitions. Then use the Shutter button to pick the hardware.

2.8.8 Shutter (RGB Color Acquire)When Use External Shutter is checked, this button lets you set up the shutter that will work in tandem with the camera.You must have already used the Device Setup command (Control menu) to set up the shutter.


Shutter Dialog Box

Please use the features from in order from the top to the bottom:

Select Device: First, select the correct shutter hardware from the Device pop-up menu. This menu only listsshutters unless you check the Show All Devices in List checkbox.

Select Position: Next, choose the correct positions to use for the Open and Closed positions. For mosthardware, this will be obvious, but it is also possible to assign an empty filter wheel positionto Open and an opaquely blocked filter wheel position to Close.

Try Now: You can click the Try Now buttons to test the shutter (or other hardware).

Extra Open Time: In the rare case that the camera has a time lag before it starts to capture an image, increase theamount in the Extra Open Time field. This will keep the shutter open longer to account forthe lag time and not close the shutter too early. If you think you need to change the ExtraOpen Time, add time to it until the sum intensity of the image does not increase.

Keep Open… Clicking Keep Open... saves time by keeping the shutter open during capture of multipleframes. Otherwise, the shutter would have to close and open again between each imagecapture. However, this exposes your sample to more light, which may damage some samples.

2.8.9 Create Timing Window (RGB Color Acquire)This option will create a text window to document the relative time at which each image was grabbed, starting from thefirst capture. The text window will be named Timing Window.

2.8.10 Scripting the RGB Color Acquire Command.You can record the RGB Color Acquire command in a script. This lets you quickly use the same settings repeatedly,and it also lets you repeatedly use the command within a larger protocol.


2.9 Multi Dimensional AcquireMulti Dimensional Acquire allows you to capture multi-spectral and three-dimensional data:

• Capture images of one wavelength or up to seven different wavelengths (multiple spectra)

Multi-spectral data are images of the same sample acquired at different wavelengths. Data for each wavelengthis stored in a separate image sequence. iVision acquires these by switching filters in the light path, using amotorized filter switcher (e.g. filter wheel, slider, or liquid crystal filter) and then grabbing the images.

• Capture sequences of three-dimensional data

3D sequences are linear series of images where each image is taken at a different height. When viewed as amovie (using the Animate command in the View menu), the image appears to be panning up and down throughthe sample. (These are also called through-focus images, or Z-series.)

• Capture 3D sequences of multi-spectral data

This is an extremely versatile command, giving you a lot of control over the collection of a large amount of data.

Using Multi Dimensional Acquire:

1. First, you must have selected a camera using the Device Setup command (Control menu) and the Select Cameracommand (Camera menu), described on page 5

2. Set up your filter-switcher and/or focus motor using the Device Setup command (Control menu) and the SelectDevices command (Control menu), which is described on page 71.

• For multi-wavelength acquisitions, you need the filter wheel, liquid crystal tunable filter, or other filterswitcher.

• For 3D acquisitions, you need the focus motor.

3. Select the Multi Dimensional Acquire command and fill in its options. Pages 30 through 43 describe the options indetail.

• You can choose to begin the acquisition immediately by unchecking the Preview option.

• You can also choose to begin the acquisition interactively, using the Preview dialog box to set up 3Dacquisition and other parameters while viewing the live image.

When using the Preview control panel, click the Continue/Pause button to toggle the continuous acquisitionmode on and off. Acquisition will be paused when the preview mode first begins, to prevent unnecessaryexposure of the sample.

During the preview, you can interactively set the starting and ending positions for your 3D stack image andoptimize the exposure times for each filter position.

4. When you are ready to acquire the data, click OK, and the full set of data will be collected for you automatically.The information related to the acquisition parameters is displayed in a separate table and is also assigned to specificiVision variables.

Results produced by Multi Dimensional Acquire:

Multi Dimensional Acquire produces one image sequence for each color acquired. If the use Z step was selected, theneach sequence will contain a 3D volume of data. In addition to the image data, It generates a table of values with all theparameters of the acquisition experiment.


You have three options:

Use Z Step Use Timelapse Use Z Step AND use Timelapse

Will generate one 3D sequence foreach filter position used.

Will generate one Timelapse sequencefor each filter position used.

Will generate N 3D sequences foreach filter position used.

N corresponds to the number of timepoints in the Timelapse.

This allocation is appropriate for performing post-processing steps such as 3D deconvolution and 3D rendering. Eachsequence will hold the data acquired with a different filter. Please note that this can amount to a lot of data requiring alarge amount of RAM.

If you do not use the Z-Step (3D) or Time Lapse features, then each image "sequence" will contain one frame. If you doacquire a 3D or time-lapse sequence, then each image sequence will contain multiple frames. Click apple arrow keys toview each frame and step through the sequences. You can also use the Animate command (View menu) to observe yourdata in motion.

2.9.1 Multi Dimensional Acquire: General TabWe suggest you pay particular attention to the Preview option, as it gives you powerful, interactive control over yourdata acquisition.


General Tab of the Multi Dimensional Acquire Dialog Box

Preview: Checking this box turns Multi Dimensional Acquire into an interactive command.

When the user checks Preview and clicks OK, the Preview dialog box appears and previewmode begins. The Preview dialog lets the user alter the multi-D acquisition parameters whilelooking at the image on screen. These include the exposure for each filter position, thebinning, and the region of interest to be captured.

The Multi Dimensional Acquire Preview dialog is described in detail starting on page 39.

Scripting: If you are scripting the Multi Dimensional Acquire command, you should probablyleave Preview unchecked.

Experiment Name: The name you type in the Experiment Name dialog box will be given to the image windows.When using a filter-switching device, the name of the filter used will be appended to theexperiment name, and this will become the name of the window (for example, "Nematodesectioning - Dapi").

CreateTiming Window: When you check this box, iVision will record the time after acquiring each set of colors.

For example, when acquiring red, green, and blue channels, iVision will record the time afterit grabbed the image of the blue channel. When acquiring 3D sequences, iVision will record atime for each Z-step. The times will be recorded into a window named with the experimentname and the word "_Timing."

Display


After Each Grab: When you check this box, each frame of a multiple image acquisition will be displayed as it iscaptured.

Speed: In most cases the time-limiting step is the mechanical filter switching. However,when you are concerned about acquisition speed, do not check Display After EachGrab. Then the program will not have to waste acquisition time by updating thedisplay on the screen.

2.9.2 Multi Dimensional Acquire: Size TabThe main dialog's Size tab controls which portion of the CCD will be read and put into the window, and how muchbinning will be used.

Size Tab of the Multi Dimensional Acquire Dialog Box

Full Frame: Click on this radio button if you want the new image to be the full size that the camera canproduce.

ROI on Window: This radio button enables you to acquire only part of the image's full frame (i.e. a sub-regionof the CCD).

1. To do this, draw a ROI on an image. If you have already acquired an image, draw a ROIaround a cell or feature.

2. Next, select the Multi Dimensional Acquire command.

3. Click on this radio button.


4. Select (or type) the name of the existing image from the drop-down box.

When you use this feature, the newly acquired image will only contain the cell or feature youselected. You can use an ROI drawn on any image.

If you draw the ROI on a binned image and acquire the sub-region with the same bin size,then the new image will be the same size as the ROI drawn. If you draw the ROI on anunbinned or newly created image, then the new image will be binned according to theAcquire command’s Bin Width and Height parameters.

Acquiring using the ROI on a window is very handy when you are acquiring a lot of imagesand want to keep the image sizes small. Grab one full-size image first and draw a ROI aroundthe important details. Next, acquire images with the size set to ROI on Window. Youracquisitions will proceed much faster and without taking up as much hard drive space.

Custom Rect: The Custom Rectangle radio button lets you specify the exact borders of the image you wantto acquire. The Left, Right, Top, and Bottom values are the locations of the edges of therectangle. These locations are measured in pixels from the upper left corner of the CCD.

Binning: Binning affects both the resolution of the image (the number of pixels that represent the data)and the exposure time needed to acquire the image.

A bin is a group of pixels made by counting a rectangular group of pixels out as if they wereone. If two pixels side by side were counted out as if they were one pixel, we would say thatthey were binned 2x1, and the resultant image would be half as wide as normal, and brighter,too.

Because light from multiple pixels is being counted as one value, binning reduces theexposure time dramatically.

2.9.3 Multi Dimensional Acquire: Shutter & Filters TabThe main dialog's Shutter & Filters tab lets you pick the motorized shutters and filter switchers for the MultiDimensional Acquire command to control.

1. Choose motorized shutters in the top portion of this tab.

You can use two shutters for switching between two light sources, such as multi-mode imaging, acquiring bothfluorescence and bright field data.

2. Pick the filter switcher in the tab's bottom section.

3. If you don't have one or the other, simply choose "None" for that device.


Shutter & Filters Tab of the Multi Dimensional Acquire Dialog Box

For easiest use, fill in these options in the order they are described here:

Shutters:

Shutter 1, 2;Open, Close Pos: Pick the motorized shutter that protects the sample from the light. Also choose the

appropriate open and closed positions.

Keep OpenDuring Z / Preview: Check this box to keep the shutter open during preview mode or while acquiring a Z

sequence.

When doing multi-mode imaging (using two light sources), you should not checkthis box for the bright field shutter.

Shutter 1, 2Open Delay: This extra delay may be needed to better handle cameras with a slower initialization

rate.

The range of delays is usually in the range of 5 ms to 120 ms (typical 30ms); themaximum allowed is 5 seconds. If you think you need to change the Shutter OpenDelay, add time to the Delay until the sum intensity of the image does not increase.

Filter Switchers:


Filter Wheel: Pick the appropriate wavelength-changing hardware. The typical choices areexcitation filter wheels, monochromators, emission filter wheels or a completemicroscope filter cube.

Filter Name: Pick the filters from the Filter Name boxes in the order you want them used.

Shutter: Pick whether shutter 1 or 2 will be opened when iVision switches to this filter. Thiscontrols which light source is used. What mechanical shutter corresponds to number1 and 2 is specified in the shutters section of the dialog.

Exp. Time: You can use a different exposure time (in milliseconds) with each filter toaccommodate your sample.

If you set the #/Var icon to Var for any of the parameters, that parameter value willcome from the specified variable.

AE: The Autoexposure function will calculate optimized exposure times for this filterwhen this box is checked. The Exp. Time: value will be used as the initial guess.

Tar. Max: The Target Maximum is the maximum intensity value for which the autoexposurefunction will aim. Set this about 2/3 or 3/4 as high as the camera's maximum output.You want to approach but not reach the camera's maximum value.

Enable: Use the buttons remove/add each filter you want to use. You can enable up to 7different filters.

AE Retries: This is the number of times iVision will try again to calculate a good autoexposuretime. You may set it higher than one for brightfield images, but it is usually set tozero for fluorescence in order to avoid unnecessary photo damage to the sample.

To do multi-mode imaging (grabbing both fluorescence and bright field images, including phase or DIC), please followthese steps:

1. Assign the fluorescence and bright-field shutters to Shutter 1 and Shutter 2.

For this example, please assume that Shutter 1 is your fluorescence excitation shutter, and Shutter 2 is your brightfield shutter.

If your system does not include a motorized, bright-field shutter, you may be able to use a computer controlled lamppower supply or a bright-field filter wheel as Shutter 2. You may need to set the Shutter 2 Open Delay to accountfor the duration of the lamp power-up, however.

2. Do not check the Keep Open During Z / Preview box for the bright field image.

This is because if the bright field shutter remained open, the light would wash out the fluorescent signal.

3. Choose the Shutter to determine which lamp will be used for each filter (and therefore for each image).

If the filter wheel is mounted on the fluorescence excitation path, and you are using the brightfield shutter, the actualposition of the filter wheel does not matter; pick anything for the Filter Name.

If you are switching a complete microscope cube set, you may switch an analyzer for DIC imaging in and out of thebrightfield light path. You can also do DIC-fluorescence imaging with a set of linked Filter wheels (see the DeviceSetup section on page 68).

4. Click OK or set something else within Multi Dimensional Acquire.


2.9.4 Multi Dimensional Acquire: Z-Steps TabThis tab lets you acquire 3D sequences (through-focus images). If you are also using the Time Lapse tab, then you cangrab multiple 3D data sets within one experiment.

All of these positions and distances are measured in microns.

Z-Steps Tab of the Multi Dimensional Acquire Dialog Box

Use Z Step: Check the Use Z Step box to acquire 3D sequences. Leave this box unchecked if you onlywant images of a single plane.

Use Current Pos. asZero (relative move): Check this box to set the starting and stopping positions relative to the Z-motor's current

position. For example, setting Start Pos. to -5 and Stop Pos. to 5 would image a 10-micronthickness around the current position. If unchecked, it will measure the starting and stoppingpositions from the motor's origin (absolute movement).

In both cases you can review/change the actual start and end positions within the previewdialog before capturing the data.

Start, Stop Pos.: Use the Start Pos. and Stop Pos. fields to enter the starting and stopping positions for the 3Dacquisition. These are the initial and final distances of the focus motor from its zero (home)position. We recommend moving upwards, to avoid the backlash effects of the microscope'sfocus mechanism.


Step by: In the Step by field, enter the interval distance between image planes. This is also called theZ-step size.

Note: Multi Dimensional Acquire captures one image at the bottom of the sample, before stepping upwards. Thus, ifyour starting position is at 0 µm, your stopping position, is at 10 µm, and your step size is 1 µm, you will geteleven images, not ten: one image at the bottom of the sample, and ten more images above that, taken at 1 µmintervals.

If the Use Current Pos. as Zero box is checked, the values in this dialog will not be updated by the values enteredduring the Preview process. If unchecked (Absolute move), then the values in this dialog will reflect the actual range oftravel captured in the last Multi-D acquire command. This allows you to capture 3D stacks one wavelength at a timewhile changing the filter position manually.

Use Current Pos. as Zero is: MultiD (Zstage) values :

Checked: Will Not be updated, they reflect relative movements.

Unchecked: Will be updated, they reflect absolute movements.

2.9.5 Multi Dimensional Acquire: Time Lapse TabThis tab lets you acquire time-lapse sequences. If you are also using the Z-Steps tab, then you can grab multiple 3Dimages (One image window per time point).

Time Lapse Tab of the Multi Dimensional Acquire Dialog Box

Use Time Lapse: Click in this checkbox in order to perform a time lapse acquisition.


Experiment Setup: iVision requires three values for the experiment: Frames, Interval, and Experiment Length.Click the radio button next to the value you want iVision to calculate for you; that value willbecome grayed out. Then type in values for the other two.

Frames: This is the number of frames you want to acquire for each filter position.

Interval: Interval is the length of time taken by one iteration. Interval can also be thought ofas the time between starting to grab one set of images (one at each filter position)and starting to grab the next set.

Experiment Length: Experiment Length is the total amount of time taken by the time lapse experiment.

For example, clicking on Experiment Length will gray it out. You would then enter thenumber of Frames and the length of each iterations into the Interval field. The dialog boxwould automatically calculate the Experiment Length.

Changing the units used for Interval and Experiment Length does have an effect on thecalculated values. The available units of time are milliseconds (ms), seconds, minutes, andhours.

You can choose for any parameter (except the one to be calculated) to be taken from anumeric variable. Do this by toggling the # button so it reads Var. Then enter the number ofthe variable where the parameter is stored. When a variable has been given for a parameter,the grayed out calculated value field will disappear. This is because the result of a calculationdependent on a variable cannot be displayed in real time.

Grab to Disk: The Grab to Disk option automatically saves each image immediately after it is acquired(“grabbed”). Images will be saved as indexed files, with the experiment name (General Tab)followed by a number. The numbered file names allow you to open the images sequentially ata later time. You can then convert them into a single-file sequence of images, or you canprocess each image in turn. You must be have checked the Time Lapse checkbox to be ableto check the Grab to Disk checkbox.

Saving images to disk can be important if you are acquiring data for a long time. A poweroutage or computer malfunction could cause all data stored in memory to be lost. Any datasaved on disk, however, would be safe.

If you are trying to acquire data as fast as possible, you would not want to use this option.You would not want to take the time to save each image immediately after acquisition.

Browse: Click the Browse button and use the standard file management dialog box to select adestination folder.

File Type: Select with which file type you want to save the images: iVision format or TIFF.

To use this Time Lapse tab:

1. Check the Use Time Lapse checkbox.

2. Select one of the three radio buttons: Frames, Interval, or Experiment Length. iVision will calculate this value foryou.

3. Fill in the other two values.

4. If you want to save your data to disk during the experiment, check the Grab to Disk checkbox.

a. Click Browse to select the directory for saving your data.


b. Select a File Type for your data: either iVision or TIFF.

2.9.6 Multi Dimensional Acquire Preview: Preview DialogThis Acquire Preview dialog box appears when you check the Preview box and click OK in the main dialog box of theMulti Dimensional Acquire command. This dialog controls many aspects of the acquisition, letting you control theprocess while seeing the effects of your actions in the live image. Only one of the image windows will display a liveimage at any one time, as selected by the Filter Position list.

The main feature of this Acquire Preview dialog, however, is the ZStage tab and its options for interactively setting upthe multi-D acquisition. This lets you scan through the sample on-screen, and then find the start and end points of theacquisition.

2.9.6.1 Multi Dimensional Acquire Preview: Camera Tab

The following features are always displayed on the Acquire Preview dialog box:

Exposures: This value is the exposure time for the filter shown in the Filter Position field. You canchange the exposure by clicking the up and down arrows or type in a new value and press theApply button, Times are measured in milliseconds.


AE: Click this autoexposure button to calculate and use the optimum exposure time for thecurrently selected filter. The autoexposure function uses the Exposures value as the initialguess.

AE Target Max: When performing an autoexposure (using the AE button), the target maximum is themaximum intensity value for which the autoexposure function will aim. Set this about 2/3 or3/4 as high as the camera's maximum output. You want to approach but not reach the camera'smaximum value.

Filter Position: By selecting a filter from this list, you will switch filter positions and choose which windowwill be affected by the exposure time and normalization settings. You also bring thecorresponding window to the front.

Continue/Pause: The Continue button at the bottom of the dialog starts the acquisition. The Pause buttontemporarily halts the acquisition. You can use this button to halt the acquisition while youchange the parameters in the Preview control panel.

Acquisition will be paused when the preview mode starts. You must click Continue to seeyour live image.

Gain, e Gain, Offset: These controls are identical to the ones in the standard camera Preview dialog. They affect theimage returned by the camera.

Shutter: Click the Keep open while live box to hold the shutter open. Use the Open and Close buttonsto manually change the shutter.


2.9.6.2 Multi Dimensional Acquire Preview: Size Tab

The Size tab's features let you quickly change the image's size:

Multi Dimensional Acquire Preview Dialog Box,Showing Size Tab

Resize Image To: Use the Full Frame and ROI options to change the image size and the corresponding area ofthe CCD being read out to the computer.

Full Frame: When you click this button, the full size image will be acquired, using the wholeCCD.

ROI: When you click this button, only the selected portion of the CCD will be acquired.To use this, select part of the image by clicking and dragging the mouse over theimage. Then click this ROI button. The image will be resized to this region ofinterest. The rest of the CCD will be quickly read out and disposed of, making for afaster acquisition.

Binning: Binning reduces the image size while increasing the image intensity. Please read thedescription of binning on Size section of the Multi-D Acquire command.


2.9.6.3 Multi Dimensional Acquire Preview: Norm Tab

The Norm (normalization) tab lets you improve the display of each image window without changing its data. Thesesettings will affect whichever data channel is selected in the Filter Position field.

Norm Tab ofMulti Dimensional Acquire Preview Dialog Box

Sliders: These sliders select the data values at which pixels will be displayed as white and black. Thetop slider selects the minimum (black) and the bottom slider selects the maximum (white) Alldata between these values will be shown as shades of gray. The Auto-Enhance Contrastoption disables these sliders.

Show Sat. Pixels: When Auto-Enhance Contrast is checked, showing the saturated pixels will highlight pixelsthat have the minimum or the maximum intensity values (e.g. 0 or 4095 for a 12 bit camera).You may want to adjust the exposure times to bring these "saturated" pixels back within thecamera's data range.

Otherwise, this option will highlight pixels that have values above the white point and belowthe black point. High values are displayed as red; low values are displayed as blue.

Auto-Enhance Contr.: When this option is checked, the minimum and maximum data values in the image will be usedas the white and black points. The values are continuously recalculated to adjust for imageintensity changes.


2.9.6.4 Multi Dimensional Acquire Preview: ZStage Tab

Use the Z Stage tab to enter the locations of the end points of your Z series and to calculate how many planes will makeup the series. You can use the Relative Movement arrows or the Position controls to move the focus motor up anddown to the start and stop positions. In most cases you will move the Z Stage with the knob of a motorized microscopeor a joystick. Then set the step size (Step by) and the # of Steps will be calculated automatically.

ZStage Tab ofMulti Dimensional Acquire Preview Dialog Box

Relative: Use the relative movement controls to move the focus motor up or down by increments of 1micron or 0.1 micron. The size of the move will be determined by which radio button isselected. The stage will move when the up and down spinner arrows are clicked. You will seethe change in the current position in the Position text box.

Poll Stage: Use this box to continuously monitor the Z stage position. If the Use Current Position asZero box was checked in the main Multi-D dialog, then all reported position values will berelative to the starting position when this command was called.

Position: The Position box reports the current position of the focus motor. The position is alwaysreported in microns from the motor’s zero point. This box is also used as the text entry box button, described next.


This button will move the stage to the value typed in the Position field. You can also use theother buttons next to the Start and End to move the stage to the respective Start and Endpositions. The motor will start moving as soon as you click the button

Use as Zero: Click this button to define the focus motor’s current position as its zero point. Afterwards, allabsolute movements and locations will be measured from this point. This definition of thezero point is only used within the Multi Dimensional Acquire command. We recommendthat users of piezo-electric devices not use this function.

Offset: This is the distance, in microns, between the focus motor's original origin and the pointcurrently set as the zero point, using the Use as Zero button.

Start: When the focus motor is at the starting position of the Z stack, click the Start button to recordthe position for later use by the Multi Dimensional Acquire function. We recommendstarting the Z series at the lowest point in the Z stack.

End: Click the End button to record the ending position of the Z stack.

Start andEnd: If the Use Current Position as Zero box was checked in the main Multi-D dialog

(Z-Step Tab), then all reported position values will be relative to the starting positionwhen this command was called.

Record the endpoints of the focus motor's movement for later use by the MultiDimensional Acquire function when you are focused on the starting position of theZ stack, click the Start button to record the position. We recommend starting the Zseries at the lowest point in the Z stack. Then focus on the ending position of the Zstack and click the End button.

Step by: Enter the amount by which the motor should step, which is the distance between acquisitionplanes. Please give this in units of microns.

Recommended: The theoretically best step size based on the filter and objective settings.

# of Steps: Displays the number of images that will be acquired. After entering the step size in the StepBy box, the number of images that will be automatically calculated. If the number calculatedis not satisfactory, enter a new step size and click this button again.


2.10 Set Color BalanceSingle-pass color cameras (with a built-in mosaic filter) collect red, green, and blue data in one exposure. iVision'sFocus, Single Exposure, and Full Acquire commands will scale the individual values for each channel to produce themost accurate colors. The Set Color Balance command stores the scaling values used by the acquisition commands.These values are multipliers that modify the data collected by the image. These values will remain active for allsubsequent images captured.

The Set Color Balance command stores scaling values that iVision uses to produce the most accurate colors. iVisionuses these scaling values because single-pass color cameras (with a built-in mosaic filter) collect red, green, and bluedata in one exposure. iVision's Focus, Single Exposure, and Full Acquire commands will scale the individual valuesfor each channel

The Set Color Balance command stores scaling values used to optimize the colors grabbed by single-pass colorcameras. Because these cameras collect red, green, and blue data in a single exposure, the colors cannot be balanced byadjusting the exposure times for each channel. Instead, each channel must be weighted to produce the most accuratecolors.

