quick start tutorials - emory university department of...

Quick Start Tutorials

Imaris 6.3

Bitplane AGBadenerstrasse 682

CH-8048 Zurich

[email protected]

Table of Contents

1 Introduction 1

......................................................................... 311.1 Reference Manual

2 Visualize Data Set 4

......................................................................... 512.1 Imaris Main Screen

......................................................................... 722.2 Open Data Set

......................................................................... 932.3 Slice View

......................................................................... 1142.4 Surpass View

......................................................................... 1352.5 Volume

......................................................................... 1462.6 Select and Navigate

......................................................................... 1572.7 Rotate Image

......................................................................... 1682.8 Translate Image

......................................................................... 1792.9 Scale Image

......................................................................... 18102.10 Save Scene File

......................................................................... 19112.11 Practice Makes Perfect

......................................................................... 22122.12 Change Background Color

......................................................................... 24132.13 Change Channel Color

3 Generating Movies 25

......................................................................... 2613.1 Load Scene File

......................................................................... 2723.2 Key Frame Animation

......................................................................... 2933.3 Shoot and Play

......................................................................... 3243.4 Save Movie

4 Imaris and QuickTimeVR 34

......................................................................... 3514.1 Basic Principles

......................................................................... 3624.2 Generate File

......................................................................... 3834.3 Interactive Display

......................................................................... 3944.4 Optimized Settings

5 Design Mixed Model Rendering 41

......................................................................... 4215.1 Add Volume

......................................................................... 4425.2 Display Adjusment

......................................................................... 4735.3 Add Surfaces

......................................................................... 5045.4 Change Surface Color

......................................................................... 5255.5 Add Spots

......................................................................... 5565.6 Change Spots Color

......................................................................... 5675.7 Final Image

6 Measure Structures 57

......................................................................... 5816.1 Line or Polygon

......................................................................... 6026.2 Grid and Scale Bar

......................................................................... 6236.3 3D Measurement

7 Track Particles 64

......................................................................... 6517.1 Visualization

......................................................................... 6627.2 Segmentation

......................................................................... 6937.3 Tracking

......................................................................... 7047.4 Filter Tracks

......................................................................... 7257.5 Displacement

......................................................................... 7367.6 Additional Example

8 Define Region Seed Points 77

......................................................................... 7818.1 Visualization

......................................................................... 7928.2 Create Surfaces

......................................................................... 8138.3 Define Region Growing and Display Surfaces

9 Analyze Neuron 84

......................................................................... 8519.1 Visualization

......................................................................... 8629.2 Change Channel Color

......................................................................... 8739.3 Automatic Detection

......................................................................... 9149.4 AutoPath Mode

......................................................................... 9359.5 Filament Tool Kit

10 Volume of Interest 97

......................................................................... 98110.1 Visualization

......................................................................... 99210.2 Add Contour Surface

......................................................................... 101310.3 Verify Planes

......................................................................... 102410.4 Draw Border Lines

......................................................................... 104510.5 Edit Polygon

......................................................................... 105610.6 Calculate Contour Surface

......................................................................... 106710.7 Create Surface Object

......................................................................... 107810.8 Mask Channel

......................................................................... 109910.9 Display Masked Channel

11 Volume over Time 111

......................................................................... 112111.1 Visualization

......................................................................... 113211.2 Add Surfaces

......................................................................... 117311.3 Time Concept

......................................................................... 119411.4 Calculate Statistics

......................................................................... 122511.5 Present Results

12 ImarisXT 123

......................................................................... 124112.1 ImarisXT Features and Advantages

......................................................................... 126212.2 Programmable Interface

......................................................................... 130312.3 InvertMiddleSlice

......................................................................... 132412.4 Create the M-File''InvertMiddleSlice.m''

......................................................................... 133512.5 Embed the new Function as XTension

......................................................................... 135612.6 Matlab Shortcut to Start Imaris

136Index

1 - Introduction

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1 Introduction

Why should you read and practice the Imaris Quick Start Tutorials?

Quick Start Tutorials is the fastest way to understand and masterImaris. It offers as a series of examples and supporting commentariesdesigned to quickly acquaint new user with the structure, possibilitiesand the most compelling features of the Imaris, furthermore it mayshow yet unrecognized new features of the software to the advanceduser.In the Quick Start Tutorials, you will follow a series of straightforward,step-by-step instructions. The tutorial is organized into 11 lessons. Eachlesson introduces you to several of the unique features of Imaris.Thetutorials are designed to be followed sequentially, but if you are alreadyfamiliar with Imaris the basic lessons may be skipped. The tutorials arecross-referenced by hyperlinks highlighted in blue underlined font. TheTable of Contents and the Index provide further support for navigationin the tutorials. At any time during the tutorial, you can accessReference Manual for additional information and an in-depthexplanation of specific features of Imaris.Each tutorial is a hands-on seminar, therefore open Imaris on the lefthand side of the screen and adjust the online Tutorial on the right handside of the screen and practice step-by-step. If you prefer to use aprintout, a copy of this document is also provided in PDF format.When you finish the tutorial, you'll have a greater understanding of howto implement Imaris in your research and be ready benefiting from ofuser friendly, fast, and powerful Imaris in processing microscopicimages.

Imaris Start Screen

Introduction - 1

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Information in this online document is subject to change without noticeand does not represent a commitment on the part of Bitplane AG.Bitplane AG is not liable for errors contained in this online document orfor incidental or consequential damages in connection with the use ofthis software.This document contains proprietary information protected by copyright.No part of this document may be reproduced, translated, or transmittedwithout the express written permission of Bitplane AG, Zurich,Switzerland.

For further questions or suggestions please visit our web site at: www.bitplane.com or contact [email protected].

Bitplane AGBadenerstrasse 6828048 ZurichSwitzerland

April 2009, Bitplane AG, Zurich. All rights reserved. Printed in Switzerland.Quick Start Tutorials V 6.3.0

1 - Introduction

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1.1 Reference Manual

The Reference Manual provides a description of all menu entries,display modes, functions and parameters. To open the onlineReference Manual click on the menu Help and select ReferenceManual.

The Imaris Reference Manual displays.

On the left hand side, click on a chapter in the Table of Contents todisplay a list with the available subchapter.

Visualize Data Set - 2

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2 Visualize Data Set

In this first tutorial you will learn the Imaris basic working steps. Thedemo image is a Pyramidal cell, which you will Open to create a 3Dvolume visualization. In the chapter Select and Navigate you willlearn about the two different mouse pointer modes in the Surpass view.You will learn how to manipulate the view by Rotating , Translating

or Scaling the image. Finally, you will save the view to disk asScene File for later use. At the end of this tutorial you will also learnhow to change the Imaris Background Color .

Visualized Data Set, Pyramidal Cell

User Level: BeginnerModule: Imaris

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2 - Visualize Data Set

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2.1 Imaris Main Screen

PC: Double-click on the Imaris shortcut on the desktop of yourcomputer to open the program.Mac: In the folder Applications double-click on Imaris to open theprogram.

After installation, upon the first program start Imaris is in the Surpassview.

Switch to the Slice view by clicking on the Slice button.

PC Main Screen

This is the Imaris Slice view on a PC.


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Mac Main Screen

This is the Imaris Slice view on a Mac.

See also: Slice view and Surpass Tree9 11


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2.2 Open Data Set


Open a Data Set in the Slice View

Switch to the Slice view by clicking on the Slice button in the main toolbar of Imaris and then click on Open. The Open File selection windowdisplays on the screen.

Select the data set Pyramidal cell and click on Open.\ProgramFiles\Bitplane\Imaris\images\PyramidalCell.ims

This is the Mac File Open dialog:


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Select the data set Pyramidal cell and click on Open./Applications/Imaris.app/Contents/SharedSupport/images/PyramidalCell.ims

The Slice View

The Slice view is a 2D display mode, in the viewing area the middleslice (number 35) of the Pyramidal cell displays. You scroll the slicesalong the z-axis by dragging the slider handle in the left control bar upand down.


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2.3 Slice View

In Slice the following standard views: Slice, Selection, Gallery andEasy 3D are provided.

Slice view for individual slices:

Selection view for XY; XZ; YZ views and navigation to any position in3D:


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Gallery view for viewing individual slices simultaneously:

Easy 3D for 3D projection along Z


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2.4 Surpass View

The Imaris is built around the three-panel workspace: View Area,Object List and Object Properties Area.

View Area is a display area for an image presentation and facilitateimage navigation, selection and interaction.

Object List

Simultaneous combination of various visualization techniques isachieved by Object List making understanding of the complex dataeasier.


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The default name of the folder is SurpassScene. The Scene includesthe standard Light Source 1, Frame display and the additional Objects.The Object List displays a list of all Surpass objects you added to theviewing area. To build a surpass scene click on Surpass creatingbuttons and follow creation process in creation/editing panel. The list isautomatically generated and updated when you add or delete an object. Each tree item includes a check box. Check the box to make the objectvisible in the viewing area or to make the object invisible un-check it.The currently active object is highlighted in the Object List and theappropriate properties show up.

Object Properties Area

In Object Properties Area you can find all parameters for the selectedObject.

See also: Design Mixed Model Rendering -Volume Rendering , AddSurface and Analyze Neuron

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2.5 Volume

Now you will create a 3D reconstruction of the Pyramidal cell.

Select Volume in the Surpass View

Click on Surpass in the main toolbar of Imaris.

The Pyramidal cell displays in the viewing area.

See also: Design Mixed Model Rendering -Surpass View , AddVolume and Analyze Neuron

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2.6 Select and Navigate

In the Surpass view of Imaris there are two different mouse Pointermodes - Select and Navigate.

You select the respective mode in the Pointer selection on the righthand side of the screen.

Appearance of the Mouse Pointer

Depending on the pointer mode the symbol on screen changes.

Select Navigate

Tip: You can easily switch between the two pointer modes using theESC-Key. The effect is directly visible on screen by the altered mousepointer display.

When to Use Select?

You use the pointer mode Select whenever you want to marksomething in the image, e.g. to set some measurement points on theobject surface.

When to Use Navigate?

You use the pointer mode Navigate to move or rotate the image in theviewing area.

See also: Visualize Data Set - Rotate Image 15


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2.7 Rotate Image

Rotating an image allows to change the viewing angle on athree-dimensional object.

How to Rotate an Image?

1. Choose the mouse pointer mode Navigate. 2. Click with the mouse in the image and hold the button down whilemoving the mouse (hold + drag). The image on screen is rotatedtowards the direction the mouse is dragged. Be sure to hold the mousebutton down during the whole rotation.3. Stop moving the mouse and release the mouse button to stop therotation.

How to Keep the Image Continuously Rotated?

1. Choose the mouse pointer mode Navigate.2. Click with the mouse button in the image and hold the button downwhile you move the mouse (hold + drag). The image on screen isrotated towards the direction the mouse is dragged.3. Release the mouse button while still dragging the mouse. The resultis a continued rotation (speed of the rotation according to prior mousemotion). 4. To stop the continued rotation re-click in the image area.

See also: Visualize Data Set - Select and Navigate 14


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2.8 Translate Image

1. Choose the mouse pointer mode Navigate.2. To move the image within the Surpass view (pan the object) hold theright mouse button and drag the image to the chosen location (if youuse a Mac with a one button mouse use: ctrl + click & drag to pan).3. Release the right mouse button to place the image.



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2.9 Scale Image

In the Surpass view you zoom the image either by using the mouse orby selecting one of the buttons at the bottom of the screen.

Using the Mouse

1. Choose the mouse pointer mode Navigate.2. To zoom in on the image hold the middle mouse button and drag it

towards you (if you use a Mac with a one button mouse use: shift + ctrl+ click & drag to zoom). 3. To zoom out from the image hold the middle mouse button and dragit away from you.

Using the Buttons at the Bottom of the Screen

Zoom Enter the zoom factor.100% Rotate image to original position, center in the middle and set

zoom factor to one pixel per voxel.Fit Pan position to best fit in the window and adjust the zoom

factor.FullScreen

Maximize the viewing area to full size of the monitor.

Navi Toggle display of navigation window (upper right corner of theviewing area).



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2.10Save Scene File

You can store the actual configuration in a Scene File. After saving aScene File, you can restore the configuration at any time by reloadingthe Scene File.

Tip: Saving a Scene File is the ideal way to save intermediate data inImaris.

Rename the Scene

Double-click on the entry SurpassScene and enter the new namePyramidalCellSceneA.

Save the Scene as SceneFile.imx

To save the Scene File open the menu File and select Save Scene ...

As name type in the new name of your Scene. The extension of anImaris Scene File is *.imx. To save the Scene FilePyramidalCellSceneA.imx click on Save.

See also: Design Mixed Model Rendering - Surpass View ,Generating Movies - Load Scene File

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2.11Practice Makes Perfect

The mouse handling to navigate the image in the viewing area needssome practice but once you master the mouse it is a powerful tool toanalyze your data. Start with a center position (Home Position), thenrotate the image backwards and then rotate it clockwise.

Home Position

Use the buttons at the bottom of the viewing area, click first on 100%,then on Fit.

The Pyramidal cell displays centered in the viewing area.

Tip: Whenever you lose orientation, re-center the view to the HomePosition by clicking the two buttons again.

Rotate Backwards


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Now rotate the image backwards. Choose the mouse pointer modeNavigate. Click on the base of the Pyramidal cell, hold the button downwhile you move the mouse upwards, stop the mouse movement andrelease the mouse button.

This is the result of the rotation.

Move the Image Clockwise

In the next step rotate the image clockwise. Click on the position shownabove, hold the button down while you move the mouse to the left, stopthe mouse movement and release the mouse button.


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This is the result of the rotation.

See also: Visualize Data Set - Select and Navigate , Visualize DataSet - Rotate Image , Visualize Data Set - Scale Image

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2.12Change Background Color

The standard background color in Imaris is black. To change thebackground color go into the Imaris Display settings.