These commands store their scaling values within Set Color Balance:• Preview• Single Exposure• Full Acquire• RGB Color Acquire

Set Color Balance Dialog Box

There are two ways you can color balance the images from your single-pass color camera:

The Easy Way: Choose an acquisition command and display the preview. At the bottom of the Previewpalette, click the Color Balance button. This will calculate the color balance values and usethem upon the image. If you want to see the values, choose the Set Color Balance command.

The More-Control Way: Enter your own color balance values in the Set Color Balance dialog box. This option is forusers who want the highest level of control over the color balancing.


2.11 Set Gain and OffsetGain magnifies the camera's sensitivity. The signal-to-noise ratio will drop with increase in gain. If the camera is notreceiving enough light, you can increase the gain to acquire an image. Offset raises the level of the signal. The meaningsof offset and even of gain can be different for some cameras, so please check your camera's documentation for moredetail.

Set Gain and Offset Dialog Box

A setting will be grayed out when a camera does not support it.


2.12 Camera SettingsThe Camera Settings command configures the currently selected camera. This replaces the More dialogs that used to bein each acquisition command. Configuration settings depend on the model of camera.

Example of a Camera Settings Dialog Box

Please read the manufacturer's documentation for information about the camera-specific features offered by this dialogbox.

Acquisition Hardware Specifics - Cooke-PCO Cameras 49

3 Acquisition Hardware SpecificsMost acquisition hardware consists of a camera, a controller board, cables, and a power supply. Some systems also havea separate controller box. Here are instructions for installing a generic camera system:

A. Install the controller board into your computer.

This is the computer interface board, or frame grabber.

1. Shut down your computer.

2. Open the computer's case (see your computer manual for instructions on how to do this).

3. Ground yourself before touching the board or the inside of the computer.

4. Insert the board into any open PCI slot. Be sure it is completely seated.

5. Close the case.

B. Hook up the cables.

1. The board you just installed in your computer needs to be connected to the camera (or external controller if,any).

2. On most systems, the cables can only be connected in one way.

C. Connect the power cord or power supply.

1. If your camera has a separate power supply, connect it to the camera, and then plug the power cable into an ACoutlet.

2. If your camera has a built-in power supply, just plug its power cable into an AC outlet.

D. Start your computer.

E. Turn on the camera and/or controller.

F. Start iVision.

50 Acquisition Hardware Specifics - Cooke-PCO Cameras

3.1 Cooke-PCO Cameras

3.1.1 Supported CamerasiVision supports the KL, KQ, KS and EM SensiCam cameras.

3.1.2 Hardware InstallationThe SensiCam hardware consists of the camera, power supply, controller board, and camera cable. To install thehardware, do the following:

Connecting the camera to the controller board:

1. The board has three BNC connectors: Rx (receive), Tx (transmit), and Trig. Attach the connectors at one end ofthe cable to Rx and Tx on the board.

2. Attach the connectors at the other end of the cable to the camera, making sure to swap the connectors. Thecable attached to Rx on the board should be attached to Tx on the camera, and the cable attached to Tx on theboard should be attached to Rx on the camera.

LED indicator:

There is an LED next to the power switch. It has three states:

Red: Solid red means that the cables are not connected properly, or that the computer is off.

Flashing green-red; Flashing green-red means that everything is connected properly, but the camera has not yetreached its cooled temperature (images may be grabbed when the camera is in this state).

Green: Solid green means that everything is connected correctly and the camera has reached itscooled temperature.

3.1.3 Possible Settings for Cooke CamerasData Acquisition: The SensiCam grabs data from the CCD in 32-pixel blocks. If each coordinate of the area

being grabbed is not a multiple of 32, image data is grabbed from the camera and placed in atemporary block of memory. iVision then copies the requested area from this block into theimage. This extra step uses more memory and takes a little more time.

The fastest performance can be attained by making the coordinates of the area being grabbedmultiples of 32 (0, 32, 64, 96, etc.).

Exposure Time: The SensiCam does not allow an exposure time of zero. The minimum exposure time for theSensiCam is 1 msec.

3.1.4 Camera Settings CommandUse the Camera Settings command (Camera menu) to configure your camera. You only need to use this commandonce, but if you like you can change these settings whenever you like.

When controlling acquisition with a TTL signal, use the External Trigger Mode to pick the trigger polarity. You canacquire images when the TTL signal rises or falls.

Acquisition Hardware Specifics - Cooke-PCO Cameras 51

The Gain setting magnifies the camera's sensitivity. The signal to noise ratio will drop with increase in gain. If thecamera is not receiving enough light, you can increase the gain to acquire an image. The SensiCam QE has three gainsettings (1, 2, 3). Gain 3 enables the camera's low-light mode. Other SensiCam models only have two gain settings (1, 2).

52 Acquisition Hardware Specifics - Diagnostic Instruments Cameras

3.2 Diagnostic Instruments Cameras

3.2.1 The Different Cameras and How to Use ThemThe supported Diagnostic Instruments cameras can be divided into four families of cameras:

Spot RT-SE & RT-KE Includes the monochrome, monochrome IR, color mosaic, slider, and 3-shot colorSpot Insight & Insight QE Includes the monochrome, monochrome IR, and color mosaicSpot Insight 1394 Includes the monochrome, monochrome IR, and color mosaicSpot RT Includes the Spot RT monochrome, color, and sliderSpot Includes Spot II, Spot, and Spot Jr.

Note: In the rest of section 3.2, "Spot" will refer to all four families of the Diagnostic Instruments cameras. Forexample, the Spot White Balance command works for the Spot RT-SE as well as the Spot II.

The cameras can also be grouped according to the type:

Monochrome: These cameras capture grayscale images.

• In the Camera Settings command (Camera menu), choose Image Type: Grayscale andthe desired Bit Depth (either 8 or 12 bit).

• Use the Preview, Single Exposure, or Full Acquire command from the Camera menu.

Color Mosaic: These cameras have filters attached to the CCD, allowing them to acquire a color image witha single exposure.

• In the Camera Settings command (Camera menu), choose Image Type: Color and thedesired Bit Depth (either 8 or 12 bit).

Your images will have the data type Color 24 or Color 48 depending on the bit depth.

• Use the Preview, Single Exposure, or Full Acquire command from the Camera menu.

3-Shot Color: These cameras have a built-in liquid crystal filter. They acquire a color image using threeexposures: one each for red, green, and blue. They can also acquire a grayscale image by notchanging the filter. You can use them as either three-pass color cameras or single-shotcameras.

• For single-shot color image capture:

• In the Camera Settings command (Camera menu), choose Image Type: Color andthe desired Bit Depth (either 8 or 12 bit).

Your images will have the data type Color 24 or Color 48 depending on the bitdepth.

• Use the Preview, Single Exposure, or Full Acquire command from the Cameramenu.

• For single-shot grayscale image capture:

• In the Camera Settings command (Camera menu), choose Image Type: Grayscaleand the desired Bit Depth (either 8 or 12 bit).

Acquisition Hardware Specifics - Diagnostic Instruments Cameras 53

• Also in the Camera Settings command, set the Filter option to the filter color youwant used. For example, you could set this to Clear.

• Use the Preview, Single Exposure, or Full Acquire command from the Cameramenu.

• For three-pass color image capture:

(Use this mode only if you require different exposures for each color channel.)

• In the Camera Settings command (Camera menu), choose Image Type: Grayscaleand the desired Bit Depth (either 8 or 12 bit).

Your images will have the data type Color 24 or Color 48 depending on the bitdepth.

• Set the Filter option to As Is in the Camera Settings dialog box.

• Use the RGB Color Acquire or Multi Filter Acquire command from the Cameramenu. Your Spot filter must be selected within Device Select (Control menu).

3.2.2 Possible Settings for Spot CamerasExposure Time: Possible exposure times for the original Spot ("Spot I") camera range linearly upwards from

80 milliseconds.

Exposure times for all other cameras range linearly upwards from 1 millisecond.

Gain: The gain values will vary depending on the camera model and the bit-depth being used.

Also, the Spot Realtime Focus command uses a different range of gains than are used bynormal acquire commands.

3.2.3 Camera-Specific Data FilesSome Spot cameras come with a camera-specific data file that has a description of the "bad pixels" in your particularcamera. This file can be found on the CD that came with your camera.

This file must be placed in the "iVision 3.9 Folder" at the same level as the iVision application. As analternative, it can be placed inside the "/Library/Preferences/Spot Prefs" folder that will exist if you haveinstalled the Spot application.

• Spot Enhanced:

The file is named "chipinfo.dat".

• Spot SE and KE:

In this case, the file name contains the serial number of the camera and ends with .cif(for example: "D123456.cif").

• Spot RT:

These cameras do not need such a file; the camera itself stores the information.



Setup Dialog Box for All Spot Cameras

Image Type: Choose Color if you are going to acquire color images using the Preview, Single Exposure,or Full Acquire command.

Choose Grayscale if you are going to grab grayscale images or if you are going to use theRGB Color Acquire or Multi Filter Acquire command.

Bit Depth: Select 12-bit to acquire 4096 levels of data per pixel. This is the most desirable option forcollecting quantitative data. If you are collecting color images, then this will result in 12 bitsof data per channel in a Color 48 image.

Select 8-bit to gather 256 levels of data per pixel. If you are collecting color images, then thiswill result in 8 bits of data per channel in a Color 24 image.

Filter: The Filter option controls the liquid crystal filter, if the camera has one. Use this pop-up boxto control which filter is used when acquiring grayscale images.

For example, if you choose Blue, then the filter will change to the color blue. If you are usingthe Focus, Single Exposure, or Full Acquire command, then you'll acquire a grayscaleimage of the light passed by the blue filter.

When using the RGB Color Acquire or Multi Filter Acquire command, set Filter to As Is.

TTL Lag Time: This is useful if you are reading the TTL signal from the BNC port on the Spot PCI board.The TTL lag time is the number of milliseconds from when the TTL signal is high to whenthe acquisition starts.


3.2.5 Spot-Specific Commands

3.2.5.1 Spot Realtime Focus

Spot Realtime Focus provides speedy method for focusing your microscope or lens in real-time, without delay.

This produces a lower-quality image. iVision does not normally keep it, but you can use this command to capture data(by clicking the Capture button on the preview control palette).

Spot Realtime Focus Dialog Box The Dimensions Dialog Box

Image Type controls whether you focus using a slow, color image, or a faster, grayscale image. When you chooseGrayscale, you can set the Filter to a specific color (including Clear or None).

The Rotate and Flip X/Y options let you re-orient the image to make focusing easier. Rotation: Left rotates the image90° counter-clockwise.

The Exposure options set the exposure time in milliseconds. Click the Auto option to use an automatically-calculatedexposure time; click Manual to use the time that you enter.

The Mag option resizes the window to fit your screen; this does not alter the data.

The Gain option intensifies the data. Gain settings depend upon the hardware. The Gain numbers here differ from whatiVision normally uses.

Click White Balance to automatically correct the colors within a color image.

Click Dimensions to set the size and region of the focus window, using the dialog box shown on the right.


When you click OK in the setup dialog box, the following preview control palette appears. It lets you control theacquisition options while you focus your lens or microscope.

Real-time Focus Control Palette

Click Snapshot to acquire an image. The image will be placed within a new window and then the preview will continue.When you just click Done, the preview window will close and no image will be saved.

3.2.5.2 Spot Set Filter Position

This command lets you control your camera's liquid crystal filter, if it has one.

Spot Set Filter Position Dialog Box


3.2.5.3 Spot White Balance

This command is the best way to color balance images for Spot cameras that contain liquid crystal filters. Use thiscommand to scale the red, green, and blue intensities to produce the best color pictures. After using this command, thecamera will continue using these balancing multipliers for subsequent images.

For cameras that have Bayer-mosaic filters, please use the Set Color Balance command (Camera menu).

Spot White Balance Dialog Box

Grab Order: Choose the order in which the camera will acquire the color channels. Normally, the channelsare grabbed in the order: red, green, blue. You can change this to, for example, blue, red, andthen green.

Store Results… Check this to store the scaling values in variables. Then type the variable numbers to be used.By using this option, you can see exactly how iVision is correcting your data.

OK: When you click OK, iVision will begin calculating the scaling values to be used to whitebalance the camera.

58 Acquisition Hardware Specifics - Hamamatsu Cameras

3.3 Hamamatsu Cameras

3.3.1 Supported CamerasiVision supports Hamamatsu's Orca cameras, such as the Orca-285-1394, Orca-ER-1394, Orca-II-ER-1394, Orca-AG-1394 and Hamamatsu C9100EM.

3.3.2 ConflictsThe Orca driver has an apparent conflict with QImaging cameras. To work around this, do not enable both the QImagingand the Orca device modules at the same time. Keep the device module for the Orca camera in iVision's "DeviceModules" folder, and move the QImaging device module to the "Device Modules - Disabled" folder.

3.3.3 Camera Setup

Example Setup Dialog for Orca-DCAM

The Allow Fastest Capture option allows the camera to preview and capture as fast as the camera can deliver frames.However, on some machines the FireWire bus can not handle the amount of data being sent and the image will not becorrect. Turn this off if the image is broken into pieces, or otherwise does not show a complete frame at all times.

The DIG63 Board button shows a picture of the DIG36 PCI board and how to position the switch blocks on it.

Acquisition Hardware Specifics - Hamamatsu Cameras 59

3.3.4 Camera Settings CommandUse the Camera Settings command (Camera menu) to configure your camera. This command is scriptable.

Orca-DCAM Camera Settings

Bit Depth: The menu will list the available bit depths for the currently selected camera.

External Trigger: This lets you set the edge of the signal on which to trigger.

Light Mode: For cameras with an enhanced sensitivity mode. Low Light enhances the sensitivity bydisabling the anti-blooming feature. The High Light mode enables it.

Target Temperature: For cameras with programmable cooling, the target temperature may be set.

Acquisition Hardware Specifics - Roper Photometrics Cameras 61

3.4 Roper Photometrics Cameras

3.4.1 Supported CamerasiVision supports Roper Scientific's Photometrics PVCam cameras, which include the various CoolSNAP cameras, theQuantix, and the Sensys.

3.4.2 Possible Settings for the Photometrics CamerasImage Size: The width of the image will always be even. If you set the acquisition command to capture an

image with an odd width, then the resulting width will be one pixel smaller.


Camera Settings Dialog Box for Photometrics PVCam Cameras

Camera Mode: Choose Color if your camera has Bayer color-mosaic filters attached to the CCD.

Choose Grayscale when you have a grayscale camera.

Bit Depth: Select 16-bit or 12-bit (depending on the camera) to acquire the full range of data availablefrom the camera. This is the most desirable option for collecting quantitative data. If you arecollecting color images, then this will result in 16 or 12 bits of data per channel in a Color 48image.

62 Acquisition Hardware Specifics - Roper Photometrics Cameras


Scale Factor: This factor controls how iVision will convert the camera's raw data from 16- or 12-bit to 8-bitdata. When using this feature, iVision will divide each pixel by the Scale Factor. Use smallerScale Factors in dimmer lighting. Use high factors for bright pictures to avoid saturating theimage.

Enhanced Sensitivity: This option is only available when using a CoolSNAP ES or CoolSNAP HQ camera.

Shutter Mode: Lists the shutter options available for the current camera.

Clear Mode: Lists the clear options available for the current camera.

Readout Port: This only applies to cameras, such as the Cascade series, with multiple readout ports on thechip. As of this writing Roper Photometrics has not yet enabled this feature in their driver forOS X.

Temperature Setpoint: Allows you to set the temperature of the CCD, which the controller will attempt to maintain.

Motion Control - Control Menu Commands 63

3.5 QImaging Cameras

3.5.1 Supported CamerasiVision supports all models of Quantitative Imaging's Retiga, QICam, and MicroPublisher,

While QImaging cameras can draw their power from the FireWire connection, some come with an optional powersupply. You may want to use the external power supply if the camera draws too much power from other devices hookedto the computer, or if the other devices draw too much power away from your camera.

3.5.2 ConflictsThe Hamamatsu Orca driver has an apparent conflict with QImaging cameras. To work around this, do not enable boththe QImaging and the Orca device modules at the same time. Keep the device module for the QImaging camera iniVision's "Device Modules" folder, and move the Orca device module to the "Device Modules - Disabled"folder.

3.5.3 Possible Settings for QImaging CamerasExposure Time: Exposure times for QImaging cameras can range from 1 millisecond to 15 minutes.


QImaging Camera Settings Dialog Box

Image Type: Choose Color if your camera has a color CCD, or if you have a monochrome model with aQImaging RGB filter attached to it..

64 Motion Control - Control Menu Commands

Choose Grayscale when you have a grayscale camera.

Bit Depth: Select 12-bit to acquire 4096 levels of data per pixel. This is the most desirable option forcollecting quantitative data. If you are collecting color images, then this will result in 12 bitsof data per channel in a Color 48 image.


High Sensitivity: For cameras that support this option, turning it on will enhance the sensitivity of the CCD bydisabling the anti-blooming feature.

External Trigger: A signal from external hardware can trigger acquisition. Use this option to control whetheracquisition is triggered by a high or a low signal.

Turn Off Lighted Panel: For cameras with an LCD information panel mounted on the camera body, this box will turnthe panel off, keeping it dark.

Use Cooling: Check the Cooling option for better quality, if you have a cooled camera. Uncheck this optionto turn off cooling and require less power. You may need to do this to power a camera off of alaptop computer's FireWire port.

Fan Speed: For cameras that allow it, you may change the fan’s speed in increments of 1/4, 1/2, 3/4 andFull speed.

Regulated Temperature: For cameras that support it, you can set the temperature to a specific value. Choose the UseMinimum setting to set the temperature to whatever the camera defines as its lowest.


3.6 Virtual Camera

3.6.1 PurposeThe Virtual Camera allows you to work with the Camera Control commands without having a physical cameraconnected to the computer. In particular, you can write and test scripts using the Virtual Camera to provided sample data.

The Virtual Camera returns a dummy image consisting of either a horizontal ramp of pixel values, running low to highfrom left to right, or an image consisting of randomly selected pixel values. The values are limited by the DynamicRange selected in the Camera Settings command. The actual range will vary, based on the exposure time, and the gainand offset settings.

3.6.2 Camera Settings CommandUse the Camera Settings command (Camera menu) to configure the virtual camera’s output.

Virtual Camera Settings Dialog Box

Dynamic Range: This allows you to limit the pixel values that the Virtual Camera will return, in order to mimicthe possible results from a real camera.

Type of Image: The Virtual Camera will return a dummy image consisting of either a horizontal ramp ofvalues (low to high from left to right), or an image of randomly selected pixel values. In eithercase the minimum and maximum values will not fall outside of the range given in theDynamic Range setting.

Color Camera: When checked the virtual camera returns RGB values for each pixel.

Enable Fast Sequence: When checked the virtual camera simulates cameras that can grab an entire sequence at once,rather than by being prompted for each frame by iVision.

Click OK when you are done.


Part IIMotion Control

4 Control Menu Commands

4.1 IntroductionThe Control menu contains all commands that control microscope hardware. The commands described below are deviceindependent, meaning that they and the dialog boxes for using them remain the same regardless of the identity of thehardware. Shutters, filter changers, and microscope devices (apart from stages) can all be controlled using the commandsstarting with “Microscope.” Motorized XY stages and focus (Z) motors for microscopes can be controlled with theControl menu commands starting with “Stage.”

Control non-stage microscope devices:• Microscope Control• Microscope Variable• Microscope Lamp• Microscope Speed

Control XY and Z stages:• Stage Control• Stage Set Zero Position• Stage Record Position• Stage Move to Recorded Pos• Stage Z-Sections Setup• Stage Speed

Filter changers include liquid crystal tunable filters (LCTFs), filter wheels, and other wavelength switchers. MotorizedXY stages move in the horizontal, or X and Y, plane. Focus motors, also known as Z motors or Z stages, move yourstage or your objective up and down.

4.2 About Device ModulesFiles called device modules handle the control of devices connected to your Macintosh by iVision. These files reside in afolder named “Device Modules” within the iVision folder. There is usually one file per device. These files are added andupdated with installers that you receive from BioVision, and you do not need to deal with these files directly.


4.3 Device SetupThe Device Setup command is used to tell iVision what hardware is available and how it is set up. The Device Setupdialog box lists all of the devices found in the “Device Modules” folder, along with the version number of the DeviceModule. It groups these under headings for each type of device, such as Camera, Stage and Positioners.

Device Setup Dialog Box

The symbols to the right of the listed devices describe the status of that piece of hardware:

Disabled: If a piece of equipment is temporarily missing, you can disable it so that iVision does not tryto connect to it. Do this by clicking on the device name (e.g. Prior Stage) and clicking Setup,and then uncheck the Enabled checkbox.

Needs Setup: This indicates that the piece of equipment has not yet been set up, and cannot be used.Double-click the device name, or use the Setup button to change this.

Not Connected: This indicates that the computer and the hardware are not communicating. If the hardware ison (it should be on before starting iVision) and the communications cable is connectedproperly, then just click Connect or Connect All. This icon should change to the Connectedcheck mark. If it does not, return to the Setup dialog.

Connected: This indicates that the computer and the hardware are communicating properly, and thedevice is ready to be used.

In Use: This indicates that a camera is currently in use and can not be Disconnected.


4.3.1 The Setup ButtonSelect a device and click Setup to specify which port (e.g. serial port) the hardware is using. Alternatively, you candouble-click the name of a device module to open the same dialog. The Setup dialog also includes options and settingsspecific to the hardware. For example, you can identify the model of hardware, calibrate XY stages, and name thepositions on a filter wheel or objective turret (e.g. "Dapi Filter", "40x oil").

Before using the Setup dialog, the symbol beside the device name in the Device Setup dialog is probably a triangle forNeeds Setup. After using the Setup dialog, this should change to the Not Connected symbol.

Every Setup dialog box has these features:

Example of a Device Module Setup Dialog.

Enable: This checkbox indicates whether or not the device should be used by iVision. Whenunchecked, the device name listed on the Device Setup dialog will have an X beside it, toindicate its disabled status. The benefit of unchecking this box is that iVision will not attemptto connect to a device which may have been temporarily removed or powered off.

Connect/Disconnect: This is the same as the Connect/Disconnect button in the main Device Setup dialog.

Port: This menu applies to devices that connect to your Macintosh through a serial port. This pop-up menu is initially set to Not Assigned. You must choose the correct port to which youconnected your hardware. You can add serial ports to your computer by installing PCI cardsor USB-to-Serial adapters.

Status information: When the device is not connected, Not connected to ... will appear below the pop-up.Otherwise the model name or firmware version of the device will be listed to indicate aconnection has been established. Some devices never send information back to the computer,in which case the status will say Connection assumed.

For more details about the Setup dialog box for specific hardware, please see the appropriate part of the Motion ControlHardware Specifics section in this manual, starting on page 68.

4.3.2 The Connect and Connect All ButtonsThe Connect buttons initiate communication between the hardware and the computer. The Connect button works on thedevice selected. The Connect All button works on all enabled devices. When a connected device is selected, theConnect button becomes the Disconnect button, while Connect All becomes grayed out. These actions toggle the NotConnected and Connected symbols.


4.3.3 Serial StatusThis button opens a window which shows you the state of the first eight serial ports on the machine. The name of theport is listed to the right of each button. Clicking a button causes the information for that port to be listed at the bottom.iVision may be able to indicate that another program is using a serial port, but further details (such as the name of theprogram and the port settings) will not be available.

Example of the Serial Status Dialog Box.

4.3.4 DoneClick Done when you are finished. Because these actions take effect immediately, there is no Cancel button.

4.3.5 Determining Step Size for StagesIn almost all cases, the step sizes of your stage motors will be the defaults given in the Motion Control HardwareSpecifics chapter, on page 103. Use these step sizes and move the stage a measurable distance, such as 1 cm for XYstages and one rotation of the focus knob (usually 100 µm) for Z motors. Only if the stage moves an unexpected distancemight you need to determine the step size. The step sizes may also be given by the documentation provided with yourhardware. (A step is the smallest movement the motor can make, so the step size is the number of steps needed to move amicron.) The mechanics of the stage motors determines the step size.