Open the Preferences - Display Window

Select the menu Edit - Preferences ... - Display. On the right handside you find the Colors dialog.

Change Background Color

To change the Background Color click on Select... to open the SelectColor window.

Select the desired color and click on OK. The preview changesaccordingly.


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In the window Preferences - Display click on Ok. The result is in the viewing area. Please close Imaris before you startwith the next tutorial.

See also: Design Mixed Model Rendering - Change Spots Color 55


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2.13Change Channel Color

Now you change the color of the the display Channel 1 from yellow tored. To open the Display Adjustment window select in the menu Edit - Show Display Adjustment.

Click on Channel 1 to open the color selection. In the color table selectand adjust the color and click on OK. This is the result in the viewingarea:

MIP Display, Channel Color Red, Pyramidal Cell

See also: Design Mixed Model Rendering - Display Adjustment ,Change Spots Color , Visualize Data Set - Change BackgroundColor

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3 - Generating Movies

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3 Generating Movies

Animated visualizations of three-dimensional data sets can be saved asanimations in Imaris. They can also be exported as movies forconvenient display in standard movie players. First of all, you Load theScene File of the processed data set generated in the previoustutorial Visualize a Data Set . This tutorial requires the understandingof the Key Frame Animation function in Imaris. You will practice tosubsequently Shoot , Play , and Save a Movie .

Movie Sequences, Pyramidal Cell


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Generating Movies - 3

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3.1 Load Scene File


To make a movie please load the Scene File PyramidalCellSceneA.Click on the menu File and select Load Scene or click on the iconLoad Scene.

In the dialog window select the file PyramidalCellSceneA.imx and clickon Open.

Click on Yes to exactly reconstruct the Scene.

The Pyramidal cell displays in the viewing area and in the propertiesarea you find the corresponding Object List.

See also: Visualize Data Set - Save Scene File , Visualize Data Set -Surpass View

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3.2 Key Frame Animation

The key frame Animation function defines several subsequent views(key frames) of your image, which will be used to create an animation.Imaris generates smooth transitions between the selected views byinterpolation.

Click on the icon Animation in the toolbar. You find the Key FrameAnimation window at the bottom of the screen. It is divided in fiveparts, the Key Frame window, the Rotations window, the Animationwindow, the Play Back State window, and the Strip window.

The Key Frame Window

In the Key Frame window you define and manage the key frames ofyour movie. The three buttons in the first row are to Add, Modify orDelete key frames in the movie strip. Use the Delete All button todelete all key frames. With the Arrow buttons you step to the Previous(arrow to the left) or Next (arrow to the right) user-defined key frame.

The Animation Window

In the Animation window you can enter the number of movie Frames.


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There is the button to open the Animation Settings... as well as thebuttons to Play (arrow to the right) and Record (red dot) the movie. Topause the animation re-click on Play. Rotation panel allows scrollthrough the pre-set rotations and Custom button allows to insert youown degree of rotation in any direction.

The Film Strip Panel

The Strip window provides the working area. A thick blue line indicatesa user-defined key frame. A white line represents the active key frameand a thin blue line stands for an interpolated frame. Click on a line todisplay the corresponding image view in the viewing area. A click on theblue line will display the image view corresponding to the view inSurpass view area.

Buttons Overview

Add-addition of new key frame (the imagepresented on the screen will appear as a keyframe)

Modify-modification and adjustment ofhighlighted frames in the film strip

Delete active key frame

Delete All key frames

Play/Pause to preview or to pause of animation

Record animation to movie file

Go to Previous key frame

Go to Next key frame


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3.3 Shoot and Play

In this section you will learn how to:

Shoot a movie with 20 frames. Add the first and last key frame to the movie strip. Insert additional key frames. Play and pause the movie. Modify a user-defined key frame. Please remember: Each time you click on the button +Add in the KeyFrame window the camera captures the next key frame of your movie.

Movie Length

In the field Frames type in 20. The strip will indicate these frames by 20thin blue lines which will define the duration of animation.

Adjust and Capture the First Key Frame

Turn the image view to the first position (click on the bottom of the


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screen on the button 100%, then on the button Fit). Capture the firstview with a click on the button +Add in the Key Frame window. Bydefault, the actual image position is taken as first and last key frame. Inthe movie strip the first line turns white (active key frame), the last oneturns thick blue (user-defined key frame) and the other lines stay thinblue.

Add Additional Key Frames

Move the image to the next viewing position. Click on the base of thePyramidal cell, hold the button down while you move the mouseupwards, stop the mouse movement and release the mouse button.The view should be similar to:

Click on the button +Add in the Key Frame window. The first key frameturns thick blue (user-defined key frame). In the movie strip anadditional white line (active key frame) is inserted on the right hand sideof the last active key frame (in the example at position 10,5).

Move the image to the next position. Click on the base of the Pyramidalcell, hold the button down while you move the mouse downwards, andthen move the mouse to the left hand side. Stop the mouse movementand release the mouse button. The view should be similar to:


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Click on the button +Add in the Key Frame window. In the movie stripan additional white line (active key frame) is inserted on the right handside of the last active key frame. All user-defined key frames areautomatically distributed equally.

Tip: To get a first impression about the movie click in the Strip window,hold the mouse button down and drag the mouse to the right or left,respectively. The corresponding image views display consecutively inthe viewing area.

Play the Movie

Click in the Animation window on Play (arrow to the right) to displaythe movie in the viewing area. To pause the animation re-click on Play(arrow to the right).

Modify Key Frame

Click on any thick blue line in the strip to display the correspondingimage view. Rotate the image in the viewing area to a new position andclick in the Key Frame window on Mod. (Modify) to save the newimage position.

See also: Generating Movies - Key Frame Animation , Visualize DataSet - Rotate Image , Visualize Data Set - Scale Image , VisualizeData Set - Practice Makes Perfect

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3.4 Save Movie

There are two possibilities to save the movie. Either as an Imaris SceneFile or as a movie-file (*.avi).

In Imaris as Scene File

Whenever you save the Scene File in Imaris, the actual animation issaved automatically as part of the Scene File. Open the menu File andselect Save scene... and click on Save.

Save Different Animations in Different Scene Files

Saving different animations of the same data set requires saving ofdifferent Scene Files. After generating a new movie by the key frameAnimation, rename each time the Scene File, e.g. add "Movie" at theend of the Scene File name. Open the menu File and select Savescene.... In the dialog window type in the new Scene File name (e.g.PyramidalCellSceneAMovie.imx) and click on Save.

Record the Animation as AVI Movie

To save the animation as AVI movie click in the Animation window onthe button Record (red dot). The Save As Movie dialog windowdisplays on screen.


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As File name type in the corresponding Scene File name, use thedefault Movie Settings and click on Save. The file extension for theAVI movie is *.avi.

Please close Imaris before you start with the next tutorial.

See also: Visualize Data Set - Save Scene File , Generating Movies- Key Frame Animation , Imaris and QuickTimeVR

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Imaris and QuickTimeVR - 4

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4 Imaris and QuickTimeVR

To export the visualized results of your investigation Imaris supports theQuickTimeVR file format. In the first section you will learn about theBasic Principles of QuickTimeVR - the file generation mechanismand the default movie settings. Then you will Generate a QuickTimeVRFile in Imaris to Visualize the Results interactively inQuickTimeVR. Optimized Settings for a presentation and a webpage are described in detail in the last chapter of this tutorial.

QuickTimeVR Display, Pyramidal CellUser Level: BeginnerModule: Imaris

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4 - Imaris and QuickTimeVR

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4.1 Basic Principles

Sometimes it is very convenient to show the data visualized in Imaris inan alternative, independent program. Therefore Imaris supports theQuickTimeVR allowing user to show complex scenes outside Imarispacking. Imaris automatically takes several snapshots of your image inthe viewing area using different, predefined camera positions.Depending on the settings more or less snapshots are saved in thecorresponding file (*.mov). The actual image size on the computerscreen and the compression factor have an impact on the file size andthe quality of your QuickTimeVR.

Please note: QuickTimeVR is not automatically installed on yourWindows PC when you install Imaris. Please install QuickTimeVRbefore you continue this tutorial.

QuickTime Default Movie Settings

Compression Factor: Select a compression factor between 0 (HighQuality) and 100 (Low Quality), the default setting is 5.Degrees between two Frames: Here you define the degrees betweentwo frames, the default setting is 10.Total Angle Horizontally: Defines the angle of the horizontalmovement, the default setting is 360.Total Angle Vertically: Defines the angle of the vertical movement, thedefault setting is 180.

See also: Imairs and Quick Time VR - Optimized Settings 39


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4.2 Generate File


Load your data set or Scene File (e.g. PyramidalCellSceneA).

Adjust the Viewing Area

Adjust the size of the viewing area to the desired size of theQuickTimeVR viewing area, the display is 1:1.

Save the QuickTimeVR File

In the Surpass view click on the icon QuickTimeVR on the right handside of the screen to open the Save as Movie dialog window.


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Type in the File name, select QuickTimeVRMovie (*.mov), use thedefault Movie Settings and click on Save.

What Happens in the Imaris Viewing Area?

Automatically Imaris starts to turn around the image in the viewing areaand takes a snapshot of each image position. The degree between twoframes is 10, the angle horizontally is 360 and the angle vertically 180.Down to the right you find a progress bar. Depending on the settings,this procedure will take several seconds or even minutes.

See also: Visualize Data Set - Open Data Set , Generating Movies -Load Scene File

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4.3 Interactive Display

Double-click on your QuickTimeVR file to show the results.

The image displays in the QuickTimeVR viewing area. You navigate theScene using the mouse. With the standard settings you rotate theobject 360 horizontally and 180 vertically. The angle between twoimage frames is 10.


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4.4 Optimized Settings

If you want to save a movie for a presentation on your computer the filesize is according to experience is not the limitation. To put the movie onyour web page the file should not be too large.

Please note: If you increase the degree between two frames lessimages are stored in the file. The effect is directly visible during imagenavigation in QuickTimeVR. The step between two images is bigger,the navigation becomes fragmentary.

Please note: If you reduce the total angle horizontally from 360 to 160 itis no longer possible to turn the image around the axis.

Adjust Settings for a Presentation

Adjust the size of the viewing area in Imaris, the display inQuickTimeVR is 1:1. In the Surpass view click on the icon QuickTimeVR on the right hand

side of the screen to open the Save as Movie dialog window.

Use the Compression Factor 5. Set the Degrees between two Frames to 5.


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Set the Total Angle Horizontally to 360 and the Total AngleVertically to 360. Check Play Movie with default Media Player when finished. Click on Save.

Adjust Settings for a Web Page

Adjust the size of the viewing area in Imaris, the display inQuickTimeVR is 1:1. In the Surpass view click on the icon QuickTimeVR on the right hand

side of the screen to open the Save as Movie dialog window.

Use the Compression Factor 50. Set the Degrees between two Frames to 20. Set the Total Angle Horizontally to 160 and the Total Angle

Vertically to 160. Check Play Movie with default Media Player when finished. Click on Save.


5 - Design Mixed Model Rendering

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5 Design Mixed Model Rendering

This tutorial is focused on the different visualization options in theSurpass mode of Imaris to gain visualization control of the objects. ThePtK2 cell is particularly suitable for a mixed model rendering. You willstart with a Volume rendering for all channels and you will adjust the contrast, brightness and transparency in the Display Adjustmentwindow. The microtubules remain Volume rendered but for thechromosomes you will choose the Surfaces mode in Imaris. For thekinetochores you select the Spots display and in the next step youChange the Spots Color . The Object List displays a list of allobjects in the viewing area and is the ideal instrument to handle theFinal Image .

Mixed Model Rendering, PtK2 CellUser Level: BeginnerModule: Imaris

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Design Mixed Model Rendering - 5

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5.1 Add Volume


Open the demo image PtK2 cell. PC:\ProgramFiles\Bitplane\Imaris\images\PtK2Cell.imsMac:/Applications/Imaris.app/Contents/SharedSupport/images/PtK2Cell.ims

Volume

To open the data set select the Surpass mode. Click on the iconSurpass in the main toolbar of Imaris.

On the left hand side in the Volume - Properties you find the Modeselection window.

Select as display mode Blend. In the blend mode all values along theviewing direction including their transparency are used for thecalculation.


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This is the PtK2 cell display in the viewing area.

See also: Visualize Data Set - Open Data Set 7


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5.2 Display Adjusment

The objects rendered as Volume are usually quite dark and have a lotof noise. To optimize Volume rendering the image contrast, brightnessand blend opacity will be adjusted. These settings are in the DisplayAdjustment window. The Display Adjustment function lets you interactwith each channel individually, control Channel Visibility and Color,Intensity Range Min / Max, Blend Opacity and display Channel IntensityHistogram.

Adjust All Channels Simultaneously

To open the Display Adjustment window select in the menu Edit - ShowDisplay Adjustment.

In the upper part of the window each channel is represented by achannel bar. In this case the red channel corresponds to thekinetochores, the green channel represents the microtubules and theblue channel is the DAPI stained DNA. Click on Advanced to open theadvanced settings.Usually the color contrast values of the voxelsstretch over a wide range (e.g. 0-255).


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In this multi channel dataset check the box Select all Channels andset Min: 28.000 and Max: 200.000. The effect on all channels isdirectly visible in the viewing area.

Adjust Individual Channel

To tune the green channel (microtubules) individually.To focus on thegreen channelun-check the red and blue channels. Selecting orde-selecting channels controls their visibility. Select the green channelby clicking on the channel bar. In the viewing area only the greenchannel is now displayed.

Set the Range - microtubules Min: 20.000 and Max: 45.000. To tune the red channel individually.To focus on the red channel(kinetochores) check the check box for the red channel and un-checkthe boxes for the green and blue channel to control channel visibility.Inthe viewing area only the red channel histogram is displayed. Changefor the selected red channel the value Min to 20.000 and the value Maxto 100.000. The transparency of a channel is altered by adjusting theOpacity to 64%.