If the default setting does not match your stage, use the following steps to determine your motor’s step size:

1. Choose Device Setup from the Control menu. Select the stage in question (for example, the Prior Stage) and clickSetup. There, set the Steps / Micron to 1. This will force the motor to move one step for every micron, and to reportthe distance moved in steps.

2. Now choose Stage Record Position from the Control menu. Check Increment After Record. Record the position.


3. Using the stage’s joystick, move XY stages one centimeter in one direction (using a ruler) or move Z motors one fullrotation. If you do not have a joystick, use the Stage Control command to move the stage to the right position.Record the position again.

4. Comparing the two measurements, you can determine the number of steps the motor moved.

XY stage step size:

€

# steps moved cm10,000 microns cm

Z motor step size:

€

# steps moved rotation100 microns rotation

Most focus knobs are marked out as 100 microns per rotation. Replace this figure to suit your microscope.

5. You can now enter these numbers into the Steps / Micron edit boxes in Device Setup.

4.4 Device SelectYou must use the Device Select dialog box to identify the hardware you will be using. This, for example, lets you attachmore Z stages and filter wheels to your computer than you will be using at any one time. This dialog box also lets youname the microscope-control devices.

The Device Select dialog contains two tabs, Stages and Microscope:

The first tab, labeled Stages, allows you to select which installed stage devices, if any, to use for the XY Stage and ZStage. Next to the popup menus, the dialog will mention whether or not the selected device is connected. If it is notconnected, you will need to use Device Setup to establish a connection. Any devices selected here will appear in theDevice Toolbar.


The second tab, labeled Microscope, allows you to select other devices, like filters, lamps and shutters which you will beusing. This tab contains two tables.

The table on the left, labeled Available Devices, shows a list of the devices available for use. To declare that you wish touse a device from this list, it needs to be added to the table on the right. This can be accomplished using one of threemethods. The first method is to double click on an available device. The second method is to select one or more availabledevices and drag them to the table on the right. The final method is to select one or more available devices and press theAdd button. If a device in the Available Devices table is dimmed, it means that the device is already in the table on theright.

The table on the right displays what devices, except for stages, you will be using. This table consists of five columns.The first column, labeled Device, shows the official device name. The second column, labeled Name, shows the name ofthe device as it will appear in various places throughout the application. This name is editable by clicking once on thename you wish to change. The third column, labeled Type, indicates whether the device is a positioner or variabledevice. The forth column, labeled In Toolbar, allows you to have the device appear in the Device Toolbar. The fifthcolumn, labeled Connected, indicates whether or not iVision can communicate with the device. If the device is notconnected, you will need to use Device Setup to establish a connection. Selecting and dragging can reorder devices inthis table. The order they appear in this table is the order they will appear in the Device Toolbar. To remove one or moredevices, select them and press the Remove button.

4.5 Device ToolbarThe Device Toolbar provides readily available interfaces for the motorized microscope hardware attached to yourcomputer. Click on one of the Device Toolbar buttons to open the interface for that hardware. The interface for thathardware will contain a close button, which can be used to close the interface. You can also click on another devicebutton to switch to that device’s interface. You can use any other iVision command while leaving the interface open.


The devices, which appear on the toolbar, and their order is determined by the Device Select command.

When the N button, at the top of the toolbar, is pressed, the names of the devices are shown.

When the Poll button, at the top of the toolbar, is pressed, polling will occur for the device whose interface is beingshown. During polling, some controls on the interface for the device may be unavailable.

If scripting is turned on, actions taken on the Device Toolbar to move a device will be recorded into the scriptautomatically.

4.6 Device SpecificiVision will place special commands that are only useful for specific hardware in a sub-menu of this menu item.

4.7 Microscope ControlThe Microscope Control dialog controls all positioner devices (hardware with discrete positions). The device isidentified by the name you assigned it in the Device Select command. You can move the device/change its position byusing one of the radio buttons and then clicking Do Now or OK.


Microscope Control Dialog Box

Devices: Pick the positioner device to be moved. This pop-up menu lists all positioners selected in theDevice Select dialog box (described above on p. 71). Below the Device menu is a reminder ofthe device you are selecting.

Home: This moves the device to its Home position. The device's position #1 is commonly defined as"Home". If the device does not have a Home position, this radio button will not be available.

Position #: This allows you to specify the position as a number, where the first position is 1 and the lastposition depends on the number of filters that your specific hardware can handle. Using anumeric position is most helpful when you want to specify the position from a variable. Whenscripting, using a numeric position is also helpful when you do not know the exact device(and hence the position names) that the script user will have.

Position Name: This lets you pick the position by the name you assigned in the Device Setup dialog:

Position Name Pop-up Menu

Wait Until Done: If checked, the Microscope Control command will not return control to you until after themotion has been completed by the device. If unchecked, control is returned immediately afterthe command is sent to the controller, without waiting for the motion to complete.


The Wait provision makes it simpler to write scripts, since the commands will be executedconsecutively. You may choose to start executing another command after sending thiscommand. You should be aware of the timing of the different commands used and fine tunethe script by trial and error.

Do Now: Clicking the Do Now button will move the device (or set the delay time or whatever) withoutclosing the dialog box, so you can use it again.

Since this is an interactive option, it is not available when scripting.

OK: Clicking OK will close the dialog box and perform the command. If you had already movedthe device with the Do Now button, clicking OK will try to move it again (if you had chosenHome, the device would rotate to home again).

Cancel: Clicking Cancel will close the dialog box without doing sending any further commands.

4.8 Microscope VariableThe Microscope Variable command controls all of the smoothly-moving, variable-position devices that you selected inthe Device Select command. (Device Select is described on page 71.) The device is identified by the name assigned to itin the Device Select command. You can change its position with the scroll bar, or by typing a number directly into theedit box. You can move the scroll bar with the mouse or by pressing the arrow or Page Up and Page Down buttons onyour keyboard.

Microscope Variable Dialog Box

Devices: Pick the variable device to be moved. This pop-up menu lists all variable devices selected inthe Device Select dialog box. Below the Device menu is a reminder of the hardware you willbe controlling.

Scrollbar: The scrollbar will show the current value of the device’s position. The names above thescrollbar, at each end, indicate the meanings of the minimum and maximum positions. Thenumbers below the scrollbar show the minimum and maximum values for those positions.The current value is shown in the edit box. If the edit box is set to a variable box (Var), thenthe value will be shown above the center of the scrollbar.

Cancel: For this command, the Cancel button will return the device to its original position. If youhave changed devices with the Device menu, only the last device will be returned to itsoriginal position.


4.9 Microscope LampThis command is used to control microscope lamps.

Microscope Lamp Dialog Box

All lamp devices that you selected in Device Select are listed in the popup menu at the top of the dialog. The on/off stateof the currently selected lamp and it’s voltage (or other measure of its brightness) is indicated with the slider. You canmove the slider or type a number and click the Set button to change the brightness.

4.10 Microscope SpeedThis command can control the movement rate of some microscope hardware. Only some devices allow their speed to bechanged through computer control.

Microscope Speed Dialog Box

Choose the hardware from the Device pop-up menu. Then move the slider to set its speed.

To reset the slider to the current speed, click the Current button.

To use the slider's setting as the device's speed, click OK.


4.11 Stage ControlThis dialog box controls the X, Y, and Z movements of the stage motors. You can move by relative amounts or toabsolute positions measured from the motors’ origins. You can also move to distances or positions specified byvariables. All distances are measured in microns. Current stage positions, if reported by the hardware, are shown in thetop-right quarter of the dialog.

The Stage Control Dialog

X, Y, and Z: Click the radio button beside Z to move the focus motor. Click the radio button beside X ifyou want to move the XY stage. Type the distance to be moved (in microns) into adimension’s text box. XY stages move in both X and Y directions at the same time. The pairof arrows beside the Z text-entry box will increment or decrement the entered amount by 0.5microns.

Clicking on the # button changes it to Var, for Variable. When this is done, the number in thebox refers to a variable in the Variables window, where the actual number to be moved isstored. For more information on using variables, please read the iVision Variables section ofthe Should-Read chapter in the User's Guide.

Home XY Stage: When this radio button is clicked, the action will be to move the stage to the corner with the“lowest” position values. The values for this position are then set to zero. The stage is usuallydriven against the limit switches at the “top-left” of the stage.

Center XY Stage: When this radio button is clicked, the action will be to move the stage to the center of itstravel limits. This is commonly done by moving the stage to each limit switch on each axis(nearest and farthest corners). The mid-point on each axis can then be determined and thestage will be moved there. The final position values depend on the equipment in use.

Absolute: When this radio button is clicked, the values represent an absolute position. This position ismeasured from the stage’s origin point, which is different depending on the particularhardware used. The origin can be set with the Stage Set Zero Position command.

Relative: When this radio button is clicked, the values represent a specified distance from the currentposition. The minimum or range of distances that can be moved is displayed above theCancel and OK buttons. The range of relative Z motion is limited in order to help preventaccidental collisions between objective and sample.


Do Now: Click the Do Now button to move the stage without closing this dialog box. In this way youcan move the stage repeatedly without calling the command over and over.

Cancel: Clicking Cancel will exit the dialog without performing any movements described in the text-entry boxes. This will not undo any movements already performed with the Do Now button.

OK: Clicking OK will move the stage as set, and then close the dialog box. If you click OK whenthe Relative Movement radio button is selected, then the stage will move even if you alreadymoved it with the Do Now button.

4.12 Stage Set Zero PositionThe Stage Set Zero Position command allows you to set the currently reported position of either stage to be zero. Thiscan simplify both relative and absolute positioning, since you do not have to take into account the stage’s currentlyreported position (which may be negative or very large) when calculating a new one. Only the checked stages will beaffected.

The Stage Set Zero Position Dialog Box

4.13 Stage Get PositionThe Stage Get Position command puts the current X, Y, and/or Z position(s) into an iVision variable. The right side ofthe dialog box displays the current positions of the XY and Z stages.

Stage Get Position Dialog Box

Choose the axes and enter the variable numbers.

Axis: Check the boxes for the axes whose positions you desire. The Z stage is the focus motor.

Put into Variable: Type the number (the index) of the variable that will store the position.


Do Now: Click the Do Now button to get the position without closing this dialog box. With this button,you can store multiple positions (while moving the stage with a joystick or other control)without calling the command repeatedly.

4.14 Stage Record PositionThis will record the stage positions to a table for later use. This can be used for repeated stage movement or to record thelocations of objects of interest within your sample. All positions are recorded in microns. The current positions of theXY and Z stages are displayed above the Cancel and OK buttons.

The Stage Record Position Dialog Box

Into the Window: The stage positions will be recorded into the window listed here. The names of existingwindows can be selected by typing them in or by selecting them from the pop-up menu (byclicking on the arrow button to the right). Windows must be at least three columns wide andof floating point type. If the window does not already exist, Record Position will create it.

Record to Row: Record the stage position to this row of the window selected above. If the # button is clickedand changed to Var, then that variable will determine which row is recorded to. The pair ofarrows to the right of the text-entry box increments and decrements the row number. Thearrow buttons do not function with variables.

IncrementAfter Record: When this is checked, the row number in the Record to Row text-entry box will increase by

one each time the position is recorded. This means that the next recording will be placed inthe next line of the table. Otherwise, the line selected is replaced with the new information.

Record: XY, Z: These check boxes let you choose which dimensions will be recorded. The stage positions foreach dimension will always be recorded to the same column in the table, whether the otherdimensions are being recorded or not.

Do Now: Click the Do Now button to record the position without closing this dialog box. In this wayyou can record multiple positions (while moving the stage with a joystick or other control)without calling the command over and over.


4.15 Stage Move to Recorded PositionAfter you have recorded a stage position, this command lets you move the stage back to that same position.

Move to Recorded Position Dialog Box

In the Window: Select the table that contains the positions to which to move. You can type the name of thewindow or select it from the pull-down menu on the right.

Using Row: Select the desired row in that window. The arrows increment and decrement the row numberby one. The number of a variable representing the row number can be entered in the text-entrybox if the # button is clicked and changed to Var.

IncrementAfter Move: When this is checked, the row number in the text-entry box above it will increase by one each

time this command is called.

Move: XY, Z: Click one or both to choose which motors will move.

Wait Until Done When checked, the command will not return control to iVision until the motor has completedits travel. When not checked, the command will return immediately, while the motor is stillmoving.

Do Now: Click the Do Now button to move to the indicated position without closing this dialog box. Inthis way you can move to multiple positions without calling the command over and over.


4.16 Stage Z-Sections SetupUse this command to define parameters to position the Z-axis at fixed increments between two points. This command,combined with an acquisition step, can be used to produce a stack of optical sections. You must specify the start and stoppositions and either the number of planes desired or the vertical separation between the planes.

The Z-Axis Sections Setup Dialog

The scroll bar on the left, as well as the arrow, Page Up and Page Down buttons, can be used to move the focus motor tothe start and stop positions. The current focus motor position is displayed to the right of the scroll bar. The highest andlowest possible positions (shown here as 0 to 100 µm) change depending on the hardware. All distance values are shownin microns.

A table of the parameters required to section a sample is shown to the right of the scroll bar. The right-most columnholds the names of the variables that the parameters will be stored in. You can change these to suit your situation.

In order to find the top and bottom of your stack of Z-planes, you will need to put light to the microscope eyepieces.Focus the microscope on the bottom (or top) of the volume of interest. Using the bottom plane as the starting position isusually recommended, so that the motor is working against gravity as it moves, which helps to insure accuracy. Be waryof driving the objective into the sample! You can use the scroll bar (or the arrow or page buttons) to adjust the Zposition of the objective. When ready, click the Record Start Position button.

Next, find the top (or bottom) of the volume, and click the Record Stop Position. Once both values have been entered,the Total Thickness in microns between the two position is automatically entered into the dialog.

Next, the Number of Z Planes and the Plane Separation must be determined. To do so, choose which one you wish tocontrol and click on that item. Then enter the value; the other value will be calculated from the one you enter. Forexample, when the Number of Z Planes is entered, the Plane Separation is calculated.

This calculation is also done when you click the OK button. Clicking OK also writes all five values to the specifiediVision variables so that they can be used by other commands. Remember that dividing a 40 micron-thick sample into 40slices of 1 micron each requires 41 Z-planes: 1 for the bottom plane and 40 on top of that.

In brief, the Z-Sections Setup procedure is:

1. Move to the bottom plane and click Record Start Position.

2. Move to the top plane and click Record Stop Position.

3. Enter either the Number of Z Planes or the Plane Separation.

4. Click OK.


You are now ready to use a script or another extension to capture a stack of optical sections. Here is the basic layout of asimple script to capture a stack of optical sections:

1. StageZ-Sections Setup: As described above, this command records the locations of the upper and lower planes of your

Z-stack and helps figure out the number and thickness of optical sections. These parametersare recorded to the Variables window.

2. New Image: This makes a new image sequence with one frame for each optical section.

3. Select Frame,Stage Control: These set the starting conditions. The Select Frame command sets the image back to the

previous frame. The Stage Control command makes certain that the Z motor is at the startingposition, which it reads from the appropriate variable.

4. Top of Loop: This label marks the top of the acquisition loop.

5. Select Frame: This sets the image to the next frame. The first time through, this sets the image to framenumber 0, the very first frame in the sequence.

6. Acquire: The camera’s image capture commands go here.

7. Stage Control: This steps the Z motor up (or down) the thickness of one optical section. The distance is readfrom the appropriate variable.

8. Loop: This returns the progress of the script to the Top of Loop label. The script will loop for thesame number of times as the number of Z-planes. This number is, again, pulled from thevariables recorded by Z-Sections Setup.

9. Animate: This will display your images in order from front to back.

Sample Z-Section Acquire Script


4.17 Stage SpeedSome devices allow the user to set the speed at which the device moves. The Stage Speed command lets you specify thisspeed as a percentage of the fastest speeds for the device. If the device does not support speed control, the matchingslider is disabled.

The Stage Speed Dialog Box

Check the checkbox for the axis that you want to change. Press the Current button to set the slider to the current speedof the device. Move the slider to set the stage's new speed. The setting takes effect after the OK button is clicked.

Note that most devices automatically accelerate and decelerate the motor at the start and end of the travel, respectively. Ifthe travel distance is short, you may reach the top speed for only a moment. It is possible for the controller to support ahigher speed than the actual stage motor can handle. In that case the motor will move and then appear to get stuck;reduce the speed setting by half and try again.


4.18 StageScan Array Calibrate The StageScan Array Calibrate command records and calculates the corner positions of multi-well plates and otherarrays of samples on a motorized XY stage. The StageScan Array Setup command, described on page 88, uses thiscalibration data to create a locations window, which contains the locations of all wells/samples. The StageScanMove/Iterate command (page 95) can then move the XY stage to each and every well or sample.

(An alternative to using StageScan Array Calibrate and StageScan Array Setup is the StageScan Mosaic Calibratecommand, which uses a different method to create a locations window.)

StageScan Array Calibrate Dialog Box for a Single Array


4.18.1 Calibration MenuThis menu contains all of the calibration files contained within the StageScan Preferences folder inside of the iVisionapplication folder. The directory path is:

Your Computer: Users: YourUserName: iVision 4.0 User Folder:iVision Preferences: StageScan Preferences:

If iVision cannot find a calibration file, it will create a default, untitled calibration.

Switching between calibration files saves the initial calibration file. The buttons at the bottom also affect the calibrationfiles:

Duplicate: Pressing the Duplicate button copies the current calibration file. To make a newcalibration file, click Duplicate and enter a new file name. You can then edit thesettings for this new calibration file.

Save: The Save button saves changes to the current calibration file. It becomes enabledwhen the calibration settings have changed. If the Calibration menu contains nocalibration files, then a small dialog box will ask you for the file name.

OK: Pressing the OK button will save the current calibration file and close the dialog.

Cancel: Pressing the Cancel button will close the dialog without saving any changes to thefile.

4.18.2 Single Array TabSelect this tab if there is only a single array of samples. Page 84 shows a picture of the Single Array tab.

First, enter the Dimensions of the array. These values can either come directly from the user or through a variable. Forthe Rows and Columns fields, the minimum value is 1 and the maximum value is 999.

When the dialog first appears, iVision will poll the stage for its current position. If you check the Poll Stage Positionbox, iVision will poll the stage about once every second.

You can view the coordinates as either Micrometers or Millimeters by selecting from the Units menu.

To set a coordinate, select its radio button and move the stage to that corner. Press the Set button, which you will find inthe bottom-right corner of the dialog box. iVision will read the stage position again and store that position. Black dotswill appear next to coordinates that have been set. If the current stage position does not match the currently selectedcoordinate, the position will be italicized instead of normal text.

To clear the settings of a coordinate, select its radio button and press the Clear button. Pressing the Clear All button willclear all of the coordinates.

Note: When creating a new calibration file, click Clear All to reset the positions of all coordinates.

To move to a coordinate that has been set, select its radio button and press the Go button (in the dialog's bottom-rightcorner). The Go button will be dimmed if the coordinate has not been set.

Once at least three coordinates or two coordinates that are diagonal from each other have been set, the Calculate AllPositions button will be enabled. Press the Calculate All Positions button to calculate the remaining coordinates.


4.18.3 Multiple Arrays TabSelect this if there are multiple arrays of samples.

The Multiple Arrays Tab of the StageScan Array Calibrate Dialog Box

When the dialog first appears, iVision will poll the stage for its current position. If you check the Poll Stage Positionbox, iVision will poll the stage about once every second.

1. First, enter the dimensions of the super array, and then enter the dimensions of each array.

a. On the # of Arrays line, enter the number of Rows and Columns of arrays within the super array.

b. On the Dimensions line, enter the number of Rows and Columns of samples within each array.

You can enter each value as a number or a variable. The value for these fields can range from 1 to 999.

2. Next, choose how many coordinates to set for each array. Set more coordinates to calculate the best locations for thewells within the arrays. The Information Provided options are:


Minimum: Use Minimum if your plates are rectangular, withoutsignificant deviations, and are parallel and lined-up with eachother.

This option will only require the positions of the top-left andbottom-right corners of the first plate, and of the top-leftcorner of the plate below and to the right of that. That lastposition tells iVision the distances between the arrays.

If you check Distance Between Arrays, then only the top-left and bottom-right coordinates of the first array will berequired.

Remember that the distance between arrays is the distancefrom the centers of the last wells in one array to the firstwells in the next array.

Medium: Use Medium if your plates are rectangular and parallel witheach other, but are not lined up.

This option will require the top-left and bottom-left cornersof the first plate, and the top-left corner of every other plate.The positions of the top-left corners tell iVision where tofind each plate.

Complete: Use Complete if your plates aren't rectangular and / or arenot parallel with each other.

To completely define the positions of multi-well plates onyour XY stage, you must record the position of every cornerof each plate.

3. To set a coordinate:

a. Click on its red dot within the superarray diagram. (The squares represent arrays, and the dots represent thecorners.)

b. Move the stage to that position.

The required coordinates are shown in red.

c. Click the Set button.

Once the required coordinates have been set, they are shown in green. iVision displays the array number, row,column and corner below the Clear button.

To clear the settings of a coordinate, select its radio button and press the Clear button. Pressing the Clear All buttonwill clear all of the coordinates.

Note: When creating a new calibration file, click Clear All to reset the positions of all coordinates.

4. To move to a coordinate that has been set, select its radio button and press the Go button (in the dialog's bottom-right corner). The Go button will be dimmed if the coordinate has not been set.

Click the triangle to see more detail on the coordinates for the currently selected array.


4.18.4 Calculate All PositionsOnce the required coordinates have been set, the Calculate All Positions button will be enabled. Click Calculate AllPositions to determine the locations of all remaining corners of the array(s). They will turn from gray to black when youdo so.

Important: Remember to press this button! The whole point of this command is to calculate and recordthe positions of all array corners in the calibration file.

4.18.5 Duplicate, Save, DeleteThese buttons work on calibration files. They are described above in the Calibration Menu section, on page 85.

4.18.6 Scripted BehaviorWhen scripted, the StageScan Array Calibrate command will do one of three things: Calculate All Positions, Go to aposition, or Set a coordinate.

When you script the StageScan Array Calibrate command,

1. Choose a calibration file from the Calibration pop-up menu.

2. Choose an array (when using the Multiple Arrays tab) and pick a coordinate (a corner of the array).

3. Click one one of the three buttons: Calculate All Positions, Go or Set, and then click OK.

When scripted, those buttons act like radio buttons: only one of them can be pressed at a time. The pressed buttondetermines what happens when you run the script.

Set: Click on a coordinate and press the Set button. Running this scripted command will set thecoordinate to the stage's current position.

Go: Click on a coordinate that's been set and press the Go button. Running this scripted commandwill move the stage to the coordinate's position.

CalculateAll Positions: After your script has set the required coordinates, record the command with this button

pressed. Running this scripted command will calculate all remaining coordinates.

4.19 StageScan Array SetupThe StageScan Array Setup command creates a "locations window" that stores both the positions of the array's wells orsamples and the path along which they should be scanned. StageScan Array Setup uses the calibration file created bythe StageScan Array Calibration command.

These two commands make automatic image acquisition possible by telling the StageScan Move/Iterate commandwhere the wells are placed on the XY stage. You will next use the StageScan Move/Iterate command within a script toautomatically move to and acquire images of every well or sample within the array.

The StageScan Array Setup command records and calculates the absolute positions of the array elements. That meansthat the positions are measured from the stage's origin. When you use the StageScan Move/Iterate command's Iteratetab, choose the Absolute Moves option.


(An alternative to using StageScan Array Calibrate and StageScan Array Setup is the StageScan Mosaic Calibratecommand, which uses a different method to create a locations window.)

Note: You must have already clicked Calculate All Positions in StageScan Array Calibrate, and saved yourcalibration data, in order to use this command.