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The other option for Display Adjustment modification is clicking directlyon the color channels and changes will be immediately visible in imageview area.

Reset -reverts values to defaultsAuto -selects min and max valuesAuto Blend -selects optimized min/max values



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5.3 Add Surfaces

You will now add to the Volume rendered microtubules a Surfacesreconstruction of the DNA.

In the Objects toolbar of the Surpass view click on the icon to adda new Surfaces item.

Creation Wizard - 1/6 Algorithm

Select segment a Region of Interest (ROI) and Process entire Image

finally and click on (Next).

Creation Wizard - 2/6 Region of Interest (ROI)


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In a viewing area a rectangle-bordered section overlaid on image isrepresenting the Region of Interest (ROI).Entering the values in the corresponding x-, y-, and z- fields (min/max)modifies the size and the position of the ROI. The size and position ofROI can be also modified directly by click on the arrows in the previewrectangle (change cursor to Select mode). To move the ROI, clickinside the rectangle, hold the mouse button down & drag the entire ROIaround. The value in x, y, z fields are updated automatically. Click on

(Next).

Creation Wizard - 3/6 Source Channel

Select as a Source Channel the Channel 3 (blue) and Absolute Intensity

as a option for Threshold. Click on (Next).


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Creation Wizard - 4/6 Threshold

For the Threshold adjustment select Manual option and set a value to30. Click and drag into the histogram can also change the thresholdvalue.

Click on (Next).

Creation Wizard - 5/6 Classify Surfaces

On the tab Classify Surfaces you can sort and filter the resultingSurfaces by various filter criteria. In this example, a sorting is not

necessary. You can delete all Filters and click on (Next).

Creation Wizard - 6/6 Complete ROI

To complete Surface creation click on (Finish).


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5.4 Change Surface Color

Color Properties

In the properties panel click on the tab Color (rightmost in the tabselection) to open the Color Properties dialog box. Change the colorfrom gray to a dark blue by click on the color wheel. The Surface in theviewing area is displayed the chosen color.

Additional to the Volume rendered microtubules the Surfacesreconstruction of the DNA displays in the viewing area of Imaris.


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In the Object List now un-check the box for the item Volume and newcreated surface is displayed in viewing area.

See also: Visualize Data Set - Change Background Color 22


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5.5 Add Spots

The kinetochores are small, globular and fairly spherical, and so ideal toshow how Imaris detect Spots automatically. We will now add a Spots

object to automatically segment the kinetochores.

In the Objects toolbar of the Surpass view click on the icon to addnew Spots.


Leave the boxes Segment only a Region of Interest and Different

Spot Sizes (Region Growing) un-checked and click on (Next).



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Select as Source Channel the Channel 1 - (kinetochores) and set the

Estimated Diameter to 0.500 um and click on (Next).

Creation Wizard - 3/3 Classify Spots

Imaris detects an automatic threshold to insert the Spots. You see atthe same time the gray Spots and the Volume rendered red channel.

Verify the Spots

With the automatic threshold detection some Spots are not identifiedbecause they are under the threshold limit.

To change the lower threshold select Manual and type in 10.000.Alternatively you can left click on the yellow line and drag the mouse.Move the line to the left to decrease the threshold and get additional


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objects with a low intensity value. The effect is directly visible in theviewing area. Compared to the first image display (automatic threshold

12.885) there are additional Spots. Click on (Finish). This is theresult in the viewing area.

See also: Measure Sstructures - Line and Polygon (to define theminimum spot diameter), Design Mixed Model Rendering - ChangeSpots Color

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5.6 Change Spots Color

Color Properties

In the properties panel click on the tab Color (rightmost in the tabselection) to open the Color Properties dialog box.

Select the Tab Color and change the color from gray to a red by click on the color wheel. The Spots in the viewing area is displayed thechosen color.

Now un-check in the Object List the box for the item Volume.

You see only the red Spots In the viewing area.

See also: Visualize Data Set - Change Background Color , ChangeSurface Color , Change Channel Color

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5.7 Final Image

The purpose of this complex tutorial is to get an idea about the differentrendering possibilities in Imaris. To display the composed image checkall items in the Object List. In the Display Adjustment window checkonly the green channel.

The microtubles display in green as Volume rendering, thechromosomes in blue as Surfaces and the kinetochores in red asSpots.

Tip: A Scene File stores a configuration of Imaris and Surpass andallows you to restore the work at any time by loading the Scene Fileagain. For details please refer to chapter Visualize Data Set - SaveScene File and Generating Movies - Load Scene File .


See also: Visualize Data Set - Surpass View , Design Mixed ModelRendering - Display Adjustment

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6 Measure Structures

In each Imaris view there are several measurement options. Thistutorial will focus on different measurement functions in the Slice view

. Depending on what you want to measure in the image you eitherchoose as interactive measurement type Line or Polygon . If youwant to estimate distances in the image you can lay a Grid over yourimage. The interactive Scale Bar also helps in estimating sizes anddistances. In the last chapter you will learn how to measure distancesbetween different slices (3D Measurement ).

Grid and Scale Bar Display, PtK2 Cell


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6.1 Line or Polygon


Open the demo image PtK2 cell in the Slice view. In the slice selectionon the left hand side of the screen select slice number 17 (type in 17and press Enter).

PC:\ProgramFiles\Bitplane\Imaris\images\PtK2Cell.imsMac:/Applications/Imaris.app/Contents/SharedSupport/images/PtK2Cell.ims

Point to Point Distance Measurement

In the PtK2 cell the kinetochores are labeled in red. You will measurethe diameter of a kinetochore using the point to point measurementfunction in Imaris.

On the right hand side of the screen you find the Measure window.Choose the measurement Type Line. Set the two measurement pointswith two consecutive clicks. The result displays in the status field


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Distance: 0.32 um. Click on Delete to clear the status field and start anew measurement.

Measure Perimeter

Now you would like to know the perimeter of the blue labeled DNA.

Choose the measurement Type Polygon. All measurement points areconsecutively connected by lines and the distance is the sum of thedistances between the points. Draw a polygon around the DNA like inthe image above. The result displays in the status field Distance 9.21um. Click on Delete to clear the status field and start a newmeasurement.



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6.2 Grid and Scale Bar

Display a Grid

One way to estimate distances in the image is to use the grid display.

On the right hand side in the Measure window check the check boxGrid. This is the result in the viewing area:

Adjust the Interactive Scale Bar

You can adjust the line length, line width, location and font size of theinteractive scale bar. Move the mouse pointer over a drag region. Theshape of the mouse pointer indicates the interaction.

Line Length and Line Width


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To adjust the length of the scale bar move the mouse to one end of thescale bar until the pointer symbol alters (vertical dash and arrow). Nowclick on the scale bar end and hold the button down while you drag themouse to the left or right, respectively. The grid width changesaccordingly. In the same way you adjust the thickness of the scale bar.

Location

To translate the scale bar click on it (pointer symbol cross), hold thebutton and drag the mouse.

Font Size

Move the mouse pointer over the legend until the pointer symbol alters(horizontal double dash and double arrow). Click and hold the buttondown while you drag the mouse up (enlarge size) or down (reducesize).

Please close Imaris before you start with the chapter 3D measurement.


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6.3 3D Measurement


Open the demo image Pyramidal cell in the Slice view.

PC: \ProgramFiles\Bitplane\Imaris\images\PyramidalCell.imsMac:/Applications/Imaris.app/Contents/SharedSupport/images/PyramidalCell.ims Measure Distance Between Different Slices

In the slice selection on the left hand side of the screen select slicenumber 21 (type in 21 and press Enter).

On the right hand side in the Measure window select as measurementType Polygon.


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The first picture is slice number 21, the second picture is slice number25 and the last picture is slice number 32.

Set the first point with a click on the upper right end of the filament.Change to the next slice number (edit the field or use the arrow keys onthe keyboard) and set the next measurement points with consecutiveclicks. Follow the filament through the slices. The dashed line indicatesconnecting lines between two slices. You find the distance between thefirst and the last measurement point in the status field Distance on theright hand side of the screen.


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7 Track Particles

ImarisTrack is a separate Imaris module to follow 3D-objects over time,display their paths and analyze their movements. The demo imageshows the movement of the algae chlamydomonas reinhardtiizoozpores. The first step is the Visualization of the data set and theautomatic Spot Detection (Segmentation ) over time. With theautomatic Tracking you link consecutive time points and the result isa colored Track - the motion path of a single object over time. You willlearn how to show the Track Displacement . Using the Filterfunction you can group and analyze the Tracks depending on variouscriteria.

Spots & Tracks, Swimming Algae

User Level: AdvancedModule: Imaris, MeasurementPro, ImarisTrack

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7 - Track Particles

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7.1 Visualization


Open the demo image Swimming Algae in the Slice view. PC: \ProgramFiles\Bitplane\Imaris\images\SwimmingAlgae.imsMac:/Applications/Imaris.app/Contents/SharedSupport/images/SwimmingAlgae.ims

Add Volume

To create a Volume reconstruction of the data set select the Surpassmode. Click on the icon Surpass in the main toolbar of Imaris andselect Volume. In Mode selection select the option Blend (blending allvalues along the viewing direction and including their transparency). To open the Display Adjustment window select in the menu Edit -Show Display Adjustment. Click on Advanced to open the advancedsettings. Set the threshold Range for Channel 1 (green) to Min: 90 and Max: 230.

This is the result in the viewing area.

See also: Visualize Data Set - Open Data Set , Design Mixed ModelRendering - Add Volume

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7.2 Segmentation

An automatic image segmentation and object tracking methods will beexplained by analyzing image Swimming Algae. First you will identifyand create spots. Then you will establish a time dependant relationshipbetween spots (track them over time) and create Tracks . Finally youwill sort the Tracks based on the Track duration and visualizetracking results.

In the Objects toolbar of the Surpass view click on the icon to addnew Spots.


In the Algorithm Setting check the Track Spots (over Time).

Click on (Next).


Select as Source Channel the Channel 1 (green) and set theEstimated Diameter to 4.000.

Click on (Next).

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Select as Filter Type: Quality, select Manual and set Lower Threshold

to 15.000. Click on (Next).

You see at the same time the gray Spots and the Volume renderedgreen channel.

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Creation Wizard - 4/6 Edit Spots

Do not edit any Spots and click on (Next).

See also: Design Mixed Model Rendering - Add Spots , DesignMixed Model Rendering - Change Spots Color , Measure Structures- Line and Polygon (to define the minimum spot diameter)

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7.3 Tracking

Creation Wizard - 5/6 Tracking

A Track is a component within the Surpass view that describes thebehavior of an object over time. (To automatically create all Tracks youdid check the box Track Spots (over Time) on the first window of theCreation Wizard.)

As Algorithm select Autoregressive Motion and as MaxDistance set

10.000 um, and for the MaxGapSize set 3. Click on (Next).

This is the result in the viewing area.

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7.4 Filter Tracks

Creation Wizard - 6/6 Classify Tracks

The sorting of the Tracks is essential for the further analysis. In thefollowing you sort the Tracks by their length.

Choose as Filter Type: Track Length. Select Manual and enter asvalue 145.000 um. You can also the move the yellow lines by using themouse.

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The effect is directly visible in the viewing area. Click on (Finish).

This is the Spots and Tracks display in the viewing area.

See also: Visualize Data Set - Surpass View , Volume over Time -Time Concept

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7.5 Displacement

This visualization mode shows the displacement of a Track as an

arrow. Select the tab Settings .

In the Tracks Path adjustment window check the respective check boxDisplacement.

The displacement arrows display in the viewing area.


Please close Imaris before you start with the next chapter.

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7.6 Additional Example

In this additional example for particle tracking you will analyzedeveloping thymocytes in an intact thymic lobe. You will visualize and segment the cells. Then you will create theTracks over time and sort the Tracks based on the Track duration.

In Imaris open the demo image R18Demo in the Surpass view. PC: \ProgramFiles\Bitplane\Imaris\images\R18Demo.imsMac:/Applications/Imaris.app/Contents/SharedSupport/images/R18Demo.ims

Visualize, Segment and Track the Cells

As Initial Scene select Volume. In the Objects toolbar of the Surpass

view click on the icon to add new Spots.


Check the Track Spots (over Time).

Click on (Next).

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Select as Source Channel the Channel 1 (green) and set theEstimated Diameter to 5.000. Click on the box for the Background

Subtraction option. Click on (Next).


Select for the parameter Filter Type: Quality, select Manual and setLower Threshold to 10.000. As an alternative you can left click into thehistogram and drag the yellow line left or right to change the thresholdvalue.

Click on (Next).

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Creation Wizard - 4/6 Edit Spots

No Spots editing is needed, so click on (Next).

Creation Wizard - 5/6 Tracking

As Algorithm select Autoregressive Motion and as MaxDistance set

10.000 um, and as MaxGapSize set 3. Click on (Next).

Creation Wizard - 6/6 Classify Tracks

The Tracks will be sort by the track duration.

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Adjust the parameter Filter Type: Track Duration. Select Manual, setLower Threshold to 2.000. The effect is directly visible in the viewing

area. Click on (Finish).



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8 - Define Region Seed Points

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8 Define Region Seed Points

In this tutorial you get familiar with the region growing to segmenttouching objects. First you Visualize a plant cell with chloroplasts(green) and cell wall components (red). Then you detect thechloroplasts and define seed points for the Create Surfaces SeededRegion Growing . When you start the region growing a workingchannel displays in the viewing area. The seed point regions will growuntil they reach the defined border. The result of the region growing inthis example are Display Surfaces .

Surfaces, Region Growing, Plant Cell


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8.1 Visualization


Open the demo image PlantCell.ims in the Surpass view.