StageScan Array Setup Dialog Box for a Single Array

4.19.1 Setup MenuThe setup file identifies the wells to be scanned and the path along which they will be scanned.

The Setup pop-up menu contains all of the setup files contained within the StageScan Preferences folder inside of yourhome folder. The directory path is:

Your Computer: Users: YourUserName: iVision 4.0 User Folder:iVision Preferences: StageScan Preferences

If iVision cannot find a setup file, it will create a default, untitled calibration.


Switching between setup files saves the initial setup file. The buttons at the bottom also affect the setup files:

Duplicate: Pressing the Duplicate button copies the current setup file. To make a new setup file,click Duplicate and enter a new file name. You can then edit the settings for thisnew setup file.

Save: The Save button saves changes to the current setup file. It becomes enabled when thesettings in this dialog box have changed. If the Setup menu contains no setup files,then a small dialog box will ask you for the file name.

OK: Pressing the OK button will save the current setup file and close the dialog.

Cancel: Pressing the Cancel button will close the dialog without saving any changes to thefile.

4.19.2 Calibration DataUse this pop-up menu to select a calibration file created by the StageScan Array Calibrate command. A diagram ofyour array will appear in one of the tabs below. The proper tab will automatically activate based on the chosenCalibration Data file.

This menu will be disabled if the command cannot find any setup files in the "StageScan Preferences" folder. In thiscase, please run the StageScan Array Calibrate command. Make certain to press the Calculate All Positions buttonand save your calibration data.

4.19.3 Path From StartThe path determines the order in which every selected element in every array will be visited.

The green dot in each path icon, and the green dot on the array diagram, represents the origin at which the path starts.Press the Option key and click on a well to move the origin.

The eight paths, from left to right, are:

Move to the left, down to the next row and then to the right

Move to the right, down to the next row and then to the left

Move to the left, down to the next row and to the left again

Move to the right, down to the next row and to the right again

Move up, over to the next column and up again

Move down, over to the next column and down again

Move up, over to the next column and then down

Move down, over to the next column and then up


4.19.4 Single Array TabThe Single Array tab, shown on page 89, displays a diagram of the array. Select the array elements (wells or samples)that you want scanned.

Selected elements will be hilited blue, and will contain a green dot or arrow. The StageScan Move/Iterate commandwill move to these array elements. Unselected elements will have gray backgrounds, and the StageScan Move/Iteratecommand will ignore them.

The green dot indicates the origin, or starting point, for the scanning path. You can move the origin by Option-clickingon another array element. The green arrows indicate the directions in which the stage will move.

Press the Select All button to select all of the dots in the array. Press the Clear Selection button to deselect all of the dotsin the array.

You may want to clear the entire selection first, and then select the specific rows, columns, and dots that you desire:

• Hold down the shift key and click in two spots to select a range of rows, columns, or dots.

• Hold down the command key (Apple key) and click in two spots to select only those two rows, columns, ordots, without the elements in the middle.

• After clearing the entire selection, you can also simply click-and-drag to select a rectangular region of elements.

When you finish selecting elements, remember to click Duplicate or Save (in the bottom-left corner of the dialog box) tosave your setup.

4.19.5 Multiple Arrays TabThe Multiple Arrays tab displays a diagram of one array at a time. Select the array elements (wells or samples) that youwant scanned. Then use the other controls on the Multiple Arrays tab to select the elements on the other arrays withinyour super-array (your collection of arrays). The Path from Start setting applies to all of the arrays; each array does notget its own path.

To select array elements (that is, wells or samples), please follow the instructions given for the Single Array tab (page91).

To select a specific array within your super-array, set the Array Row and Array Column values. These values can bespecified directly or through a variable.

You may select a different group of elements for each array. If you would prefer to select the same group of elements forsome or all arrays, use the buttons at the bottom of the Multiple Arrays tab:

• Select All selects all of the dots in the current array.

• Copy copies the current selection, including the location of the origin.

• Paste applies the previously copied selection to the current array.

• Paste All applies the previously copied selection to every array.

• Clear deselects all of the elements in the current array.

So if you want to scan the same group of elements on every array, select them on one array, press Copy, and then pressPaste All.


When you finish selecting elements, remember to click Duplicate or Save (in the bottom-left corner of the dialog box) tosave your setup.

4.19.6 Create Locations WindowClick this button to create a window containing all the information needed to scan your array(s).

When a valid setup file has been specified, the Create Locations Window button will be enabled. This button will bedisabled if the current calibration file is not complete. In that case, please return to the StageScan Array Calibratecommand and make certain to click the Calculate All Positions button and to save your calibration data.

When you click the Create Locations Window button, a small dialog will ask you to name the new locations window.Click Create in that dialog to make the new window. Remember to save the locations window so you can use it later.

A locations window has the following properties:

1. It is a floating-point window containing six frames (0-5) and at least 5 columns.

2. Frame #0 contains the absolute X coordinates for stage positions

3. Frame #1 contains the absolute Y coordinates for stage positions

4. Frame #2 contains the absolute Z coordinates for stage positions

5. Frame #3 describes the path the stage will follow when moved by the StageScan Move/Iterate command

6. Frame #4 is for the user to use as they wish

7. Frame #5 is reserved for use by iVision.

Important: Remember to press this button! The whole point of this command is to create this locationswindow to be used by the StageScan Move/Iterate command.

4.19.7 Scripted BehaviorThe scripted Stage Array Setup command will choose the setup (the path and selected elements) and the calibration file,and create the location window.

When you script this command,

1. Choose a Setup file. If you have not already saved an appropriate setup, choose a path and select the desiredelements on the array(s). Click the Save or Duplicate button to save your setup.

2. Choose a Calibration Data file.

3. Click the Create Location Window button, and then click OK.


4.20 StageScan Mosaic Calibrate The StageScan Mosaic Calibrate command helps you acquire a mosaic, or rectangular array, of images. Place yourarea of interest in front of the camera and then run this command. You will set the number of images to capture and thedistance between the centers of each image. Your script will then move to each position and captures an image there. Thescript will use the StageScan Move/Iterate command to move the stage to each position.

The StageScan Mosaic Calibrate command calculates the capture positions based upon the camera's initial position. Ifthe camera is looking at the center of the sample, then this command will calculate all positions relative to the center. Ifthe camera is looking at a corner of the sample, then this command will calculate all positions relative to that corner.

The StageScan Mosaic Calibrate command creates a locations window containing relative positions, whereas theStageScan Array Calibrate and StageScan Array Setup commands record absolute positions. So when you use theStageScan Move/Iterate command's Iterate tab, choose the Relative Moves option.

StageScan Mosaic Calibrate Dialog Box

The features of this dialog box are described below:

4.20.1 Capture DimensionsThe Rows and Columns fields set the width and height of the mosaic, measured in the number of images. In the exampledialog box, above, the mosaic will be eight images tall and twelve images across. These values can be entered directly orthey can come from a variable.


4.20.2 Initial Camera PositionClick a radio button to set the camera's position relative to the mosaic at the beginning of the image capture. The mosaiccan be created with the camera starting out in the center or at any corner.

The grid in this box loosely represents the images that will be acquired.

A gray box around one of the radio buttons represents the camera's top-left corner. This helps keep track of how thecamera is turned in respect to the stage. In the example dialog box, above, the gray box is in the mosaic's upper-rightcorner, showing that the camera is turned 90° to the stage.) Account for the camera's rotation with the Reverse...Direction buttons, described below.

4.20.3 X/Y MovementThese two fields determine the distance between the centers of images. The stage will move this relative distance whenmoving into position for the next image. These values can be entered directly or they can come from a variable. Theunits can be entered in micrometers or millimeters, depending on the selection in the Units menu.

If your camera is rotated with respect to the stage, click one or both of the Reverse... Direction buttons. A gray boxaround one of the radio buttons represents the camera's top-left corner. This helps keep track of how the camera is turnedin respect to the stage.

4.20.4 Create Locations WindowClick this button to create a window containing all the information needed to capture your mosaic image.

When you click the Create Locations Window button, a small dialog will ask you to name the new locations window.Click Create in that dialog to make the new window. Remember to save the locations window if you want to use it againlater.

A locations window has the following properties:

1. It is a floating-point window containing six frames (0-5) and at least 5 columns.

2. Frame #0 contains the absolute X coordinates for stage positions

3. Frame #1 contains the absolute Y coordinates for stage positions

4. Frame #2 contains the absolute Z coordinates for stage positions

5. Frame #3 describes the path the stage will follow when moved by the StageScan Move/Iterate command

6. Frame #4 is for the user to use as they wish

7. Frame #5 is reserved for use by iVision.

Important: Remember to press this button! The whole point of this command is to create this locationswindow to be used by the StageScan Move/Iterate command.


4.20.5 Scripted BehaviorThe scripted StageScan Mosaic Calibrate command will record all of the dialog's settings. The Create LocationsWindow button becomes an on/off button so that you can record its position.

When you script this command, set up the dialog as you normally would:

1. Set the Capture Dimensions

2. Choose the Initial Camera Position

3. Enter the X and Y Movement, and perhaps reverse the direction of movement

4. Turn on the Create Locations Window button by clicking it down, and then click OK.

4.21 StageScan Move/IterateThis command uses the data stored in a locations window to move the stage. StageScan Move/Iterate can move thestage to multiple positions in multiple arrays, in an order of your choosing. Scripts using StageScan Move/Iterate canquickly move to all the desired positions within an array or mosaic, acquiring images and performing other actions ateach position.

Move Tab of StageScan Move/Iterate Dialog Box

4.21.1 Position WindowPick the locations window that describes the array(s) you want to scan and the path along which you want the stage tomove. The Position Window menu contains all locations windows currently open in iVision. (That is, all windows thatare open and that have all of the properties of a valid locations window, as described on page 92.)


4.21.2 Move TabUse the Move tab (shown above) to move to a specific element within a specific array. Use the Iterate tab to move to thenext element along the chosen path (as recorded in the locations window).

The Array Row and Array Column fields identify the desired array. The Row and Column fields identify the desiredelement (well or sample) in the array. These values can be entered directly or they can come from a variable. You canalso select the Row by choosing a letter (A through P) from its pop-up menu:

The Row Pop-Up Menu, Showing Row Letters

Press the Move button to move the stage to the chosen array element. In addition, clicking on Move sets the followingvariables:

Variable Value stored in the variable

235 array row value (the row the array occupies within the super-array)

236 array column value (the column the array occupies within the super-array)

237 row value (the row the well or sample occupies within the array)

238 column value (the column the well or sample occupies within the array)

Below this tab, the dialog shows the stage's Current Position in micrometers.


4.21.3 Iterate TabUse the Iterate tab to move to the next element along the chosen path (as recorded in the locations window). Use theMove tab to move to a specific element within a specific array.

Iterate Tab of the StageScan Move/Iterate Dialog Box

The Movement Type option informs iVision whether the element positions were recorded relative to the stage's origin(absolute positions) or relative to the last position (relative positions).

• Use Absolute Moves if the StageScan Array Setup command created the locations window.

• Use Relative Moves if the StageScan Mosaic Calibrate command created the locations window.

Press Reset to initialize the iteration process. It sets the index to 0 (as shown at the bottom of the dialog, along with thestage's current position). The index tells StageScan Move/Iterate where it is along the scanning path, which tells it theposition to read from the locations window. Every time the stage is moved, the index increases by one.

Press Move to Next to move the stage to the next position along the chosen path. At the bottom of the dialog box, youwill see the Current Position change and the Current Index increase by one. When StageScan Move/Iterate reachesthe end of the path through the array(s), the index will be set to zero and the scan will be ready to start again.

Pressing either Reset or Move sets the following variables:

232 the current index

233 the total number of positions

4.21.4 Scripted BehaviorScripting this command records the name of the locations window and all settings within either the Move or Iterate tab.In addition to picking the Position Window from open windows, you can type in the window name (since the windowmay not yet exist when the script is recorded)

When scripting the Move tab, click the Move button to make the stage move when the scripted command runs.

When scripting the Iterate tab, click either the Reset or the Move to Next button.


4.22 Define User DevicesThis command allows you to define two types of custom devices.

Serial devices are devices that are controlled by sending data (generally character strings) to them over a serial port.

Linked Devices are “pseudo-devices” where the positions consist of combinations of real devices. For example if youhave two filter wheels, one built into a microscope, and one attached to an external controller, you could build a linkeddevice where each position combines the two wheels. Moving the Linked device to a position would cause both wheelsto move to whatever position you defined for that linked position.

4.22.1 Using Device DefinitionsThis command allows you to create, edit, and test the definitions. To make use of them in the rest of iVision’s devicecommands, you must follow two steps:

1) Add the definitions you wish to use to the Generic S&F device module (see page 111). Do this from the DeviceSetup command in the Control menu. This makes them available to the Device Select command.

2) Select the matching devices from the Device Select command (Control menu), as with any other devices.

4.22.2 Managing Device DefinitionThe device definitions are files that store the Serial or Linked settings and commands or positions. The DeviceDefinition files are listed in the column on the right side of the Define User Device dialog. The buttons to the left of thelist allow you to manage the files.

You can rename a definition by clicking on the name in the list and waiting for it to become editable. Then type the newname and press Return or Enter to complete the change.

Device Definitions portion of the Define User Devices dialog.

New: Creates a new definition file and makes it the active definition. It will be named “Untitled”, or“Untitled x”, where x is a number that makes the name unique.


Revert: Once a definition has been saved you can undo all unsaved changes by clicking Revert torestore the last settings that were saved to disk.

Save: Saves the current settings for this definition to disk.

Duplicate: This will duplicate the current definition into a new definition with the same name and anumber appended to the end (e.g. “EXFO Lamp 1”). This is convenient when two devicesshare many but not all settings.

Delete: This will delete the definition file and remove it from the list. It is permanent, so be carefulthat you really mean it.

When a device definition is selected from the list on the right, that device's settings will now be listed in the left side ofthe dialog. You can now edit those commands by editing the fields within the Device Type, Port Setup and Commandsor Positions tabs. Click Revert to restore the serial definition to its last saved state. When you are done, click Save tosave these changes to a file.

4.22.3 Device Type TabThis tab allows you to specify the kind (Serial or Linked) and type of device you will be defining. The type of deviceselected will determine the icon associated with the device in the Device Toolbar and how many commands one candefine for the device.

Define Devices Dialog Box: Device Tab

The six types of devices one can define are Simple, Shutter, Port Select, Filter, Objective and Lamp. If you are unsure ofthe type you should select, the Simple type is the most generic and should be used. If the Shutter or Lamp devices areselected, only open/on and close/off commands can be defined. The remaining devices allow up to 12 commands to bedefined.

4.22.4 Port Setup Tab (Serial)This tab only applies to Serial device definitions, and is dimmed for Linked Devices.


Define Devices Dialog Box: Port Setup Tab

Each serial device has specific port settings. For example, most hardware communicates at 9600 baud, while some olderhardware communicates at 300 baud. Entering the correct port settings allows iVision to speak to the hardware.

You should consult your hardware's manual to learn the communication settings.

4.22.5 Commands Tab (Serial)The column on the left lists twelve commands (two for Lamp and Shutter types) that can be defined. You may leavecommands undefined. While the commands usually represent “positions”, such as the positions on a filter wheel, theycan also represent actions that iVision does not usually support (for example, “Start Pump” and “Stop Pump”). Use adevice type of Simple for those commands. To edit a command, select it from the Commands table.

The Commands Tab for Serial Devices

Except for Lamp and Shutter devices, the name of the command can be changed. To name the name, simply click on thename of the command in the Commands table and it will change into an editable text field. Changes to the commandname will take effect when you select a different command, save changes to the serial definition or press return. Eachcommand can be programmed to perform one or more the following tasks, in the following order:


1: Output Message:The output message is constructed by pressing the Add button which will place editable itemsin the Output Message table. Each item consists of a popup menu, which will contain thevarious types of information that can be sent, and, depending on the type, the text to be sent.There are six types of information that the output message can consist. They are ASCII Text,ASCII Integer from Variable, Single Byte, Carriage Return, Line Feed and Carriage Returnfollowed by a Line Feed. For ASCII Text, ASCII Integer from Variable and Single Byte,clicking in the cell can change the information.

2: Wait for Input Message:iVision will expect to receive this response from the hardware. Usually, this is a success orfailure message. Consult your hardware's manual to learn its response codes.

Message Type: The popup menu declares what type the response will be. For ASCII Text, ASCIIInteger from Variable and Single Byte, the field to the right of the popup menushould be used to enter in the expected text.

Result Variable: iVision will set the value of this variable based on whether the hardware's responsematches your message text. A value of 0 means the hardware gave the expectedresponse, and a value of 1 means it gave a different response. A value of 2 means itdid not respond within the time out period.

Time Out: Enter how long the computer must wait for a response. If the desired response is notreceived within the time out period, an error is generated and iVision displays analert box.

3: Delay: Wait for a specified period of time after receiving the response (an optional time delay). Thisis a convenient feature when you are sending commands to move motorized devices, whichmay require additional time to perform their task.

You can use the Do Now button to test the format of each command before storing it in a serial definition. Set UsingPort to the hardware's serial port.

4.22.6 Positions Tab (Linked)The column on the left lists twelve positions (two for Lamp and Shutter types) that can be defined. You may leavepositions undefined. A linked position is defined as a combination of one or more positions of other devices.


The Positions Tab for Linked Devices.

Except for Lamp and Shutter devices, the name of the position can be changed. To name the name, simply click on thename of the position in the Positions list and it will change into an editable text field. Changes to the position name willtake effect when you select a different position, save changes to the linked definition or press return. Each position canbe programmed to perform one or more position movements by other devices.

Use the Add button to add device positions to the definition. The menu on the left will list the devices currently selectedin the Device Select command. The menu on the right will list all of the positions for that device. The Wait columnindicates whether the linked device will wait for that movement to complete before performing the next move in the list.The Do Now button allows you to test the current definition.

Motion Control Hardware Specifics 103

5 Motion Control Hardware Specifics

5.1 Common Device SetupMost devices which are controlled through a serial port connection have the following controls in common:

Example of Port Selection and Connect Button

The Enable Device Module checkbox can be used to make iVision ignore the Device Module without having to moveor delete the Device Module file. When the box is un-checked, iVision will not try to connect to the device. This is mostcommonly done when the device is physically disconnected or otherwise not available.

The Port menu will list the names of all of the serial ports available on the machine. In addition, a Not Assigned entry isincluded for use when you have not yet assigned a port for the device, or if there are no serial ports installed. You mustselect the port to which the device is connected before the Connect button is highlighted.

Example of Port Selection and Disconnect Button

Once the Connect button is pressed, the Device Module will attempt to establish communication with the device, and ifit succeeds, the button will change to Disconnect. Usually some information about the device will also be displayed,such as the version of firmware that it is using. This can be useful for cases where a support person needs to knowexactly which model of device you own.

5.2 Common Device InformationEach Device Module provides a dialog that allows you to enter information about the components that the modulecontrols. There are several common types of components that iVision controls, and the device modules use commondialogs to enter information for them.


5.2.1 Filter Information

The Filter Device Information Panel

For filter devices you may set the name of the filter, its wavelength (WL) in nanometers, and a representative color foreach position.

5.2.2 Microscope Information

The Objective Device Information Panel

For objective turrets you may enter several pieces of information for each installed objective. The magnification must benumeric (10, 40, 100, etc.) The suffix may be an arbitrary string, or empty. The magnification (followed by “X”) and thesuffix are combined to form the name of the objective used in other dialogs. For example, “10X PL FL” in the exampleabove. You also enter the Numerical Aperture (N.A.) and the Refractive Index for the objective. These are used tocalculate values used in the deconvolution of Z-stacks.

The XY Spacing is only displayed in this dialog. It is entered through the Define XY Units command in iVision.

5.2.3 Simple Positioner Information

The Simple Positioner Information Panel

If the device is any other simple positioner, you only need to enter a name for each position. For example, a microscopemay have a prism that can be moved between Left, Right, and Bottom positions.


5.3 Applied Scientific Instruments Filter Wheel andShutter

5.3.1 ASI S&F SetupThe ASI S&F device module can support two filter wheels, plus a “linked” wheel which moves both wheels to the sameposition. In addition, the three shutters are also supported.

Within Device Setup, select ASI S&F and click the Setup button to open this dialog box:

The ASI S&F Device Setup Dialog Box

The Device Setup dialog box provides the standard controls for connecting the controller to the appropriate serial port. Italso lets you name the positions for the each filter wheel.

Three filter wheels are defined. Two are the standard wheels. The ASI Linked device is a virtual, or pseudo-devicewhich treats the first two wheels on a card as though they were a single wheel. For example, setting the ASI Linkeddevice to position 3 will cause both wheels to be moved to position 3. If you use Wait Until Done the wait will only bemade for the second wheel.

The ASI shutter requires the use of a fixed delay to insure that it has fully opened or closed. Enter a time, in millisecondsin the Delay While Shutter Moves field. The dialog lists the recommended delay.


5.4 ASI Stage

5.4.1 ASI Stage SetupWithin Device Setup, select ASI Stage and click the Setup button to open this dialog box:

The ASI Stage Device Setup Dialog

The number of steps per micron for the Z-axis may depend on the type of microscope to which the Z-axis motor isattached.

5.4.2 ASI Stage LockStages with closed loop encoders can be set to maintain the current stage position against drift caused by gravity,accidental jostling, gear slippage, and so on. Any movement is countered by the controller running the motor(s) in theopposite direction. If the stage is moved by a computer-issued move command, or with an attached joystick or focusknob, the lock is ignored until the new position is reached. The lock is then reestablished.

Choose the Device Specific menu item from the Control menu to see this command. It is scriptable.

The ASI Stage Lock Dialog Box


Z Axis: Only check this box if you have a Z axis device on your controller, and you want to changethe state of the lock for the Z axis.

XY Axis: Only check this box if you have an XY stage on your controller, and you want to change thestate of the lock for the XY axis.

Off: Discontinues the stage lock for this axis.

On: Turns on the stage lock for this axis


5.5 BD Biosciences CARV IIThis device module controls the Becton Dickinson Biosciences’ CARV II confocal imager. It provides access to thethree filter wheels (Excitation, Emission, and Dichroic) as well as a “linked” wheel which allows you to move all threewheels to the same position with one command. The device module also controls the shutter, the placement (in/out) ofthe spinning disk, the prism slider (camera/eyepiece), and the FRAP and Intensity apertures.

5.5.1 BD CARV II SetupThis dialog box lets you configure the serial port and name the positions of the filter wheels.

The Setup Dialog Box for the BD CARV II.

5.5.1.1 Connecting Device and Computer

1. First, make certain that the Enabled box is checked.

The Enable Device Module checkbox tells iVision to connect to the microscope when the program is started. If youlater decide to use iVision without the device for a while, uncheck this box to prevent iVision from trying to connectto it. (You will need to use the Disconnect button before you can uncheck Enable Device Module)

2. Click the Port pop-up menu and select the serial port to which you connected the microscope’s RS232 port. TheSetup dialog will be mostly dimmed until the serial port has been identified.

3. Now you can click the Connect button, which just became active. (If you already clicked OK in this dialog box, youcan use the Connect button in the Device Setup dialog box.)


If you cannot connect to the imager, make certain that:• you are using the correct cable and none of its pins are damaged,• all cables are firmly attached, and• the device is turned on and initialized before iVision is turned on.

5.5.2 CARV II OptionsOptions for the CARV are available in a separate command under the Device Specific menu, within the Control menu.This command is scriptable.

The CARV Options Command Dialog Box

Internal/External… Select External to allow the shutter to be triggered from an external signal. Otherwise, useInternal.

Adjacent…Timing: When this is Off the delay after a filter switch is the maximum delay needed for the longestpossible filter wheel movement. When this is checked On, the delay is reduced to that neededfor moving between two adjacent filter positions.

Spinning Disk: Stop Spinning lets you stop the disk any time it is moved out of the light path.Set Intensity Iris to… lets you tie an automatic change in the intensity iris aperture to theremoval of the spinning disk from the light path.

Lockout Touch Screen: Checking this prevents the touch screen from being used to make changes to the devicepositions. This can be useful as part of a script for automated image acquisition.