PC: \ProgramFiles\Bitplane\Imaris\images\PlantCell.imsMac:/Applications/Imaris.app/Contents/SharedSupport/images/PlantCell.ims

Visualize Chloroplasts

This is the volume rendering of the plant cell in the viewing area.


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8.2 Create Surfaces

In the Objects toolbar of the Surpass view click on the icon to addnew Surfaces.


Select segment only a Region of Interest (ROI) and Process entire

Image finally. Click on (Next).


A rectangle-bordered section is overlaid on image, representing theRegion of Interest (ROI). Entering the values in the corresponding x-,y-, and z- min/max fields or directly by click on the arrows to modify the

ROI size and position. Click on (Next).


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Select as Source Channel the Channel 2 (chloroplast) and check theoption Smooth and as Surface Area Detail Level use 0.250 um.Check option Background Subtraction and set as Diameter of

largest Sphere which fits into the Object value of 5um. Click on (Next).

Creation Wizard - 4/6 Threshold

In the Threshold adjustment select Manual, set threshold to 10.000and select Split touching Objects (Region Growing). The number of

steps increase from 6 to 8. Click on (Next).


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8.3 Define Region Growing and Display Surfaces

Creation Wizard - 5/8 Region Seed Points

Set as Estimated Diameter value of 4.0 um. Click on (Next).

Creation Wizard - 6/8 Classify Seed Points

Select as Filter Type: Quality, select Manual, set Lower Threshold to10.000.

This is the result in the viewing area. Click on (Next).


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Creation Wizard -7/8 Classify Surfaces

Select as Filter Type: Number of Voxels, select Manual, set Lower Threshold to 10.000.


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This is the result in the viewing area. Click on (Next).

Creation Wizard -8/8 Surface computation for the entire dataset To complete Surface creation with Split touching objects selection click

on (Finish). This is the result of the seeded region growing in theviewing area.



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Analyze Neuron - 9

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9 Analyze Neuron

FilamentTracer is a separate Imaris module to detect, visualize andmeasure filamentous structures. The demo image shows a hypocampalPyramidal cell in an organotypic cell culture. The tutorial will focus ontwo ways of filament tracing, how to automatically create filamentstructure in Automatic Detection and the semi-manual creationalternative in the AutoPath Mode . In the last chapter Filament ToolKit you will find numerous visualization filament possibilities and ashort description where to find and how-to-handle the different tracingfunctions in the complex FilamentTracer module.

Filament Structure, Pyramidal Cell

User Level: AdvancedModule: Imaris, FilamentTracer

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9.1 Visualization


Open the demo image Pyramidal cell in the Slice view. PC: \ProgramFiles\Bitplane\Imaris\images\PyramidalCell.imsMac:/Applications/Imaris.app/Contents/SharedSupport/images/PyramidalCell.ims

To select the Surpass mode click on the icon Surpass in the maintoolbar of Imaris. The Pyramidal Cell is displayed.

MIP Display, Pyramidal Cell

Analyze Neuron - 9

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9.2 Change Channel Color

Now you change the color of the the display Channel 1 from yellow tored. To open the Display Adjustment window select in the menu Edit - Show Display Adjustment.

Click on Channel 1 to open the color selection. In the color table selectred and click on OK. This is the result in the viewing area:

MIP Display, Channel Color Red, Pyramidal Cell

See also: Design Mixed Model Rendering - Display Adjustment ,Change Spots Color , Visualize Data Set - Change BackgroundColor

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9.3 Automatic Detection

In the Automatic creation method of the FilamentTracer the start pointand the end points are initially detected. Then these points areautomatically connected with lines following the image intensity and thefilament structure is created. For this neuron image there is onestart-point at the location of the soma, and many end-points located atthe dendrites and/or the spines terminal point.

Add new Filament

In the Objects toolbar of the Surpass view click on the icon to adda new Filament.


Select segment a Region of Interest (ROI) and as Algorithm select

Autopath (no loops) and click on (Next).

Creation Wizard - 2/6 Region of Interest

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In the viewing area a rectangle-bordered section overlaid on image isrepresenting the Region of Interest (ROI). Modify the position of ROIdirectly by holding the mouse button and drag the entire ROI (changecursor to Select mode). Enter the value in x, y, z fields from pictureabove. It is important that the ROI includes the filament start point.

Click on (Next).

Creation Wizard - 3/6 Points Diameter

Select as Source Channel the Channel 1 (red). Set the Starting PointDiameter to 10.000 um, the End Point Diameter to 0.455 um. Click

on (Next).

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Creation Wizard - 4/6 Classify Points

Imaris detects the filament Starting Point automatically and no StartingPoint Threshold adjustment is necessary. Select the Automatic option.For For the End Point Threshold select 35. The filament beginning point

and end points display in the image. Click on (Next).

Creation Wizard -5/6 Spines and Diameters

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Leave the box Detect Spines un-checked. Check the box Calculate

Diameter from Image and click on (Next).

Creation Wizard -6/6 ROI processing

The parameters chosen for selected ROI could be used to processFilaments either in the entire image or within a newly determined ROI.

Select option Entire Image. Click on the (Finish).

All end points are automatically connected to the start point. This is theresult in the viewing area:

.

See also: Measure Structures - Line and Polygon (to define thestarting and end point diameter)

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9 - Analyze Neuron

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9.4 AutoPath Mode

In the case that Automatic Detection method is not producing desiredresults, filament structure could be created by AutoPath method option. AutoPath method can be used for reselecting, redrawing and creatingnew filament structure. In the AutoPath mode of the FilamentTraceryou define the start point and all the end points manually. Based on theposition of the end point the AutoPath function automatically computesthe path to the starting point.

Add new Filament

In the Objects toolbar of the Surpass view click on the icon to add

a new Filament. In the Filament - Properties click on (Cancel) tostop the automatic detection of the start point and the end points. Click

on the tab Draw .

In the Method parameters select AutoPath and change to the mousepointer mode Select. A rectangle displays around the mouse pointer.

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Press Shift and right-click (if you work with a PC) on the base of thePyramidal cell to fix the starting point. (If you work on a Mac set thestarting point with: Shift + Ctrl + click.)

At the base of the cell you see the filament starting point. Now youmove the mouse cursor to an end point of a filament. The filament iscalculated automatically and displays on screen. With Shift + click youfix the filament. Move the mouse cursor to the end point of the nextfilament, press Shift + click to fix the filament. In the same way set allthe filament end points in the image.

See also: Visualize Data Set - Select and Navigate (mouse pointermode), Analyze Neuron - Filament Tool Kit

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9.5 Filament Tool Kit

In this chapter you will learn different visualization possibilities andwhere to find the different selection windows in the moduleFilamentTracer. A short, tabular overview concerning the three differentinteractive filament tracing methods will be presented.

Filament Visualization

On Filament Setting Tab change the Style to Cylinder. Checkboxes for Show Dendrites Beginning, Branch and Terminal pointsfor visualization of Filament structure.

Un-check the Volume rendering in Object List and new FilamentStructure is displayed in viewing area.