5.6 Cambridge Research Inc. RGB Liquid CrystalTunable Filter

5.6.1 CRI Filter SetupWithin Device Setup, select CRI Filter and click the Setup button to open this dialog box:

CRI Filter Setup Dialog Box

The CRI does not report any information back to the computer. As a result iVision can not verify the connection, so thedialog reports that the connection is “assumed”.

If you specify Wait Until Done when changing the filter, iVision will simply wait after the change for a specific amountof time, given in the Delay… field. The recommended delay is set for you, but you can specify a different delay if youwish.


5.7 Generic S&F Device ModuleThe Generic S&F Device Module is used to control devices which you have previously defined with the Define UserDevices command (page 98). The definitions describe the commands needed to move to each position. This DeviceModule makes those definitions available to the Device Select command, so that they may be used from any commandthat controls positioner-type devices (e.g. Multi-D Acquire).

5.7.1 Generic S&F Setup

The Generic S&F Setup Dialog Box

You may use up to 6 device definitions. For Serial devices you must indicate the port that the physical device is pluggedinto. The definitions may control parts of a single physical device (in which case they would all use the same portnumber) or they may control separate devices on different ports.

The Set button is dimmed for Lamps and Shutters because they have pre-defined positions and names (On/Off,Open/Close, respectively). For Port and Simple devices the Details button will show you the names of the positions, butyou can only edit the position names with the Define User Devices command. For Filters you can enter the wavelengthand color for each position. For Objectives you can enter the Magnification, NA, and Refractive Index; the positionname in the device definition is put in the objective suffix field.

If you change the definition of a device with Define User Devices the Generic S&F device module will automaticallyupdate itself with the changes.


5.8 Leica DM6000 Microscope (Upright and Inverted)You should turn the microscope on before you start iVision. Give the microscope time to go through its own start-uproutine. This way, the microscope will be ready to listen to iVision when the software begins communicating with it.

5.8.1 Device Setup DialogThe Leica Two microscope appears twice in the Device Setup dialog: once under Stage for the microscope’s focusmotor, and once under Positioners for the microscope’s other motorized components. Both of these entries lead to thesame dialog, and you need only select one of these to set up control of the microscope.

Within Device Setup, double-click Leica Two Stage(s) or Leica Two Light Path to open the Setup dialog:

5.8.2 Leica DM6000 SetupThis dialog box lets you configure the serial port and name the positions of the filter cube and objective turrets.

The Leica DM6000 Setup Dialog Box


5.9 Leica Two Microscope (DM IRE2, DM RXA2)You should turn the microscope on before you start iVision. Give the microscope time to go through its own start-uproutine. This way, the microscope will be ready to listen to iVision when the software begins communicating with it.

5.9.1 Device Setup DialogThe Leica Two microscope appears twice in the Device Setup dialog: once under Stage for the microscope’s focusmotor, and once under Positioners for the microscope’s other motorized components. Both of these entries lead to thesame dialog, and you need only select one of these to set up control of the microscope.

Within Device Setup, double-click Leica Two Stage(s) or Leica Two Light Path to open the Setup dialog:

5.9.2 Leica Two SetupThis dialog box lets you configure the serial port and name the positions of the filter cube and objective turrets.

The Setup Dialog Box for the Leica Two microscopes.


5.9.2.1 Connecting Microscope and Computer

1. First, make certain that the Enabled box is checked.

The Enable Microscope checkbox tells iVision to connect to the microscope when the program is started. If youlater decide to use iVision without the microscope for a while, uncheck this box to prevent iVision from trying toconnect to the Leica. (You will need to click the Disconnect button before you can uncheck Enable Microscope.)

2. Click the Port pop-up menu and select the serial port to which you connected the microscope’s RS232 port. TheSetup dialog will be mostly dimmed until the serial port has been identified.

3. Now you can click the Connect button, which just became active. (If you already clicked OK in this dialog box, youcan use the Connect button in the Device Setup dialog box.)

If you cannot connect to the microscope, make certain that:• you are using the correct cable and none of its pins are damaged,• all cables are firmly attached, and• the microscope is turned on and initialized before iVision is turned on.

5.9.2.2 Get Info

The Get Info button is enabled when a connection has been established. This button provides a list of the currentversions of the hardware devices within your microscope.

This information may be useful when getting technical support for your microscope. If the microscope doesn’t provide aversion number, or if the device isn’t present, the version number will be replaced with question marks.

5.9.2.3 Naming Positions

The lower portion of the dialog lets you edit the names of the positions of the objective turret and the filter cube turret.After connecting to the microscope the Get Names from Stand button will be enabled. If you have defined the names ofthe cubes and objectives using Leica’s software, pressing this button will read those names for the currently selecteddevice from the stand and fill in the fields for you. You can change any of those values at any time. The new names andvalues will be used in iVision, but can not be sent back to the stand.

If you have not previously defined the names, or some of the information is missing our out of date, you can enter it:

1. Select a device from the Device pop-up menu.

2. Edit the text in the Position Name boxes.

3. Pick another device from the Device menu to show and to edit its position names.

4. When you are done setting up the microscope using this dialog box, click the OK button.

If you want to leave this dialog box without saving any of the position names or settings, click Cancel.


5.10 Ludl Shutter and Filter Wheel

5.10.1 Ludl S&F SetupThe Ludl device module can support up to two Shutter & Filter cards in one controller. In addition, a second devicemodule, “Ludl S&F 2,” is supplied to allow the use of two different controllers.

Within Device Setup, select Ludl S&F (or Ludl S&F 2) and click the Setup button to open this dialog box:

The Ludl S&F Device Setup Dialog Box

The Device Setup dialog box provides the standard controls for connecting the controller to the appropriate serial port. Italso lets you name the positions for the each filter wheel.

Six filter wheels are defined; three for each of two cards. Two are the standard Main and Auxiliary wheels. The LinkedWheels device is a virtual, or pseudo-device which treats the main and auxiliary wheels on a card as though they were asingle wheel. For example, setting the Linked Wheels device to position 3 will cause both wheels to be moved toposition 3. If you use Wait Until Done the wait will only be made for the second wheel.


5.10.1.1 Get Info

After you have connected the device, you can click the Get Info button to see a report of the devices attached to yourLudl controller box. This report is read directly from the controller.

5.10.1.2 Early Model BioPoint Fix

Some early models of the BioPoint controller required that a different address be used by the software when talking tothe controller. Checking this box enables the use of those controllers. If unsure, do not check this box. Most BioPointcontrollers do not need to use this checkbox.

5.10.1.3 Switches

If you purchased the equipment from BioVision, it is already configured for your use. Otherwise, click the Switchesbutton to see a picture of how the Ludl controller's RS232 switches should be set.

The Picture Opened by the Switches button

Switches1 2 3 4 5 6 7 8

For OS 9: O O C C C C C C

For OS X: O O O O O C C C

The picture displays the above settings.

If necessary, use this information to set the switches on the Ludl controller's RS-232 (communication) board. Thesesettings should be good for MAC 5000, MAC 2002, and MAC 2000 controllers.


5.11 Ludl Stage

5.11.1 Ludl Stage SetupWithin Device Setup, select Ludl Stage and click the Setup button to open this dialog box:

The Ludl Stage Device Setup Dialog

The number of steps per micron for the Z-axis may depend on the type of microscope to which the Z-axis motor isattached.

5.11.1.1 Get Info

After you have connected the device, you can click the Get Info button to see a report of the devices attached to yourLudl controller box. This report is read directly from the controller.

5.11.1.2 Switches

If you purchased the equipment from BioVision, it is already configured for your use. Otherwise, click the Switchesbutton to see a picture of how the Ludl controller's RS232 switches should be set. Please see the description of theswitches on page 116.

5.11.2 Ludl Stage LockStages with closed loop encoders can be set to maintain the current stage position against drift caused by gravity,accidental jostling, gear slippage, and so on. Any movement is countered by the controller running the motor(s) in theopposite direction. If the stage is moved by a computer-issued move command, or with an attached joystick or focusknob, the lock is ignored until the new position is reached. The lock is then reestablished.


Choose the Device Specific menu item from the Control menu to see this command. It is scriptable.

The Ludl Stage Lock Dialog Box

Z Axis: Only check this box if you have a Z axis device on your controller, and you want to changethe state of the lock for the Z axis.

XY Axis: Only check this box if you have an XY stage on your controller, and you want to change thestate of the lock for the XY axis.

Off: Discontinues the stage lock for this axis.

On, Tolerance: Turns on the stage lock for this axis and sets the drift tolerance in steps. Once the encoderssense a change in position by this number of steps, the controller will attempt to restore theold position.


5.12 Nikon Eclipse E1000 Microscope ControlYou should turn the microscope on before you start iVision. Give the microscope time to go through its own start-uproutine. This way, the microscope will be ready to listen to iVision when the software begins communicating with it.

5.12.1 Nikon E1000 SetupThe E1000 microscope appears twice in the Device Setup dialog: once under Stage for the microscope’s focus motor,and once under Positioners for the microscope’s other motorized components. Both of these entries lead to the samedialog, and you need only select one of these to set up control of the microscope.

Within Device Setup, double-click Nikon E1000 Z-Axis or Nikon E1000 Light Path to open the Setup dialog:

Eclipse Microscope Setup Dialog Box

The Eclipse Microscope Setup dialog for the microscope provides the standard serial port connection pop-up menu.Once the connection is established, the microscope’s internal software version number is displayed below the menu.

For the Z-axis motor, the number of steps per micron of travel defaults to 20. It is unlikely that you will need to changethis. Currently, when the E1000 is first turned on, it will ignore commands from the computer to move the focus motor.The only way to enable computer control of the focus motor is to manually move the focus knob yourself. Since only thesmallest change is needed, and since you will most likely move the stage before using the computer to do so, this shouldrarely be a problem.


The Position Names box allows you to name each of the filters in the Epi-Fl cassette. By choosing Epi-Fl Shutter fromthe Device pop-up menu, you can change the names of the shutter’s two positions, though the default positions “Open”and “Close” have already been set for you.

The Reset button sends a reset command to the microscope, which is equivalent to turning the microscope’s control boxoff and back on again. You should not need this button in normal operation. If the microscope appears to fall out ofcommunication with the computer, you can test the connection by using this button. The microscope should respond withthe same beeping noises that it makes when first turned on.


5.13 Nikon Remote FocusThis device module supports the “Nikon Remote Focus Accessory”, manufactured by Conix Research Inc., which addsan external motor to the focus drive of certain Nikon microscopes.

Two models of this device have been manufactured. They require different commands to set and get the position of themotor, so you must indicate which you have. The older models have a “sensitivity” switch on the side that changes theresolution of the display on the controller. The newer models lack this switch.

5.13.1 Nikon Remote Focus Setup

Nikon Remote Focus Setup Dialog Box

Old/New Model: Indicate which model you have. If you are not sure, select one and test (without a slide!) thedistance the stage moves when commanded to move a fixed distance. If the actual distancemoved is off by a factor of 10, you have the other model.


5.14 Nikon Eclipse TE2000 Microscope ControlYou should turn the microscope on before you start iVision. Give the microscope time to go through its own start-uproutine. This way, the microscope will be ready to listen to iVision when the software begins communicating with it.

5.14.1 Device Setup DialogThe TE2000 microscope appears twice in the Device Setup dialog: once under Stage for the microscope’s focus motor,and once under Positioners for the microscope’s other motorized components. Both of these entries lead to the samedialog, and you need only select one of these to set up control of the microscope.

Within Device Setup, double-click Nikon TE2000 Z-Axis or Nikon TE2000 Light Path to open the Setup dialog:

TE2000 Setup Dialog Box

The Setup dialog for the microscope provides the standard serial port connection pop-up menu. Once the connection isestablished, the microscope’s internal software version number is displayed below the menu.

For the Z-axis motor, the number of steps per micron defaults to 20. It is unlikely that you will need to change this.

The Position Names box allows you to name the positions for the various turrets and filter wheels.


5.15 Olympus BX & IX Microscope ControlYou should turn the microscope on before you start iVision. Give the microscope time to go through its own start-uproutine. This way, the microscope will be ready to listen to iVision when the software begins communicating with it.

5.15.1 Device Setup DialogThe BX or IX microscope appears twice in the Device Setup dialog: once under Stage for the microscope’s focus motor,and once under Positioners for the microscope’s other motorized components. Both of these entries lead to the samedialog, and you need only select one of these to set up control of the microscope.

5.15.2 Olympus BX / IX SetupWithin Device Setup, double-click Olympus BX(or IX) Z-Axis or Olympus BX(or IX) Light Path to open the Setupdialog:

Setup Dialog Boxes for BX and IX Microscopes

1. First, make certain that the Enable Microscope box is checked. The Enable Microscope checkbox tells iVision toconnect to the microscope when the program is started. If you later decide to use iVision without the microscope fora while, uncheck this box to prevent iVision from trying to connect to the microscope. (You will need to click theDisconnect button in the Device Setup dialog before you can uncheck Enable Microscope.)

2. Then select the proper Port.


3. Finally, click Connect.

Next, fill in the rest of the settings:

Focus MotorSteps/Micron: The number of steps per micron of travel defaults to 100. It is unlikely that you will need to

change this.

Escape Objectivesby __ Microns: This feature protects the objective when switching to another. When you use iVision to switch

objectives, the objectives will move away from the state by the given amount, rotate, and thenmove back.

Set this distance to zero if you do not want to use the feature.

Position Names: You can name the positions for the parts of the microscope, as well as set some otherparameters.

The different controllable parts of the microscope are known as “devices.” Some devices havediscrete positions, like the filter cube turret (called “Cube” on the AX70). Once themicroscope has been connected, the software will read the position names from themicroscope. You can change these names to make it easier for you to refer to and control thedevices.

The current device is shown in the Device pop-up menu, and its positions are listed belowthat. The number of positions varies depending on the device. In the dialog pictured above,the eight cube positions are named with their Olympus model codes.

To name a position, first choose the appropriate microscope part (e.g. Cube or Objectiveturrets) from the Device pull-down box. Then select the text in the Position # box and replaceit with the correct name. Click OK when you are done to save the position names.

Variable devices that change smoothly over a large range, like aperture stops, do not haveposition names, and therefore cannot be selected in the Device menu.

When you are done setting up the microscope using this dialog box, click the OK button (shown above in the picture ofthe full dialog box). If you want to leave this dialog box without saving any of the serial port settings, click Cancel.

5.15.3 Olympus BX / IX ModeFor the computer to control the BX and IX microscopes, it must be “logged in” to the microscope. When the computer islogged in, however, the user is blocked from performing many manual actions.

iVision provides this separate, scriptable, command to give you a choice on how to manage this log in requirement.


Choose the Device Specific menu item from the Control menu to see this command.

The Olympus BX (and IX) Log In Dialog Box

If you choose Auto Log In, then iVision will log out of the microscope after each command to the microscope. Thisoption lets you use the microscope's knobs and LCD display without the additional step of logging out. However, thismay reduce the speed of automated operations.

If you choose Remain Logged In, then iVision will remain connected to the microscope between commands. Youcannot manually control the microscope while iVision is logged in, but a series of automated actions can be performedmore quickly.

In common use you would use Auto Log In most of the time, and only turn Remain Logged In on during a script thatcontrolled the scope (and then switch back to Auto Log In at the end of the script).


5.16 The Physik Instrumente Piezo-Electric Focus Device(Models 662 and 665)

The Physik Instrumente piezo-electric focus actuator is built around a piezo-electric crystal that expands under control ofelectric current. It has a 100 µm range of motion, up and down. It does not move in steps, and therefore no step sizesetting is required.

Two controllers are supported; model 662 and the newer model 665. Each model has its own iVision Device Module.

5.16.1 Setup DialogWithin Device Setup, select PI 662 Z Axis or PI 665 Z Axis and click the Setup button to open the Setup dialog:

The PI 662 and PI 665 Z Axis Setup Dialog Boxes

When connected, the area below the port selection menu shows information provided by the controller. This can behelpful if you need to identify the specific model of controller that you have.


5.17 Prior Shutter & Filter

5.17.1 Prior S&F SetupWithin Device Setup, select Prior S&F and click the Setup button to open this dialog box:

Setup Dialog Box for the Prior Shutter & Filter

Select the Port to which your Prior controller is connected, and then click Connect. When your Prior controller iscommunicating with your computer, the dialog box will display the controller's version number and serial number.

The Prior controller can drive up to two filter wheels, separately or linked. When moved as linked wheels, both filterwheels will be moved to the same position.

To name the filter positions, click on the Device pop-up box and select the filter wheel (or the linked device). Then enterthe position names.

Delay while Shutter Moves makes certain that the shutter has finished moving before iVision does anything else, suchas acquire an image.


5.18 Prior Stage

5.18.1 Prior Stage SetupThe Prior Stage device module supports the OptiScan and ProScan model stage controllers. It also offers support for theH128 and earlier models (H127 and H126). However, the earliest models did not use as many commands as the H128.As a result, some options, such as centering the stage, may not work the same as on the H128.

The Prior Stage Device Setup Dialog Box

5.18.1.1 The Prior XY Stage

The step size of the XY stage’s motors is usually 0.5 steps/µm, where a step is the smallest movement the motor canmake.

5.18.1.2 The Prior Z Motor

The Prior Z motor has an essentially unlimited range of motion in either clockwise or counter-clockwise directions.

The step size of the Z motor is usually 10 steps/µm, where a step is the smallest movement the motor can make.


5.19 Sutter Lambda DG-4 High Speed Filter ChangerThe device module for the DG-4 provides access to the four filter positions in the device. In addition, a shutter isprovided that simulates the shutter in conventional shutter/filter devices. When this shutter is closed the internal mirrorsdirect light away from the output port. When opened, the mirrors are set to the last selected filter position.

5.19.1 Sutter DG-4 SetupWithin Device Setup, select Sutter DG-4 and click the Setup button to open this dialog box:

Sutter DG-4 Setup Dialog Box

Enabled: When you first see this dialog, make certain that the Enabled box is checked. You can usethis checkbox to prevent disconnected devices from loading at program startup.

Port: The next thing you should do is to choose the serial port to which your Lambda DG-4 isconnected. You only have to do this once, unless you move the serial cable.

Device: The Device pop-up box will only list the DG-4 Filter changer.

Position 1...4: These text boxes hold whatever names you like for each filter position.


5.19.2 DG-4 CommandsBesides the filter and shutter, an additional device is provided to allow you to send a special set of commands to the DG-4. This device is named DG-4 Commands, and it provides four commands in the form of “positions.” To use these:

1. Choose the S&F Control command from the Control menu.

2. Select the Position Name radio button.

3. Choose one of the positions from the Position Name pop-up box: Blanking On, Blanking Off, Display On, andDisplay Off.

By selecting a “position," you can send the matching command to the filter changer.

Blanking On/Off: This refers to the feature Sutter calls “turbo-blanking”. When Blanking is ON, the twointernal mirrors move out of step with one another. The result is that intermediate filterpositions are not illuminated while moving between two non-neighboring filters.

For example, with Blanking OFF (the default), moving from position 1 directly to position 3will still cause light to briefly pass through position 2. With Blanking ON, this will nothappen.

For optimal speed you should keep Blanking OFF and arrange your filters next to oneanother.

NOTE: after sending this command, the display panel on the DG-4 will NOT update untilafter a new filter position is selected.

Display On/Off: When set to Display OFF, the display panel on the filter changer will not be updated. Suttersays that this improves the speed of the changer. Unfortunately, the display does not indicatethat it has been disabled, so it can appear to be displaying the wrong setting, when it isactually just out of date.

The reset button on the filter changer can be used to clear all settings back to their default values. However, if you use it,you must go to Device Setup to get the filter changer to accept further commands from the serial port. Select the DG-4device and click Disconnect, followed by Connect.


5.20 Sutter Lambda Filter Wheel Controller(Models 10-2 and 10-3)

The Sutter Lambda Device Module supports the Sutter 10-2 and 10-3 controllers. The 10-3 adds a third filter wheel and“Smart Shutters™” that can be closed a variable distance in order to act as a neutral density filter.

5.20.1 Sutter Lambda SetupWithin Device Setup, select Sutter Lambda and click the Setup button to open this dialog box:

Sutter Lambda Setup Dialog Box

When you first see this dialog, make certain that the Enabled box is checked. You can use this checkbox to preventunavailable devices from loading at program startup.

The next thing you should do is to choose the serial port to which your Lambda 10-2 or 10-3 is connected. You onlyhave to do this once, unless you move the serial cable. Then, click Connect.

Use the Device pull-down box to view and change the position names for each of the two filter wheels that the Lambda10-2 or 10-3 can control. If you identify the filters in the position names, then you can use the Microscope Controlcommand to move the filter wheels according to the position names.


5.20.2 Smart Shutters™For both the 10-2 and 10-3 controllers, iVision provides two shutter devices:Sutter Shutter A,Sutter Shutter B.These are standard shutters with Open and Close positions.

In addition, to use the Smart Shutters™ available on the 10-3 controller, iVision adds three more devices:Sutter ND Aperture A,Sutter ND Aperture B,Sutter Shutter Options.

The two aperture devices treat the shutters as though they were variable-sized apertures and allow you to open the shutterby 1 to 100%. This partial position only applies when the matching shutter device is in the Open position, When closed,the shutter is always completely closed.

The current aperture position remains in effect until either the position is set to 0%, or Fast or Soft mode (see below) isapplied to the shutter.

The Sutter Shutter Options device acts as a simple positioner and lets you control the following options:

Fast Mode: Runs the shutter at full speed. Also discontinues the ND Aperture mode, if it’s in effect.

Soft Mode: Accelerates and decelerates the shutter during its travel in order to decrease mechanical jitter,at the cost of a slower speed. Also discontinues the ND Aperture mode, if it’s in effect.

Link To Wheel: This causes the shutter to close while the filter wheel is in motion and open again once thewheel has stopped. To turn this mode off, just issue an Open or Close command to theshutter. This option does not cancel the ND Aperture mode, so you can combine the NDshutter position with the Link To Wheel option.


5.21 Vincent Associates UniBlitz Shutter ControlTwo UniBlitz device modules are provided with iVision: UniBlitz and UniBlitz 2, so that you may issue commands totwo different shutter controllers on different ports.

5.21.1 UniBlitz SetupWithin Device Setup, select Uniblitz (or Uniblitz 2) and click the Setup button to open this dialog box:

The UniBlitz Setup Dialog Box

The UniBlitz control is quite simple. Select the appropriate serial port, choose the speed of your controller, and connectthe device. Early models of the controller ran at 300 baud, later models run at 9600. The UniBlitz does not report anyinformation back to the computer, so iVision can only assume the device is connected. Testing the connection is a simplematter of using the Microscope Control dialog to open and close the shutter.

In order to support Wait Until Done, the device module will wait a specific amount of time before continuing. Theminimum delay is set in the Delay while Shutter Moves field. You may need to use a larger value, depending on theactual shutter used.


5.22 Virtual Shutter & FilterThe Virtual S&F device module is a software-only “device” that mimics the common components of a microscope. Itallows you to prototype scripts and test the control dialogs without having any actual hardware device connected to yourcomputer.

5.22.1 Virtual S&F Setup

Virtual S&F Setup Dialog Box

The Virtual Filter device module provides three eight-position virtual filter wheels: FW1, FW2, and linked filter wheels.The Linked wheel acts like the linked wheel in other device modules: it moves the first and second wheels to the sameposition. The Virtual Shutter device module also provides three virtual shutters, a virtual objective turret, a virtual lamp,a virtual port changer, and a virtual aperture device. A virtual macro lens is used to simulate a “simple” positioner, and avirtual zoom is used to simulate a generic “variable” device.

You can specify a delay period to be used when the Wait Until Done option is used in the Microscope Control dialog.

To indicate that the wheel is moving, the Virtual Filter device issues a series of click sounds, with the number of clicksmatching the “distance” (number of positions) between the current position and the new position. To indicate that theshutter is changing, the Virtual Shutter device plays one of two sounds representing the new position: Open or Closed. Ifthe shutter is already in that position no sound will be played.


5.23 Virtual StageThe Virtual S&F device module is a software-only “device” that mimics a Z stage and an XY stage. It allows you toprototype scripts and test the control dialogs without having any actual hardware device connected to your computer.

5.23.1 Virtual Stage Setup

Virtual Stage Setup Dialog Box

Start: This determines what the initial coordinates of the stages will be when iVision is first started.This lets you test scripts based on the behavior of different stages.

Travel Time: This determines if the new position is returned immediately after a move command is given,or after a delay based on the distance to be moved and the current speed of the stage. You canchange the stage’s speed with the Stage Speed command in the Control menu.

Steps/Micron: This simulates the settings used by actual stages. You can leave the default value of 1 inplace.


5.24 Yokogawa CSU-22 Confocal Scanning UnitThis device module controls the CSU-22 confocal scanning unit from Yokogawa. It provides control of the filter wheels,either individually or as a group. It also controls the shutter, the Neutral Density filter, and the speed of the spinning disk.

5.24.1 Yokogawa CSU-22 Setup

Yokogawa CSU-22 Setup Dialog Box

The CSU Combined Filters device is not like other “linked” wheel devices. There are only 5 possible positions andthese correspond to the 5 pre-determined combinations of positions provided by the device:

1) All wheels at position 12) All wheels at position 23) All wheels at position 34) EX & BA at position 2, DM at position 35) EX & BA at position 1, DM at position 3

To move the wheels to other positions, use the individual filter devices and move each one separately.


5.25 Zeiss AxioImager Microscope ControlYou should turn the microscope on before you start iVision. Give the microscope time to go through its own start-uproutine. This way, the microscope will be ready to listen to iVision when the software begins communicating with it.

5.25.1 Zeiss AxioImager SetupThe AxioImager microscope appears twice in the Device Setup dialog: once under Stage for the microscope’s focusmotor, and once under Positioners for the microscope’s other motorized components. Both of these entries lead to thesame dialog, and you need only select one of these to set up control of the microscope.

Zeiss AxioImager Setup Dialog Box

You may specify information for each of the components listed in the menu. When connected to a stand, the Get Namesfrom Stand button will be highlighted and you can fill in the names of positions from the values stored in the stand. Youcan change any of those values at any time. The new names and values will be used in iVision, but can not be sent backto the stand.


The other Positioners that are controlled by this device module are:Fluorescent ShutterCondenser ShutterHalogen LampCamera/OcularsCamera ChoicePhoto Shutter2ie Fluorescent Shutter2ie Condenser Shutter

If you have the AxioPlan 2ie Imaging model, you must use the two shutters with “2ie” in their name.

In addition, the following componentscan be controlled through the Variable Device command:Optovar ZoomAperture DiaphragmCondenser DiaphragmLuminous Field Aperture

Each of these devices can be set to one of 256 positions, in the range of 0 to 255.


5.26 Zeiss AxioPlan Microscope ControlYou should turn the microscope on before you start iVision. Give the microscope time to go through its own start-uproutine. This way, the microscope will be ready to listen to iVision when the software begins communicating with it.

5.26.1 Device Setup DialogThe AxioPlan microscope appears twice in the Device Setup dialog: once under Stage for the microscope’s focus motor,and once under Positioners for the microscope’s other motorized components. Both of these entries lead to the samedialog, and you need only select one of these to set up control of the microscope.

5.26.2 AxioPlan Setup

AxioPlan Microscope Setup Dialog Box

The AxioPlan Microscope Setup dialog for the microscope provides the standard serial port connection pop-up menu.Once the connection is established, the microscope’s internal software version number is displayed below the menu.

The number of steps per micron of travel for the Z-axis motor defaults to 20. It is unlikely that you will need to changethis. The Extra Precision Z Move checkbox can only be used with certain versions of the microscope firmware. If you


have version 1.10, with a date of 08/05/96 (this will be displayed below the Port menu), then you must uncheck this box,or you will get errors while moving the Z axis.

The Position Names box allows you to name each of the positions for the devices listed in the menu above.

The other Positioners that are controlled by this device module are:Fluorescent ShutterCondenser ShutterHalogen LampCamera/OcularsCamera ChoicePhoto Shutter2ie Fluorescent Shutter2ie Condenser Shutter

If you have the AxioPlan 2ie Imaging model, you must use the two shutters with “2ie” in their name.

In addition, the following componentscan be controlled through the Variable Device command:Optovar ZoomAperture DiaphragmCondenser DiaphragmLuminous Field Aperture

Each of these devices can be set to one of 256 positions, in the range of 0 to 255.

Processing Extensions -Fluorescence CV 141

Part IIIProcessing Extensions

6 Fluorescence CVNote: As used here, "channel" means the data for a single color within an image. Grayscale images contain one

channel; color images contain several channels (for example, red, green, and blue).

6.1 Color JoinThe Color Join command merges grayscale images together to create a color image. Each grayscale component imagegets treated as a color channel by the Color Join dialog box. Each grayscale component image contributes to the red,green, and blue components of the final, Color 24 image.

Color Join allows you to interactively set the optimal contrast and brightness for each channel and correct for smallpixel shifts among the channels. Before you choose the Color Join command, open the images to be merged. Theimages must all be the same size and have a Byte, Short Int, or Unsigned 16 data type. Images can be single frames orsequences, but all sequences must have the same number of frames. If you forget to open the images first, click Cancelon the Color Join dialog box and then open the images. The final image will have the Color 24 data type.

After you choose the command, the Color Join Controls palette and the Color Join dialog box will appear.

6.1.1 Color Join Controls PaletteThe Color Join Controls palette lets you pick the images to be merged and the colors that will represent them. It alsoallows you to create new colors for the channels. New colors can be defined to match the color of any fluorescent probe.

6.1.1.1 Images Tab

The Images tab lets you pick the images to be merged and the colors that will represent them. You can merge up to ninefluorescent images plus one brightfield image.

142 Processing Extensions - Fluorescence CV

Images Tab of the Color Join Controls Palette

Channel: Choose the color channel to represent each image. The channel's data will appear in shades ofthe channel's color. The built-in colors are red, green, blue, cyan, magenta, and yellow. Youcan also create your own colors in the Define Colors tab.

If a user-defined color is disabled or deleted, its name will be italicized in the Channel menu.Either stop using that color or reinstate it in the Define Colors tab.

You can deactivate any channel. Selecting None removes that grayscale image from the final,color image. This allows you to easily switch on and off the contribution of this channel in the

final image. To remove an image after selecting None, you must click Apply or Hide.

Image: Pick an image from the pop-up menu on the right or type the window's name. The imagesmust all be open, the same size and have a Byte, Short Int, or Unsigned 16 data type. Imagesmay either be single frames or sequences, but all sequences must have the same number offrames.

If you select a sequence with a different number of frames, its name will be underlined andcolored blue. The final image will have the same number of frames as the first sequenceselected in the Color Join Controls palette.

If an image is not the same size as the others, its name will be underlined and colored blue. Ifthe image is not open, its name will be underlined and colored red. If you pick the same imagemore than once, they will be underlined and colored green. Replace the offending image namewith a unique, open image.

If you do not want to use an image, you do not have to delete the image name. Simply changethe Channel for that image to None.

Brightfield: You can select a grayscale (Byte, Short Int, or Unsigned 16) image to use as a backdrop forthe fluorescent signals. Bright field images, particularly DIC and phase contrast pictures, areused to show underlying shape and/or structure in cells or tissue.

Final Image Name: Give a name to the merged image.

Apply: Click Apply if you want to see your chosen channels displayed in the Color Join dialog. Youmust click Apply or Hide for your changes to take effect. If you pick different images orchannels and then click OK, your change will not take effect.


Hide: Click Hide when you are done. You can show this palette again by clicking the ShowControls button on the Color Join dialog. This button will also apply if changes have notbeen applied.

When first viewing new images, Color Join will zoom out from the image such that at least one dimension fits in thedialog's available space.


6.1.1.2 Define Colors Tab

The Define Colors tab lets you create colors to use as Channels in the Images tab. For example, you can define a hueidentical to DAPI's emission color and use that for your DAPI channels. You can make new channel colors at any time.

Define Colors Tab of the Color Join Controls Palette

New: Start out by clicking the New button to create a new color.

Remove: Click Remove to delete the currently selected color. This cannot be undone.

Color Name: Give the color a name that will identify it in the Channels menu on the Images tab.

Color: Click on the color box next to the Color Name. This opens Apple's Color Picker, which givesyou a number of tools for choosing a color. Before a color can be chosen, a name must begiven for the new color.

In Menu? : Check the In Menu? checkbox if you want this color to appear in the Channels menu on theImages tab. You can uncheck colors to keep the Channels menu short, or to control whichcolors are used routinely.

Final Image Name, Apply, and Hide are described in the Images Tab section on page 142. Click Hide when you finishpicking your images and their channels.


6.1.2 Color Join Dialog BoxThe Color Join dialog lets you adjust the appearance of each image channel and then merge them together. It candisplay a preview of your changes to an image channel and the final, merged image.

Color Join Dialog Box

First, improve the display of each channel by using the Mask, Normalize, and Shift tabs. Then use the Merge tab toimprove the joining of the channels. Finally, click the OK button to create the color-joined image.

Show Controls: Click this button to choose different images or color channels from the Color Join Controlspalette (see page 141).

Cancel, OK: Click OK to merge your grayscale images into a color image. Click Cancel to close the ColorJoin dialog without creating any new windows.


6.1.2.1 Displaying images in the Dialog Box

View: Choose Split View to see two images side-by-side: one of the channels on the left, and thefinal, merged image on the right.

Pick Single View to fill the dialog box with the channel image.

Channel Buttons: The colored buttons on the top-left side of the dialog box represent your channels. The BF

button represents the bright field image. The tri-colored button ( ) below that representsthe merged, final image. Click on any of these buttons to display that image on the left side ofthe dialog box. For example, clicking on the red button (if present) will switch to the redchannel. You can also use the Channel pop-up menu.

Channel: The Channel pop-up menu lists all of the channels being merged, as well as the final(merged) image. Pick a channel to display it on the left side of the dialog box. You can alsouse the colored buttons on the top-left side of the image.

Frame: When merging image sequences, use the Frame controls to display a particular image frame.The entire sequence will be merged using the same settings regardless of which frame isshown.

You can type a frame number in the text field, step through the frames using the arrowbuttons, or scan the sequence by moving the slider. (The slider is labeled with the minimumand maximum frame numbers.) You can also toggle the # button to Var and type in avariable's index number.

Magnifying Glass: Click this tool to change the cursor to the magnifying glass. Click on the image to magnify it;hold down the Option key and click to demagnify.

6.1.2.2 Mask

Use masks to hide parts of the image channel. Hidden areas will not appear in the final image. The dialog box indicatesmasking by covering the masked areas with gray, so they appear dim.

The Mask Tab

ON / OFF: Click the ON button to create a mask for the current channel. When you first turn masking on,the entire image of this channel will be masked, removing it from the final image.


Use the Lasso (freehand) and Pencil tools to edit the mask:

Lasso Tool: Edit the mask by drawing closed loops with this tool. Hold the mouse button down and dragthe mouse in a loop. The loop will close automatically when you let go of the mouse button.

When the lasso cursor contains a plus sign, it adds to the visible regions of the image.

When you hold down the Option key, the lasso contains a minus sign, and it subtracts fromthe visible regions (by adding to the mask region).

Pencil Tool: Edit thin regions of the mask by drawing lines with the pencil tool. Hold the mouse buttondown and drag along a line or curve. If you just click the mouse button, you can draw a small,filled circle.

When the pencil cursor shows a plus sign, it adds to the visible regions of the image.

When you hold down the Option key, the pencil cursor shows a minus sign, and it subtractsfrom the visible regions (by adding to the mask region).

Pencil Sizes: These buttons control the thickness of the pencil lines.

Show All / Hide All: Click Show All to remove the entire mask. Click Hide All to mask the entire image, hiding it.

Apply: Click Apply to see the effects of your masking.

6.1.2.3 Normalize

Use the Normalize tab to select the display range of the image. Values below the minimum will be displayed as black.Values above the maximum will be set to the channel's selected color. The values in between will follow the midpointcurve described below. This does affect the data in your final, color image, unlike the Enhance menu's Normalizationcommand.

The Normalize Tab

To normalize the active image, choose the mode that the display should be Based On. Then adjust the display using theGamma, Brightness, and Contrast controls. You can also use the histogram.

Based On: The Based On options control where iVision will search for the minimum and maximum datavalues.

When the Frame or Sequence option is used, the minimum and maximum data values will befound within either the current frame or image sequence.


With the Saturated Frame and Saturated Sequence options, too, the minimum andmaximum values will be found within the current frame or sequence; however, approximately1% of the darkest and brightest pixels will be mapped to 0 and 255. This feature displaysbetter-looking images in cases where there are just a few pixels with values far from thetypical image pixel values. This might happen, for example, when you grab an image from acamera with a faulty sensor.

You use the Manual option whenever you use the Contrast, Brightness, and Gammabuttons or the histogram section at the bottom of the dialog box.

Contrast,Brightness, Gamma: These three sets of controls work in the same fashion: left arrows decrease the parameter, and

right arrows increase the parameter; double arrows make bigger changes than single arrows.The circles return Contrast and Brightness to the minimum and maximum values present inthe image and Gamma to a value of one. Changing Contrast or Brightness switches BasedOn to Manual mode.

Contrast: Increasing the contrast narrows the range defined by the minimum and maximumnormalization values, improving the display of that range of values at the expense ofthe rest of the image.

Brightness: Increasing the brightness increases the number of pixels being displayed as white (oras the saturated channel color). This occurs by decreasing the minimum andmaximum normalization values equally.

Gamma: The gamma controls how evenly the whole range of data values are normalized.Increasing the gamma decreases the contrast in the lower value range, and creates adarker background for fluorescence images.

Gamma correction is often used to correct for the effects of non-linearities in theimage acquisition or display process. Any gamma setting other than 1.00 creates anon-linear mapping of the form:

Displayed value = [(normalized value – min) / (max – min)]gamma

Mid Point: Gamma is a function of the mid point value. Mid point is expressed as a percentageof the range between minimum and maximum. The Mid Point control willimmediately apply any whole number from 5 to 95.

Histogram: The large rectangle holds a column graph of pixel intensities (on the X axis) and the quantitiesof pixels representing each intensity (on the Y axis). This histogram helps you visualize therelative amounts of intensities within the image.

Low intensities (typically black) are represented at the left of the histogram, and higherintensities (typically white) towards the right end.

The diagonal, green line on the histogram represents the gamma curve.

Type: You can view several types of histogram. Regular and Log Regular are the mostcommonly used.

From: The histogram’s information can come from the current frame or the entire sequence.

Min / Max: There are two sets of controls for manually setting the minimum and maximum datavalues that will be matched to the final values 0 and 255. The blue and red slidinghandles below the histogram and the two text fields to the histogram's right both setthe minimum and maximum values.


You can type the minimum value in the top text field, and the maximum value in thebottom text field. Hit Apply when you are done. You can also set these valuesmanually by dragging the blue and red handles back and forth across the histogram.Any pixel intensities at the blue bar or to its left will be displayed as zero, or black.Anything at the red bar or to its right will be displayed as 255 or the saturatedchannel color. Altering the minimum or maximum changes Based On to Manualmode.

Gamma: The green sliding handle in the middle of the histogram controls the gamma andmid-point. Dragging this bar will change the Gamma value shown on the left side ofthe Normalization tab.

6.1.2.4 Shift

A common problem encountered in fluorescence microscopy is the mis-registration of images captured using filters thatare not perfectly parallel. To correct for this, pick any channel and align the image using the Shift tools.

The Shift Tab

Click the arrow buttons to move the image one pixel at a time. You can also use the X and Y text fields to set the amountof the shift. Down and left are negative distances.

Click the Apply button to see the effects of shifts made using the text fields.

6.1.2.5 Merge

Use this tab to control how the channels will be merged together.

The Merge Tab

Channel Blend: At each pixel, the brightest fluorescence channel will be blended with a percentage of theintensity of the dimmer channels. Enter that percentage in the Channel Blend text field andclick Apply. Enter a lower percentage to accentuate the brightest pixels. Enter a higherpercentage to blend more smoothly. A value of 100% would provide an un-weighted blend ofcolors.


Blend n% of theFluorescent Image: Blend this percentage of the fluorescent image with the bright field image.

This feature lets you smoothly blend the fluorescence signal with the bright field background.Blending 100% of the intensities of the fluorescence channels gives you brighter color.Blending a lower percentage dims the fluorescence signal, but gives you a smoother finalimage.

6.2 ColocalizationThe Colocalization command compares two images and produces a merged image of the colocalized channels. It canalso produce data windows for further analysis of the colocalization. The Colocalization command is an important toolfor the study of fluorescence images, but it can also be used to analyze transmitted-light images.

You can use this command to statistically measure the similarity between images and to display the specific areas wherethe two probes are colocalized. The images can be a frame, a volume, or a time lapse movie.

Before you choose the Colocalization command, open the images. They can be grayscale (Byte or Unsigned 16 only) orcolor (Color 24 or Color 48) images. Either or both images can be sequences, but they must both have the same widthand height. If you forget to open the images first, click Cancel on the Colocalization dialog box and then open theimages.

After you choose the command, The I/O palette and the Colocalization dialog box will appear.

6.2.1 Important Details1. Colocalization between two channels (probes) is present when there is a proportional relationship between the

intensities at different locations within the two channels. A line in the scatter plot discussed below can represent thisrelationship.

2. You should attempt to remove any source of noise or cross talk between probes before analyzing colocalization. Thecolocalization statistics are very sensitive to background and cross talk. The colocalization coefficients are describedon page 160.

3. You should use iVision to align the images before doing colocalization analysis. The images must have identicalmagnification, translation, and orientation. The Colocalization command has Shift tools for minor realignments.

4. Keep the Intensity Delta values relatively small, so that the points between the colored, diagonal lines have similarintensity ratios ( Ri Gi ). These will be the points close to the central white line. If you make the Intensity Deltavalues too large, the colored lines will encompass outlying points caused by noise. Intensity Delta is described onpage 155.


6.2.2 I/O PaletteUse the I/O palette to pick the two images to be compared.

I/O Palette

Probe 1/2 Image: Pick images from the pop-up menu on the right or type the window's name. The imagesmust all be open and the same size. If both Probe 1/2 Use pop-up menus are set to Sequence,then both sequences must also have the same number of frames. Finally, the data in eachchannel must be the same data type. That means that you can choose a Byte image and achannel from a Color 24 image, or an Unsigned 16 image and a channel from a Color 48image.

If the data types of the two images do not match, then the image names will be underlined andcolored purple. If an image is not the same width and height as the other, its name will beunderlined and colored blue. If the image is not open, its name will be underlined and coloredred. In any case, replace that image with an appropriate one.

Channel: If you pick a color image, choose which channel (red, green, or blue) to use.

Probe 1/2 Color: Choose a color channel (red, green, or blue) to represent this image. You mustchoose a different color to represent each image, of course.

Probe 1/2 Use: If this image is a sequence, choose to use only one frame or the entire sequence.

If you use Frame, you can pick the frame by showing the image in theColocalization dialog box and changing the Frame slider or text field.

If you use Frame for one image and Sequence for the other, then the one image'sframe will be compared with each frame of the other image.


Final Image Name: Type a base name for all of the output images.

Output: Click this button to choose what types of output Colocalization will produce. Asmall list of options will appear:

Menu of Output Options

The Final Image is a Color 24 image of the merged data. This image includes theeffects of the Colocalized Pixels Tint and Non-Colocalized Pixels options. Its namewill be the name entered in the Final Image Name field on the I/O palette.

The Scatter Plot Image is a Color 48 image of the plot displayed by theColocalization dialog's Show: Scatter Plot pop-up menu item. This window will benamed FinalImageName.i.

The Statistics window stores the same statistics produced by the Show Statisticsbutton. This Floating Point image will be named FinalImageName.s. You can usethis information to reset the dialog box and reproduce the colocalization results.

The Scatter Plot Matrix contains the scatter plot's raw data in a Long Integerwindow. This window will be named FinalImageName.m.

The Colocalized Objects window is a Color 48 image of the colocalized regions.This image does not include any non-colocalized data and it does not include thecolocalized pixels tint. This window will be named FinalImageName.c.

Apply: Click Apply to display the chosen images in the Colocalization dialog box.

Hide: Click this button to apply the image selections and to hide the I/O palette. You can reopen itby clicking the Show I/O button on the Colocalization dialog box.


6.2.3 Colocalization Dialog BoxThe Colocalization dialog box's parameters let you control how tightly the colocalization will be measured.

Colocalization Dialog Box

On the left, the dialog box displays a preview of your changes to the image channel. On the right, it displays the final,merged image.

Show buttons: The two colored buttons on the top-left side of the left-hand image represent the two imagesbeing compared. The bottom button represents the scatter plot. You can switch to an image orthe scatter plot by clicking the appropriate button. You can also use the Show pop-up menu.

Show: The Show pop-up menu lists the images being compared. It also lists the scatter plot. Pick oneof the images or the scatter plot to display it on the left side of the dialog box. You can alsouse the colored buttons on the top-left side of the image (the bottom button opens the scatterplot).


Frame: When using image sequences, use the Frame controls to display a particular image frame. Ifyou set this image to Probe 1/2 Use: Frame, then the frame you display here will be used tocalculate colocalization.


Click Apply whenever you change the frame (or press a Frame control). That will update thefinal image. Moving the Frame slider will update automatically, however.

Non-ColocalizedPixels: You can Show or Hide the parts of the image that are not colocalized.

This option does not affect the scatter plot.

Colocalized PixelsTint: Check this checkbox to highlight the areas of colocalization by overlaying them with a

specific color. Click the color bar to pick the overlay color from the Apple Color Picker.

This option does not affect the scatter plot.

Max Value: This is the maximum intensity that will be considered colocalized. This is also the maximumvalue of the scatter plot.

Click Apply whenever you change these parameters. That will update the final image.

Compute Max Value: Checking this box and then pressing the Apply button will compute the bestmaximum value for the data. The maximum intensity in both images will bedetermined and a larger number equal to a power of two minus one (2n − 1) will beused as the Max Value. An attempt is made to make the # of Elements equal to 256and the bin size will be set accordingly.

Bin Size: This is the scatter plot's bin size. The scatter plot's range is divided into bins, withthis number of pixel values counted in each bin.

# of Elements: This is the number of elements, or bins, that divide the scatter plot's range. This isequal to (the Max Value +1) / Bin Size.

Threshold: This histogram controls the minimum intensity that will be considered by the colocalizationalgorithm. The area (underlined in green) to the right of the handle will be considered.Moving the handle under the histogram will apply the new Threshold immediately. You canalso use the text field to the histogram's right, after which you should click Apply.

The slider to the histogram's right scales the histogram's height. Use it to zoom into thehistogram data.

Type: Pick the kind of histogram you prefer: Regular, Log Regular, Auto-ScaledRegular, and Integrated. Regular and Log Regular are the most common choices.

From: For when you analyze sequences, the histogram can display the values from theentire sequence or from the currently displayed frame.


Shift: When filters are not perfectly parallel, or when subject matter moves, the images may not lineup. Pick a channel and align the image using the Shift tools.

Click the arrow buttons to move the image one pixel at a time. You can also use the X and Ytext fields to set the amount of the shift. Down and left are negative distances.

Click the Apply button see the effects of shifts made using the text fields and to update thefinal image. Clicking the arrow buttons automatically applies the shifts.

Since the Colocalization command measures relationships between the two channels at each(x,y,z) location, a small misalignment can affect the statistics.

Mask: If this feature has been turned on, only those pixels included in the mask are consideredduring the computations. When initially turned on, the default mask is the same as the unionof the segmentation layers for both of the images.

To mask a rectangular area, select the tool. Holding down the option key before selecting arectangular area, removes the selected area from the mask.

To mask an arbitrary area, select the tool. Holding down the option key before selecting anarbitrary area, removes the selected area from the mask.

To clear the mask, press the Clear button.

This portion of the dialog box appears when you chooseScatter Plot from the Show pop-up menu.

FrequencyCorrection: This option appears when you choose Scatter Plot from the Show pop-up menu.

This option adjusts the brightness of the points on the scatter plot. The brightness is basedupon the frequency of the intensity matches. When this option is unchecked, every point inthe plot is given the same brightness.

Force LineThrough Origin: This option appears when you choose Scatter Plot from the Show pop-up menu.

This option forces the white line which passes through the center of density of the scatter plotto also pass through the origin of the scatter plot. When turned off, the origin of the line isdetermined using a regression equation based on the method of least squares.

Intensity Delta: This option appears when you choose Scatter Plot from the Show pop-up menu.

The intensity delta component sets the tolerance factor. Increasing the intensity delta increasesthe number of pixels that are considered colocalized by accommodating noise elements andpixels (voxels) that share a wider range of proportionality (ratio of intensities). On the scatterplot, the white line in the middle runs through the plot's center of density. The colored linesrun parallel to the white line. The intensity delta is the distance between the white line andeither of the colored lines. Points close to the white line share a small range of ratio values.


Keep the value of Intensity Delta relatively small, so that the points between the colored,diagonal lines have similar intensity ratios ( Ri Gi ). These will be the points close to thecentral white line. If you make the intensity delta too large, the colored lines will encompassoutlying points.

Ratio Min / Max: These are the minimum and maximum intensity ratios (the ratios of the intensities of the twochannels) within the colocalized region.

Show I/O: Click this button to display the I/O palette. You can open the palette to change its settings atany time. Click Apply to update the Colocalization dialog box.

Show Statistics: This button opens the Statistics palette, described below on page 158.

Apply: Click this button to update the final image whenever it is active (not grayed out).

Cancel / OK: Click OK to output the colocalization results to iVision windows. Click Cancel to close thedialog without creating any windows or saving any data.


6.2.3.1 Scatter Plot

Display the scatter plot by selecting it from the Show pop-up menu.

The scatter plot is a three-dimensional histogram. The X and Y coordinates show the pixel intensities from the twochannels. Every point represents colocalized pixels whose intensities are greater than zero. The number of pixels withthose intensities is stored in the value of the scatter plot's points. (You can see this if you check the FrequencyCorrection option. The value of the scatter plot's points will be shown as their brightness.)

A

B C CD

Example of Scatter Plot The colocalized regionas represented in the scatter plot

*(iVision does not output this figure.)

This description of the scatter plot assumes that the first channel is red and the second is green.

A. This is the Threshold for the red channel. This marks the lower limit for which red pixels will be considered by thecolocalization algorithm. In the above example, only red intensities higher than 17 will be considered.

B. This is the Threshold for the green channel. This marks the lower limit for which green pixels will be considered bythe colocalization algorithm. In the above example, only green intensities higher than 24 will be considered.

C. These colored lines mark a set distance from the center of density marked by the central white line. These lines limitwhich pixels will be considered to be colocalized. The distance from the white line to a colored line is set by theIntensity Delta control (page 155). Intensity delta values correspond to the deviation of the colocalized pixels fromthe central white line. Increasing Intensity Delta increases the number of pixels that are considered colocalized, butyou should be careful not to include too much outlying noise.

D. This white line passes through the plot's center of density. All points above the threshold are used for the best linefit. In a perfectly colocalized image, all of the scatter plot's points would fall on this line. All points on this linewould be exactly yellow, indicating their perfect colocalization.

If two probes are colocalizing, then there must be a proportional relationship between the probe intensities.

The two colored lines (C) and the two Threshold lines (A and B) bound the colocalized region of the scatter plot.


6.2.3.2 Statistics

You can see these statistics in two places:

1. The Statistics palette presents the items described in this section. Open it by pressing the Statistics button

2. The FinalImageName.s text window, which is one of the Output button's options, presents an extended list ofstatistics.

The Set Variable command's Use Pixel Value option (Script menu) can pull the values from these coordinates inthe FinalImageName.s window:

Statistic (x,y)

Pearson's CoefficientOverlap CoefficientProbe 1 Overlap Coefficient (k1)Probe 2 Overlap Coefficient (k2)

(0,0)(0,1)(0,2)(0,3)

Colocalization Coefficient M1Colocalization Coefficient M2

(0,4)(0,5)

Probe 1 Colocalization RatioProbe 2 Colocalization Ratio

(0,6)(0,7)

Probe 1 MinimumProbe 1 MaximumProbe 1 MeanProbe 1 SumProbe 1 Standard DeviationProbe 1 Percent Inclusion

(0,8)(0,9)(0,10)(0,11)(0,12)(0,13)

Probe 2 MinimumProbe 2 MaximumProbe 2 MeanProbe 2 SumProbe 2 Standard DeviationProbe 2 Percent Inclusion

(0,14)(0,15)(0,16)(0,17)(0,18)(0,19)

Ratio MinimumRatio MaximumRatio MeanRatio Standard Deviation

(0,20)(0,21)(0,22)(0,23)

Probe 1 ThresholdProbe 2 ThresholdIntensity DeltaMaximum ValueBin Size

(0,24)(0,25)(0,26)(0,27)(0,28)

Probe 1 Shift XProbe 1 Shift YProbe 2 Shift XProbe 2 Shift Y

(0,29)(0,30)(0,31)(0,32)

Note: The following descriptions refer to the Probe 1 image as the red channel and the Probe 2 image as the greenchannel.

Note: "Channel" means the data for a single color within an image. Grayscale images contain one channel; colorimages contain three channels (for example, red, green, and blue).


Pearson'sCoefficient: Pearson's Coefficient (rp) provides information about the similarity of shape without regard to

the average intensity of signals.

(Equation 1)

€

rp =

Ri − Ravg( ) Gi −Gavg( )( )i∑

Ri − Ravg( )i∑

2• Gi −Gavg( )

2

i∑

Ravg and Gavg correspond to the average intensity values of the red and green images,respectively. The summation is computed over all the pixels in the images.

a. rp ranges from -1 to 1.

b. A value of 1 corresponds to a perfect correlation between both images. It can be anindicator of a high amount of overlap (or colocalization or similarity in pattern ofobjects).

c. A value of 0 indicates that the channels are not correlated and, hence, no significantcolocalization and no similar pattern of objects.

d. Negative values are difficult to interpret in terms of colocalization.

Overlap Coefficient: The Overlap Coefficient (r) represents the degree of colocalization.

(Equation 2)

€

r =

Ri •Gi( )i∑

Ri2

i∑ • Gi

2

i∑

a. The numerator can have a higher numerical value only when both channels (Ri and Gi)have significant values at the same pixel position. Hence, the numerator is proportional tothe amount of overlap or colocalization.

b. Denominator is proportional to the number of objects in both channels. It providesnormalization for the overlap coefficient.

c. The advantages of overlap coefficient are that it is not sensitive to photobleaching,differences in amplifier settings, or differences in image intensities caused by differentlabeling with fluorochromes.

d. The disadvantage of overlap coefficient is the ambiguity caused by one channel havingmore objects than the other channel. Perhaps all of the second image's objects will beoverlapped, but not all of the first image's objects will be. The overlap coefficient is onlyuseful when the numbers of objects in each channel are equal.


Probe 1,2Overlap Coefficientsk1, k2: Equation 2, above, is modified and divided into two equations to overcome the overlap

coefficient's disadvantage. This results in the split overlap coefficients (k1 and k2).

(Equation 3) r 2 = k1 • k2

so,

(Equation 4) k1 =Ri • Gi( )

i∑

Ri2

i∑

and

(Equation 5) k2 =Ri • Gi( )

i∑

Gi2

i∑

a. The coefficient k1 is sensitive to changes in the green signal intensity (Gi). Small changesin Gi will affect k1significantly.

b. Similarly, coefficient k2 is sensitive to changes in the intensity of the red signal (Ri).

c. This measure is dependent on the intensity value of both signals.

ColocalizationCoefficient,M1 & M2: The colocalization coefficients, M1 and M2, are independent of signal intensity.

(Equation 6) M1 =Ri,coloc

i∑

Rii∑

where Ri,coloc = Ri if Gi ≥ ∂2 at the corresponding pixel in the green image.∂2 is the Threshold value for the green image.

(Equation 7) M2 =Gi ,coloc

i∑

Gii∑

where Gi,coloc = Gi if Ri ≥ ∂1 at the corresponding pixel in the red image.∂1 is the Threshold value for the red image.

The colocalization coefficients have these properties:

a. These coefficients (M1 and M2) are proportional to the amount of fluorescence from eachchannel's colocalizing objects.

b. M1 and M2 are not sensitive to strong differences between the intensities of the twochannels.

c. Higher values of M1 and M2 denote a significant amount of colocalization.


Threshold 1 & 2: These values are the ∂1 and ∂2 of equations 6 and 7. ∂ is set by the Thresholdhistogram's sliding handle.

Probe 1/2Colocalization Ratio: This is the ratio of the total number of colocalized pixels to the total number of pixels greater

than the Threshold value, ∂. The numbers are counted within a single channel.

(Equation 8) ColocRatio1 =Number of colocalized pixels

Number of pixelsvalue≥ ∂1

within channel 1

where the denominator, Number of pixels, is the number of pixels in channel 1 whosevalues are greater than or equal to ∂1, the Threshold value for channel 1.

(Equation 9) ColocRatio2 =Number of colocalized pixels

Number of pixelsvalue≥∂ 2

within channel 2

where the denominator, Number of pixels, is the number of pixels in channel 2 whosevalues are greater than or equal to ∂2, the Threshold value for channel 2.

Probe 1 and Probe 2: Fluorescence CV measures the following values for each channel. It only measurescolocalized pixel values that are greater than zero.

Minimum,Maximum: The minimum and maximum values of the channel's (probe's) colocalized pixels.

Mean: The mean value of the channel's colocalized pixels.

Sum: The sum of the intensities of all colocalized pixels in the channel.

Standard Deviation: The standard deviation of the channel's colocalized pixels.

Percent Inclusion: The percentage of the channel's pixels that are greater than the Threshold value.

# of pixels over threshold for probe1 / # pixels in probe 1.

% Inclusion =# of pixels over Threshold for Probe n

# of pixels in the Probe n image× 100%

Threshold: This is the Threshold value for the channel, taken from the Colocalization dialogbox.

Ratio: Fluorescence CV measures the following values for the ratio of the intensities of the twochannels.

Ideally, colocalized pixels must have similar ratio between the intensities of the two channels.The differences in the ratios are mainly due to noise and other parameters that affect thelabelling of probes in real, live situations. These differences in intensity ratios are useful indetermining the amount of deviation (intensity delta) from the ideal colocalization slope. (Theideal colocalization slope is denoted by the white line on the scatter plot.)

Intensity Ratio (Ratio) = Ri,colocGi,coloc

Ri,coloc and Gi,coloc correspond to the colocalized pixel intensities in each channel.

The following intensity ratio measures are provided:


Minimum: The minimum ratio value within the colocalized region.

Maximum: The maximum ratio value within the colocalized region.

Mean: The mean of the intensity ratio values within the colocalized region.

Mean =

Ri,colocGi, coloci

∑

N

where N corresponds to the total number of the colocalized pixels.

StandardDeviation: The standard deviation of the intensity ratio within the colocalized region.

6.2.3.3 Interpreting Statistics

1. Pearson's coefficient provides a value for the correlation between the intensity distributions of the channels, but theoverlap of the signals is better represented by the overlap coefficient r.

2. A value of zero for the overlap coefficients means that there are no overlapping objects.

3. The overlap coefficient is useful only when the numbers of objects in the two channels are equal.

4. When the numbers of objects in the two channels are not equal, the colocalization coefficients M1 and M2 must beused to provide a better interpretation.

5. Interpretation of M1 and M2 is pretty simple, For example, when M1 = 0.25 and M2 = 1.00, it means that one quarterof the red colocalizes with green objects and all green objects colocalize with the red objects.

6. Colocalization coefficients are very sensitive to uniform and non-uniform background and cross talk. This isbecause the number of pixels with non-zero values rapidly decreases in the presence of these factors. This makes thecolocalization coefficients converge to 1.0.

Important: You should calculate M1, M2, and colocalization ratios only after the images have been corrected fornoise factors such as background and cross talk. Note that the threshold levels can be used to correct for a fixedbackground (offset) level within the colocalization command, since values below the threshold value will bedisregarded.

6.2.3.4 Possible Noise Components

• Camera dark current• Photon noise

• Non-specific binding• Cross reactivity of the fluorescent probes• Optical cross talk (bleeding)


6.3 Line MeasureUse the Line Measure command to measure intensity profiles based on fat lines. You can measure different instances ofthe same object and compare the locations of features. The Line Measure command performs two functions:

1. The dialog box displays the line's length and the min, max, and median intensities along the line.

2. The profile plot reports the pixel intensities versus the line length. The lengths of multiple lines (from multipleimages of similar objects, for example) will be averaged to make them equivalent. The lines are fat lines, so you canmeasure the sum or average of intensities across it.

Line Measure Dialog Box


6.3.1 Draw LinesYou can draw the lines at any time. You can change the Line Width after you draw the lines.

When you are displaying profile plots with % length, the Append Results option will let you make multiplemeasurements. Otherwise, you can only draw and measure one line at a time.

Magnifying Glass: Press this button to change the cursor to the magnifying glass. Click on the image to zoom in.Hold down the Option key and click on the image to zoom out.

Line-Drawing Tool: Press this button to change the cursor to the line-drawing tool. This tool can make a multi-segmented line. Click on the image at each point of the line. Double-click at the end of theline or press any key. (This is the same as the Line ROI tool, except that within this commandyou can create fat lines. Also, you should avoid clicking too quickly, which wouldprematurely complete the line.)

Use Line: When you first open Line Measure, the image's ROI will be displayed as a line. Click thisbutton to use it as a line.

A list box of lines appears to the right of the image. Display a particular line by selecting it within the list. To delete aline, select its row and click Remove Line.

If you draw multiple lines and deselect Append Results or Display as % Length, select the line that you wantmeasured. If no line is selected, then Fluorescence CV will measure the first line.

6.3.2 Pick the Window and FrameNext, pick the image you want to measure.

Window Menu: Initially, Line Measure starts out by displaying the active window. You can use the windowpop-up menu to choose any open image.

Window Pop-Up Menus

Click on the arrow button to see a list of open images. The dialog box will display the chosenimage.

Frame: If the image is a sequence, you can use the Frame controls to display a specific frame.



6.3.3 Set Up the MeasurementLine Width: Enter the line's thickness in pixels. All pixels underneath the line will be measured. Increasing

the line's thickness increases the area that will be measured.

Units: This field reports the image's units, as set by the Set XY Units command (Analyze menu).The lengths reported in the dialog's upper-right corner and in the profile plot will be given inthese units.

Using... of Pixels: Lines are measured using the Sum or the Average of the pixel intensities across the line'swidth.

6.3.4 Set Up the OutputFill the Create Profile... checkbox if you want to output the measurement results as a profile plot. The profile plot is agraph of the pixel intensities versus distance.

If you do not check this box, then look at your results in the upper-right corner of the dialog box. Clicking OK will notcreate any data windows when this is not checked.

Create ProfilePlot From...: You can output a profile plot based on the currently-displayed frame, or based upon the whole

sequence.

Display as % Length: Check this box to plot the intensities versus the percentage of the line's length. This optionnormalizes the lengths of multiple lines, so that they can be compared. (The profile plot willdisplay the length as a percentage, but you can still view the average length of all lines bychoosing View As: Text from the View menu.)

Uncheck this box to plot the intensities versus the line's actual length. When this isunchecked, you can only measure one line at a time.

Resolution: When displaying the length as a percentage, you can generate more measurements bydecreasing the resolution from 1% to 0.5% or 0.25%.

Append Results: Check this box to measure multiple lines at the same time.

If you add multiple lines and deselect either Append Results or Display as %Length, then the iVision will measure the statistics from the currently selected line.

6.3.5 See ResultsThe line measurement results appear in the dialog box's upper-right corner.

The Length will always be measured in the image's units. Use the Set XY Units command (Analyze menu) to applymeaningful units to the image.

Beneath Length, the Minimum, Median, and Maximum intensities are listed for each channel.

You can find this same information by analyzing the profile plot. When you view the profile plot in text view, you cansee the (summed or averaged) pixel intensities for each distance along the line.

Processing Extensions -MicroTome 167

7 MicroTome™

7.1 About MicroTomeMicroTome is deconvolution software that mathematically restores the quality of your microscopy images.MicroTome's commands use image restoration algorithms to recreate the original image of the sample. The resultingimage is a representation of your microscopy subject without the extra blur added by the optics.

MicroTome can deconvolve images from a standard fluorescent microscope or a confocal microscope and requires nospecial attachments. Images from bright-field microscopes can also be deconvolved, but the results are less predictable.

Rapid Deconvolution mathematically calculates and removes out-of-focus haze from microscope images. It uses a NoNeighbor (single image) technique and a Nearest Neighbor (three image) technique.

Constrained Iterative Frame is a no-neighbor deconvolution command that employs a theoretical PSF. Because it doesnot use information from neighboring frames, it is ideal for the restoration of time-lapse image sequences and quicklychanging samples.

Constrained Iterative Volume is a 3D deconvolution command that can employ a theoretical or an experimental PSFfor image restoration. Constrained Iterative Volume restores each frame in the image sequence using information fromthe volume's other frames. This command is the most appropriate for deconvolving three-dimensional sequences.

The MicroTome algorithms are based upon the UCLA Agard - Sedat algorithm (Agard et al, 1989). For a generalreference about deconvolution, please read chapter 23 of the Handbook of Biological Confocal Microscopy (Shaw,1995).

MicroTome removes the blurring haze caused by the optical characteristics of the lenses. However, like CLSM, itcannot remove scattered or reflected light produced by opaque or reflective samples.

168 Processing Extensions - MicroTome

7.2 Overview of Digital DeconvolutionIt may be helpful to review a few basic principles of optics in order to understand how MicroTome works.

A microscopy image includes in-focus and out-of-focus light. The out-of-focus light comes from above and below theplane of focus. The smear or blur produced by the out-of-focus planes is a natural consequence of the optics of themicroscope. The out-of-focus haze is added to the image in a very precise manner by the point spread function (PSF) ofthe microscope:

Object ImageBefore Convolution

by Microscope

*

PSF

=

Image Acquired byCamera

MicroTome starts out with the blurred, hazy acquired image, and works backwards to recreate the original, in-focusobject image. MicroTome does this by blurring an estimate of the original image with the PSF and comparing it to theacquired image. Then the software revises its estimate to match more closely the object image. MicroTome repeats thisprocess a number of times (iterations) until its estimate is as close as possible to the object image. This final estimate isyour deconvolved, in-focus result. MicroTome knows it is finished when it can mimic the microscope's effects byblurring the final estimate with the PSF to get the image you captured with the camera. (Wallace et al, 2001)

7.2.1 PSFs (Point Spread Functions)A point-spread function (PSF) is an image sequence that describes how a single point of light spreads out in threedimensions, in a particular optical system. This image sequence describes how the optical system (the microscope andassociated hardware) affects a point source. MicroTome then uses this information to restore the out-of-focus light backto its correct points of origin. This restoration results in an image sequence that is clear of haze and blur in every plane.

The Constrained Iterative Frame command only uses theoretical PSFs. The Constrained Iterative Volume commandcan use either a theoretical or an experimental PSF. Using a good experimental PSF produces the best results.

The experimental PSF sequence must be captured using most of the same parameters as the original, including the samemagnification and wavelength.


7.2.1.1 Theoretical PSFs

The theoretical PSF is calculated using diffraction theory. The parameters needed to calculate a PSF are:

• wavelength of light (typically the emission peak for the respective fluorescence probe)• numerical aperture of the lens• refractive index of the immersion oil• pixel dimension within a plane (dx and dy)• the distance between the acquired image planes (dz)

You must enter these parameters in the Mic. file so MicroTome can calculate the theoretical PSF from your descriptionof the optical system.

7.2.1.2 Experimental PSFs

Measured, experimental PSFs provide the best description of your microscope's real-world effect on the light passingthrough it. You must capture a good PSF, however.

The PSF for any complex optical system such as a microscope is unique in every case. The PSF's uniqueness resultsfrom its dependence on several variables, including:

• any variations in alignment of the optical components, such as in the light path;• the distortions induced by each of these components (mirrors, lenses, filters, light sources etc.);• the wavelength of light used;

and so on. Because of all of these variations, the experimental PSF captures data about the PSF that the theoretical PSFwill not.

7.2.2 Number of IterationsThe number of iterations is the number of times that the constrained iterative algorithm will process the image. Teniterations are usually appropriate for most data sets. Each iteration improves the data set by removing more haze.However, increasing the number of iterations reaches a point of diminishing returns on an exponential basis.

Using too many iterations may increase the processing time without significantly improving the image. Using too manyiterations can also cause the image to degrade. Ideally, you want to use just enough iterations to remove the appropriateamount of haze, or, in more technical terms, to stop when the error terms in the deconvolution begin to converge.

7.2.3 Background Attenuation, or Scaling the DataFor the best possible deconvolved images, your data should span the full range of values available from your camera. Forexample, the data should range from 0 to 4095 for 12-bit images and from 0 to 255 for 8-bit images. If your images donot cover the full dynamic range, you can use the Background Attenuation option to scale the data. This step is notessential, but it can improve the signal-to-noise ratio in your data set.

Background Attenuation scales the original data before deconvolution. It alters the data in memory and leaves theoriginal image window untouched.

Background Attenuation


7.3 Images MicroTome Can Deconvolve

7.3.1 Frames and SequencesMicroTome/Windows and some prior versions of MicroTome/Mac work with multiple windows, each containing asingle frame of the image. In this version of MicroTome, multiple frames of the same image must be stored within the Zdimension of a single image sequence. The same is true of experimental PSFs.

If each frame of your data is stored in individual files, you can convert them to a sequence by using the Frames toSequence command (Window menu).

7.3.2 SizeConstrained Iterative Frame can deconvolve images of any size.

Constrained Iterative Volume can deconvolve images with dimensions up to 1024x1024

Experimental PSFs used with Constrained Iterative Frame must be 128 x 128 pixels. They must also have an oddnumber of frames, but not less than three.

7.4 Using MicroTome

7.4.1 Microscope (Mic) DataAccurate information about the Microscope used to collect the data is required in order for MicroTome to remove thehaze. This data can be provided to MicoTome in one of three ways.

1. Micoscope (Mic) File

2. Image Acquire Data and Image Units

3. Manually

Each method is described below.

7.4.1.1 Microscope (Mic) Files

Mic. Files can be created using the Define Mic. File command in the 3D menu.


Define Mic. File Dialog Box

Microscope Parameters:

Lens NA: Enter the numerical aperture of the objective. You can find this printed on theobjective's side.

Oil Index: Enter the index of refraction for the medium between your objective and yoursample. This number is printed on bottles of microscope oil. Oil's index is usuallyaround 1.5. For air, you can use 1.0. For water, you can use 1.3.

Wavelength: Enter the light's wavelength in microns. This is the emission wavelength, thewavelength of the light being deconvolved. Use 0.570 for white light.

Microns / Pixel:

DX, DY: Enter the distance in microns between pixels.

You will need to measure DX and DY for each objective and each camera you use.(The image's bin size will affect this, too.) To measure DX and DY, capture animage of a stage micrometer or an object of known size. Divide the known distanceby the number of pixels used to display it. Most cameras have square pixels.

DZ: Enter the distance in microns between image planes. This is the same as the step sizeused by the focus motor when you acquired the data.

If you are deconvolving a single image, set DZ to 1.0. Changing DZ can improve theimage quality when using the Constrained Iterative Frame command.

Color Image Type,RGB Wavelengths: Rapid Deconvolution uses this information for removing haze from color images. The

Constrained Iterative Frame and Volume commands do not deconvolve color images, sothese fields are not used for those commands.

Signal to Noise Ratio:Increase the range of intensities within your deconvolved data by raising the SNR. You canset the ratio between 1 and 30, inclusive.

Open, Save, Save As:Mic. files get stored in your home directory, in the iVision Preferences folder:


Users: YourUserName: iVision 4.0 User Folder: iVision Preferences: Mic Files

The MicroTome commands will look in this folder for all Mic. files, so make sure to saveyour Mic. files here.

7.4.1.2 Providing Microscope (Mic) Data with Image Acquire Data and Image Units

The Edit Acquire Info and Set XY Units commands from the file menu and Analyze menu can be used to provide therequired information as well. The relevant acquire fields are:

Interval_ZWavelengthObjective_NAObjective_RIDeconv_SNRDeconv_ColorTypeDeconv_Red_WavelengthDeconv_Green_WavelengthDeconv_Blue_Wavelength

Setting the Deconv_ColorType field to a value of 0 corresponds to a Fluorescence image and a value of 1 corresponds toa Brightfield image.

7.4.1.3 Manually providing the Microscope (Mic) Data

The MIC Data menu has a Custom option. When this option is selected, the Mic Data is being provided manually.

Mic. Data menu and editing

By pressing the Edit button, a drawer opens up which allows you edit the current Mic values.


Mic. Drawer

If any values are changed the MIC Data menu is changed to Custom and the Save As… button lights up so the valuescan be saved into a Mic File.

7.4.2 Rapid DeconvolutionRapid Deconvolution mathematically calculates and removes out-of-focus haze from microscope images. It has twoalgorithms for removing out-of-focus haze from microscope images. They are 1) the No Neighbor (single image)technique and 2) the Nearest Neighbor (three image rapid deconvolution) technique.

You must open the image to be deconvolved before you choose this command. The image can be a single frame or asequence. Rapid Deconvolution can work with Byte, Short, and Unsigned grayscale images. It can also work with Color24 and Color 48 RGB images.

Next, pick the Rapid Deconvolution command from the 3D menu.


Rapid Deconvolution Dialog Box

Preview Frame: Previewing allows you to see the effects of your settings before you make them permanent.Use these tools to select which frame of the sequence to preview. Click on the up/downarrows or type in a new number and then click on the Go button.

Using Single/Three: Select Single Plane to use the No Neighbor algorithm; select Three Planes to use the NearestNeighbor algorithm.

% Removal: The % Removal slider sets the percentage of haze removed from the image. This setting issimilar to opening and closing the pinhole on a confocal microscope.

Gain: The Gain setting controls the image brightness during deconvolution. Usually you will adjustthe Gain setting to make the deconvolved image about as bright as the original image.

Apply Shadow: Checking the Shadow box adds a shadow function during deconvolution. The effects aresimilar to a DIC image.

Mic. Data: Pick the appropriate description of your acquisition and your system.

Noise Reduction: Noise Reduction can be applied to the image before the processing begins. If no noisereduction is required, none can be selected from the menu.

BackgroundAttenuation: Check this box to scale the data before deconvolving it.

Do All / Do Frames: Use the Do All or Do Frames options to pick which frames of an image sequence todeconvolve.

Dispose


Original Window: Check this box to close the original image after deconvolution is complete.Note: This will not save any changes you made to the original image!

Remove Haze,Before, After: A button in the lower left corner of the dialog be titled one of these three things. If the

Remove Haze text is shown, pressing the button will perform the deconvolution and thebutton will then be called Before. Pressing Before will show the image as it looked before thedeconvolution was performed. Pressing After will show the image as it looked after thedeconvolution was performed. Changing any value in the dialog will cause the button todisplay Remove Haze.

The # / Var buttons let you use values stored in variables instead of normal numbers. Set the button to # to enter a regularnumber. Set the button to Var to enter a variable's index number; the program will then use the variable's value for thatparameter.

7.4.3 Constrained Iterative FrameUse the Constrained Iterative Frame command to deconvolve images a single frame at a time. This is appropriate fortime-lapse image sequences and short-lived subjects. Constrained Iterative Frame is an image-restoring, no-neighbordeconvolution command that employs a theoretical PSF. Theoretical PSFs are described on page 168.

Constrained Iterative Frame does not use any information from neighboring frames. A rough description of theConstrained Iterative Frame method is that it creates a volume from the single frame of data, and then deconvolvesthat volume with the PSF.

MicroTome will update the image after each frame is completely deconvolved.

You can find the Constrained Iterative Frame command in the 3D menu.

Constrained Iterative Frame Dialog Box


You must open the image to be deconvolved before you choose this command. The image can be a single frame or asequence. Then pick the Constrained Iterative Frame command.

Use: Choose the image by selecting a window from the window menu or entering the name of thewindow in the field.

View Frame: Select the frame you would like to operate on while previewing the results.

Number of Iterations: Next, set the number of times that the constrained iterative algorithm will process the image.Ten iterations are usually good enough. Please see page 169 for more information.

Data Size: Set the Data Size to tell the software the range of intensities that the camera is capable ofacquiring. Common digital cameras produce 12-bit data, with data values ranging from 0 to4095. The Background Attenuation option uses the Data Size to scale the data.




Do All / Do Frames: Use the Do All or Do Frames options to pick which frames of an image sequence todeconvolve.

DisposeOriginal Window: Check this box to close the original image after deconvolution is complete.

Note: This will not save any changes you made to the original image!




7.4.4 Constrained Iterative VolumeUse the Constrained Iterative Volume command to deconvolve an entire 3D sequence (a "volume"). This methodrestores each frame in the image sequence using information from the other frames. It is more accurate for deconvolving3D images than Constrained Iterative Frame because it uses intensity information from the rest of the sequence.Constrained Iterative Volume can deconvolve the image using a theoretical PSF or a true, experimental PSF. You canread more about PSFs on page 168.

During the deconvolution, MicroTome will display the volume's middle frame. MicroTome will update this image aftereach complete iteration (that is, after all frames have completed the iteration).

You can find the Constrained Iterative Volume command in the 3D menu.


Constrained Iterative Volume Dialog Box

You must open the image sequence to be deconvolved first, and then pick the Constrained Iterative Volume command.

Use: Choose the image by selecting a window from the window menu or entering the name of thewindow in the field.

View Frame: Select the frame you would like to operate on while previewing the results.

Number of Iterations: Next, set the number of times that the constrained iterative algorithm will process the image.Ten iterations are usually good enough. Please see page 169 for more information.

Point SpreadFunction: Choose the Point Spread Function (PSF) to use for deconvolving your sequence. Read more

about PSFs on page 168.

Theoretical: MicroTome can calculate the PSF from the information in the Mic. file. The Number ofPlanes within the PSF is equivalent to the number of Z-frames. The larger the number ofplanes, the better the deconvolution, but the longer it will take. You can use from 3 to 15planes.

Experimental: An experimental PSF is a real PSF acquired under the same (or very nearly the same)experimental conditions as the sample. Your experimental PSF should be 128 x 128 pixelssquare and an odd number of frames deep.





DisposeOriginal Window: Check this box to close the original image after deconvolution is complete.

Note: This will not save any changes you made to the original image!




7.5 Troubleshooting: Image Acquisition IssuesCareful acquisition of 3D data sets produces the best deconvolved images. Poorly deconvolved images usually resultfrom poor data collection. Most problems can be solved with some adjustments in image acquisition techniques.

The typical acquisition system will include:

• a Z focus motor (or piezo-electric device) to change the focus plane in the sample• an electronic shutter in the excitation light path to minimize the exposure of the sample• a scientific-grade digital camera• iVision to control the focus motor, shutter, and camera from the computer. iVision can also control other

motorized parts of the microscope.

The microscope optics must be clean and properly aligned to minimize image problems arising from uneven illuminationor dirt.

There are several issues related to image acquisition. They include:

Choice of Camera: The use of a cooled digital camera is recommended for long exposures and a large dynamicrange.

Background noise: Avoid extraneous sources of light from reaching the sample and use a cooled digital camerafor long exposures.

Dynamic range: Increase the dynamic range of the image by increasing the exposure level for the camera.When possible, a range of gray levels greater than 3000 is recommended.

The image capture of 3D data requires a careful choice of the exposure time: A large dynamicrange will provide the best raw data to start the deconvolution process. However, a largeexposure may induce bleaching of the sample while the data is being captured.

Filters used: Use appropriate filters to avoid a high background and “cross-talk” between different probes.

Specimen thickness: Please see section 7.5.2, below. Very thin and very thick specimens can be difficult todeconvolve.


Source of the data: Image modality is described below, in section 7.5.3. Data can come from many sources,including fluorescence, transmitted, and confocal microscopy.

Slice spacing: Please see section 7.5.4, below, for guidelines on how to set the step size between images.

7.5.1 Image Capture TipsThe techniques used to capture a good image for deconvolution will vary depending on the light source involved. Fortransmitted mode, most specimens will be opaque or semi-transparent. The brightness and contrast controls of thecamera should be turned down and the transmitted light increased to a maximum. A filter should be used to eliminate asmuch background interference as possible. A 546 green filter is particularly good for the sensitivity of CCD cameras.

Fluorescent images should be captured with the correct filters and a camera that will provide the best dynamic range.You must use a camera with enough sensitivity to produce a relatively bright signal. In addition, background subtractionduring acquisition will help to eliminate some camera noise. Cooled CCD cameras will probably be necessary forfluorescent images.

7.5.2 Thickness of the SpecimenThe thickness of a specimen affects the quality of the unprocessed image. Thick specimens, particularly if they are fairlyopaque, can be difficult to deconvolve. In addition, the quality of the deconvolved image will degrade progressively asthe focus plane penetrates the specimen. (This is true of both CLSM and MicroTome.) Maximum penetration will rangefrom about 20 microns to 400 microns depending on the transparency of the tissue.

Very thin specimens (1 micron or less) also will not deconvolve well unless special care is taken during imageacquisition. Transmitted-light illumination makes very thin samples even more difficult. Specimens this thin will usuallyhave little haze to remove. If the user is working with very thin specimens, it is important to insure that the focal planesof the acquired images fall within the depth of the specimen.

7.5.3 Sources of DataMicroTome can remove out-of-focus haze from any microscope image, including DIC, phase contrast, confocal laserscanned images and polarized light images. However, the degree of improvement in these images will vary depending onthe amount of haze in the unprocessed image. This is particularly true for phase contrast, laser scanned confocal images,and polarized light images. There tends to be less haze in these unprocessed images than in fluorescent or transmittedlight images.

7.5.4 Slice SpacingThe distance between the unprocessed images (DZ, or step size) will affect the quality of the deconvolved images. Youshould determine the distance between the optical slices according to the numerical aperture you use. The following tablegives the suggested sectioning distance for varying numerical apertures:

Suggested Distance Between SlicesN.A. Microns0.50 8.00.75 2.41.25 0.51.40 0.4


7.6 AcknowledgementsPortions of this manual started out as part of VayTek's manual for MicroTome for Power Macintosh, version 3.3.

7.7 BibliographyAgard, D. A., Y. Hiraoka, P. Shaw, and J. W. Sedat. 1989. Fluorescence microscopy in three dimensions. Methods in

Cell Biology. 30:353-377.

Agard, D. 1984. Optical sectioning microscopy: cellular architecture in three dimensions. Ann. Rev. Biophys. Bioeng.13:191-219.

Brakenhoff, G. J., H. T. M. van der Voort, E. A. van Spronsen, and A. Nanninga. 1989. Three-dimensional imagingin fluorescence by confocal scanning microscopy. Journal of Microscopy. 153:151-159.

Carrington, W., K. Fogarty, L. Lifschitz, and F. Fay. 1989. Three-dimensional imaging on confocal and wide-fieldmicroscopes, p. 137-146. In J. Pawley (ed.), The Handbook of Biological Confocal Microscopy. IMR Press,Madison, WI.

Carter, K., et. al. February 26, 1993. A Three-dimensional view of precursor messenger RNA metabolism within themammalian nucleus. Science. 259:1330-1335.

Clinch, N.F., C. J. Daly, J. F. Gordon, V. A. Moss, and N. C. Spurway. Wide field volume visualisation of thickmicroscope sections by computed nearest neighbor deconvolutions. Journal of Physiol. Proceedings.

Cogswell, C. J., D. K. Hamilton, and C. J. R. Sheppard. Colour confocal reflection microscopy using red, green andblue lasers. Journal of Microscopy. 165:103-111.

Gibson, S. F. and F. Lanni. 1991. Experimental test of an analytical model of aberration in an oil-immersion objectivelens used in three-dimensional light microscopy..8 (10): 1601-1613.

Gorby, G. L. 1993. Digital confocal microscopy allows measurement and three dimensional multiple spectralreconstruction of neisseria gonorrhoeae/ epithelial cell interactions in the human fallopian tube organ culture model.Journal of Histochemical and Cytochemistry.

Harris, P.J., et al. 1994. pH, morphology, and diffusion in lateral intercellular spaces of epithelial cell monolayers. Am.J. Physiol. 266 (Cell Physiol. 35): C73-C80.

Hiraoka, Y., J. R Swedlow, M. R. Paddy, D. Agard, and J Sedat. 1991. Three-dimensional multiple wavelengthfluorescence microscopy for the structural analysis of biological phenomena. Journal of Cell Biology. 2:153-165

Inoue, S. 1989. Video Microscopy. Plenum Press, New York.

Kraig, R.P., C. Lascola, and A. Caggiano. 1994. Glial response to brain ischemi. In H. Kettenmann & B. Ransom(ed.), Neuroglial Cells, Oxford Press.

Kesterson, J. and M. Richardson. 1991. Confocal microscope capability with desktop affordability. AdvancedImaging. p. 23-26.

Minski, M. 1961 Microscopy apparatus, U.S. Patent 3,013,467.

Monck, J. R., A. F. Oberhauser, T. J. Keating, and J. M. Fernandez. 1992. Thin-section ratiometric Ca2+ imagesobtained by optical sectioning of fura-2 loaded mast cells. Journal of Cell Biology. 116:745-769.

Moss, V. A. 1992. Digital deconvolution of colour images. Proceedings 4th Amsterdam Confocal MicroscopyConference.


Rayport, S., D. Sulzer. 1995. Visualization of antipsychotic drug binding to living mesolimbic neurons reveals D2receptor, acidotropic, and lipophilic components. Journal of Neurochemistry. 65:691-703.

Richardson, M. November, 1990. Confocal microscopy and 3-D visualization. American Laboratory. 19-24.

Shaw, P. 1995. Comparison of wide-field/deconvolution and confocal microscopy for 3D imaging, p. 373-387. In J. B.Pawley (ed.), Handbook of Biological Confocal Microscopy, 2nd ed. Plenum Press, New York.

Shaw, P.J., D. J. Rawlins. 1991. The point spread function of a confocal microscope: its measurement and use indeconvolution of 3D data. Journal of Microscopy. 163:151-165.

Wallace, W., L. H. Schaefer, and J. R. Swedlow. 2001. A workingperson's guide to deconvolution in light microscopy.BioTechniques. 31:1076-1097.

Wilson, T. 1989. Three-dimensional imaging in confocal systems. Journal of Microscopy. 153:161-169.

Wilson, T. 1989. The role of the pinhole in confocal imaging systems, p. 99-114. In J. Pawley (ed.), The Handbook ofBiological Confocal Microscopy, IMR Press, Madison, WI.

Yelamarty, R., B. Miller, R. Scaduto, et. al. 1990. Three-dimensional intracellular calcium gradients in single humanburst-forming units-erythroid-derived erythroblasts induced by erythropoietin. Journal of Clin. Invest. 85:1799-1809.

Index 183

8 Index

3D3D acquire ................See Multi Dimensional Acquire

3-Shot color...............................................................52Acquire ... See also individual Camera-menu commands

Camera details .......................................................49Camera Settings....................................47, 50, 54, 61Destination ................................................ 18, 20, 25Exposure time.................................15, 23, 50, 53, 63Filters ....................................................................23Full Acquire ..........................................................11Grab to disk..................................................... 19, 26Image size ............................................17, 24, 55, 61Initial set-up ............................................................1Installing camera hardware ....................................49Preview ...................................................................6Preview palette ................................................ 12, 56Previewing .......................................2, 10, 12, 23, 56Processing (Average, Sum)....................................15RGB Color Acquire ...............................................21Select Camera..........................................................5Set Color Balance ..................................................45Set Gain and Offset ...............................................46Shutter............................................................. 20, 27Single Exposure.....................................................10Spot Realtime Focus..............................................55Spot Set Filter Position ..........................................56Spot White Balance ...............................................57Switching cameras...................................................1Time lapse ....................................................... 19, 27Timing window .....................................................21Triggering .............................................................15

Acquire menu....................................See Camera menuAcquiring images

3D images ................See Multi Dimensional AcquireApplied Scientific Instruments, Inc.....See ASI, See ASIASI.................................................................. 105, 106Auto-Exposure ..........................................................16Background attenuation...........................................169Balance Colors ........................... See Set Color BalanceBD Biosciences .......................................................108Binning .....................................................................18Calibrating stages ......................................................70Cambridge Research, Inc..................................See CRICamera menu ..............................................................5Camera Settings ........................... 47, 50, 54, 61, 63, 65CARV ............................................See BD BiosciencesChannel ........................................................... 141, 158Colocalization .........................................................150Colocalization coefficient ........................................160Color

Choose colors for Color Join ............................... 144Color Balance14, 45, See also Spot White Balance, See

also Set Color BalanceColor Join ............................................................... 141Color Join Controls palette...................................... 141Communication switches (Ludl).............................. 116Connecting hardware .......................................... 69, 70Constrained Iterative Frame .................................... 175Constrained Iterative Volume.................................. 176Contrast enhancement ............................................. 148Control menu ............................................................ 67Cooke-PCO cameras ................................................. 50CRI ......................................................................... 110Deconvolution......................................................... 168Define colors........................................................... 144Define Mic. File...................................................... 170Define User Devices ................................................. 98Desired Maximum Pixel Value ....... See Auto-ExposureDestination for new images ............................18, 20, 25Device ControlSee Microscope Control, Microscope

Variable, or Stage ControlDevice modules ........................................................ 67Device Select ............................................................ 71Device Setup............................................................. 68

Initial set-up ............................................................ 1Device Specific ......................................................... 73Diagnostic Instruments cameras ................................ 52

Camera-specific data files ..................................... 53Dimensions (Spot Realtime Focus) ........................... 55e-gain................................................. See Electron gainElectron gain............................................................. 46Enhanced sensitivity (PVCam).................................. 62Exposure time .................................... 15, 23, 50, 53, 63Fat lines .................................................................. 164FCV............................................. See Fluorescence CVFilters for acquisition ................................................ 23Fluorescence CV..................................................... 141

Colocalization ..................................................... 150Color Join ........................................................... 141Color Join Controls palette .................................. 141I/O palette ........................................................... 151Line Measure ...................................................... 163

Focus ......................................................... See PreviewFull Acquire.............................................................. 11Gain.................................................................... 13, 46Gamma ................................................................... 148Generic S&F........................................................... 111Grab to disk ........................................................ 19, 26Hamamatsu cameras ........................................... 58, 63Hardware control ...................................................... 67

184

Hardware, setting up..................................................68HazeBuster

Rapid Deconvolution........................................... 173Histogram ............................................................... 148

Colocalization scatter plot ................................... 157I/O palette ....................................................... 151, 156Image size ................................................17, 24, 55, 61Installation ................................................................49Intensity Delta ................................................. 155, 157Intensity ratio .................................................. 156, 161Leica ............................................................... 112, 113Line Measure .......................................................... 163Link to binning..........................................................13Linked Devices .........................................................98Live image .......................... See Preview or PreviewingLudl ........................................................................ 117Masks...................................................................... 146Maximum Number of Retries ..........See Auto-ExposureMicroscope control......... 73, See also Hardware controlMicroscope Lamp......................................................76Microscope Speed .....................................................76Microscope Variable .................................................75MicroTome ............................................................. 167

Constrained Iterative Frame................................. 175Constrained Iterative Volume .............................. 176Define Mic. File .................................................. 170Image Acquisition Issues ..................................... 178

Mid Point ................................................................ 148Motion Control..........................................................67

Hardware details.................................................. 103Multi Dimensional Acquire .......................................29

Acquire Preview....................................................39General tab ............................................................30Shutter & Filters tab ..............................................33Size tab..................................................................32Time Lapse tab ......................................................37Z-Steps tab ............................................................36

Multi-dimensional imagesAcquiring ..............................................................29

Multi-Filter Acquire ............................................ 22, 23Multi-mode imaging...................................... 33, 34, 35Multispectral 3D...........See Multi Dimensional AcquireNikon .......................................................119, 121, 122Normalization ................................................... 14, 147

in Multi Dimensional preview ...............................42Offset .................................................................. 13, 46Olympus.................................................................. 123Orca cameras...........................See Hamamatsu camerasOverlap coefficient .................................................. 159

Split..................................................................... 160Pearson's coefficient ................................................ 159Physik Instrumente.................................................. 126

PIFOC ...................................... See Physik InstrumentePixel shift.........................................................149, 155Point spread function ....................................... See PSFPoint-spread function (see PSF) .............................. 168Preview....................................................................... 6Preview palette ....................................................12, 56Previewing.....................................................12, 23, 56

Set Color Balance ................................................. 45while measuring colocalization ........................... 153while merging images ......................................... 145

Prior.................................................................127, 128Probe image (Colocalization) ..................... See ChannelProcessing (Average, Sum) ....................................... 15PSF......................................................................... 168PVCam cameras..........See Roper Photometrics camerasQImaging cameras ...............................................58, 63Quantitative Imaging cameras ....See QImaging camerasRapid Deconvolution .............................................. 173Ratios

Intensity ........................................ See Intensity ratioRegion of interest............................................. See ROIRegister ...........................................................149, 155Resize to ROI............................................................ 14RGB Color Acquire .................................................. 21ROI......................................................................14, 18Roper Photometrics cameras ..................................... 61RS-232 switches (Ludl)........................................... 116S&F Control ............................See Microscope ControlS&F Device Select.............................See Device SelectS&F Variable Control .............See Microscope VariableScatter plot............................................... 152, 154, 157Scripting .............................................. 6, 11, 21, 28, 78

3D-acquisition....................................................... 82Example script ...................................................... 82

Select Camera ............................................................. 5Initial set-up............................................................ 1Switching cameras .................................................. 1

Select Devices..........See Device Setup or Device SelectSensiCam cameras .................................................... 50Sequences ................................................................. 29Serial Control.....................................................98, 111

Define Devices...................................................... 98Serial Ports ............................................................... 70Set Color Balance ................45, See also Color BalanceSet Gain and Offset ................................................... 46Shutter .................................................................20, 27Single Exposure ........................................................ 10Single-shot color ....................................................... 52Snapshot ................................................................... 14Speed........................................................................ 32Spot Realtime Focus ................................................. 55Spot Set Filter Position.............................................. 56

Index 185

Spot White Balance ...................................................57Stage Control.............................................................77Stage Get Position .....................................................78Stage Move to Recorded Position ..............................80Stage Record Position................................................79Stage Set Zero Position..............................................78Stage Speed...............................................................83Stage Z-Sections Setup..............................................81StageScan Array Calibrate.........................................84StageScan Array Setup ..............................................88StageScan Mosaic Calibrate ......................................93StageScan Move/Iterate

Iterate .............................................................. 88, 93Statistics..................................................................152Statistics palette........................................156, 158, 162Step size ....................................................................70Sutter............................................................... 129, 131Switching cameras.......................................................1

Threshold.................................................154, 157, 161Time lapse acquisition......................................... 19, 27Timing window......................................................... 21Trigger

Keyboard .............................................................. 12Triggering

External................................................................. 15Uniblitz................................................................... 133User Devices............................................................. 98Vincent Associates.................................................. 133Virtual Camera.......................................................... 65Virtual shutter & filter............................................. 134Virtual Stage ........................................................... 135Yokogawa............................................................... 136Z motors

Acquiring Z series .... See Multi Dimensional AcquireZeiss ................................................................137, 139

ivision - biovis.com

Documents