Additional filament visualization is achieved by selecting different stylesof filaments (lines, cones or cylinders).

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Selection and editing of filament structure can be achieved by selectingpoint, segment or branch. Depending on selection process thedifferent image parts in viewing area are highlighted.

Image Filament structure representing point, segment or brunch asselected option.

Filament analysis and processing could be performed with automaticfilament selection choosing option Process Selection and ProcessFilament for examination filament structure features.

The Mouse Select and Process Selection Window

Click on the tab Edit .

In the window Mouse Selects you can choose between Point,Segment (filament between two branch points) and Branch (starting

9 - Analyze Neuron

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from the clicked point to all connected end points).

In the the window Process Selection you find the Delete button, andother editing functions which can be restricted to only the part(s) of theFilament which are currently selected.

The Method Window

Click on the tab Draw .

In the Method selection window you can choose between AutoPath,AutoDepth and Manual.

AutoPath

You set the starting point and the end points manually. The filament iscalculated automatically and displays on screen. The button SetSelection as Starting Point(s) is only available in the AutoPath mode.

PC: Mac:

Set the starting point ona volume object

Shift + right-click Shift + Ctrl + click

Selected segment orbranch is set as startingpoint

Left-click on a segmentor branch, then on

Click on a segment orbranch,then on

Analyze Neuron - 9

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Draw the filament Move cursor Move cursor

Fix the filament Shift + left-click Shift + click

Change the "pencil"diameter for drawing thefilament

Mouse wheel Mouse wheel

AutoDepth

You draw each filament manually from starting point to end point. Thedepth is automatically computed.

PC: Mac:

Draw a filament into thevolume

Shift + left-click +drag

Shift + click + drag

Stop drawing Stop movement and release all buttons

Stop movement and release all buttons



Manual

You draw each filament manually from starting point to end point. Youhide the Volume object and draw in one drawing plane, the depth is notautomatically computed unless Automatic Placement is enabled.

PC: Mac:

Draw the filament on theslicer

Shift + left-click +drag

Shift + click + drag

Stop drawing Stop movement andrelease all buttons

Stop movement andrelease all buttons




10 - Volume of Interest

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10 Volume of Interest

Sometimes it is useful to apply a certain function not on the entireimage but only in a region of interest (ROI). In this tutorial you definesuch a volume of interest. The demo data set is a time series of a HeLacell going through all mitotic phases in the cell cycle. In the greenchannel, the mitotic spindle is visualized by EGFP tagged alpha-tubulin.In the red channel, chromatin is visualized by tagged core histone 2B.

You will start with the visualization of the data set. First you Add aVolume reconstruction for both channels, then you manuallygenerate a Contour Surface by Drawing Border Lines in your dataset. Then you let Imaris calculate the complete Contour Surface .Based on this surface, you Create a Surface Object . This Surfaceobject is your "cookie-cutter". Duplicate the original red channel andthen use the "cookie-cutter" to Lay a Mask on this channel. Thefocus of the last chapter is the display of the Masked Channel .

Masked Red Channel, HeLa Cell

User Level: AdvancedModule: Imaris, MeasurementPro

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10.1Visualization


Open the demo image HeLa cell in the Slice view.

PC: \ProgramFiles\Bitplane\Imaris\images\HeLaCell.imsMac:/Applications/Imaris.app/Contents/SharedSupport/images/HeLaCell.ims

Select the Surpass mode. Click on the icon Surpass in the main toolbarof Imaris. To open the Display Adjustment window select in the menuEdit - Show Display Adjustment. Click on Advanced to open theadvanced settings. Set the threshold Range for Ch1 (red) and Ch2(green) to Min:45 and Max:200. The HeLa cell displays in the viewingarea.

HeLa Cell Display in the Viewing Area

See also: Design Mixed Model Rendering - Add Volume and -Display Adjustment , Analyze Neuron - MIP Display

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10.2Add Contour Surface

The Contour Surface allows you to extract a 3D object by manuallydrawing the object contours on 2D slices. You can employ this methodwhenever simple thresholding does not yield individual structures (e.g.touching cells in confocal microscopy, complex tissue recorded bytransmission microscopy).

Add Contour Surface

In the menu Surpass select Contour Surface. The new ContourSurface item displays in the Object List. All parameters on the tab

Settings display in the Contour Surface Properties (on the upperleft hand side of the screen). Adjust the following parameters andsettings.

Select Draw Board and Drawing Mode

A drawing plane displays in the viewing area.

In the Contour Surface Properties select as Draw Board XY, thatmeans you draw the first contour on the xy plane. You can draw thecontours in different draw styles. As drawing mode select Click. How-to-draw a contour is explained in detail in the chapter Volume of Interest- Draw Border Lines .

You can choose Visibility options to display previously drawn contours.Select All to show all contours.

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Change Position of the Drawing Plane/Slice Position

You can either use the slider, enter the position of the plane in therespective data field, or change the drawing plane interactively in theviewing area.

Choose the slice for the first contour by moving the Slider to the firstposition. Alternatively you can change the position of the drawing planeby mouse interaction. Switch to the pointer mode Select and move thedrawing plane by means of the mouse pointer. The border of the activedrawing plane is shown in your selected color, thus clearly indicating theContour Surface currently in use. If more than one Contour Surface hasbeen created in the Object List, the Contour Surfaces not currently inuse have a gray border.

This is the Surpass Viewing Area, Slider in First Position

See also: Volume of Interest - Draw Border Lines 102


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10.3Verify Planes

To verify the image planes you can switch to the Slice view and theGallery view by clicking on the respective icons in the main toolbar.

This is the Image Plane Overview in the Gallery View

Switch back to the Surpass view with a click on the icon Surpass.


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10.4Draw Border Lines

You can draw as many contours on a plane as required. All contoursdrawn on a plane will become part of the same object.

Draw a Polygon Line on the First Plane

Change to the pointer mode Select (press the ESC-key), hold down theShift-key and click with the mouse on the desired first position to insertthe first point. Move the mouse to the next point of the polygon, holddown the Shift-key and click with the mouse to insert the next point, andso on.

Copy and Paste Polygon

Copy or draw on each image plane a border line. If you want to copy the


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same polygon to the next image plane do the following:

With a click on the button Copy you can copy the polygon line.

Change to next plane by moving the Slider to the next position.

With a click on the button Paste the same polygon line is placed to thesecond plane.

Polygon Line on the First and Second Plane

Change to the Next Plane

Change to next plane by moving the Slider to the next position. On thenext plane you can either paste the same polygon line again, or youdraw an individual polygon line, as described on top of this chapter. Goon with the next plane, and so on until on each plane there is a borderline.



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10.5Edit Polygon

To edit the polygon lines return to the slice on which the polygon isdrawn.

PC: Mac:

Move node

To move a node you canclick on the node whileholding down the Shift-keyand simultaneously dragthe mouse.

To move a node you can clickon the node while holdingdown the Shift-key andsimultaneously drag themouse.

Insert nodeinto polygon

Hold down the Shift-keyand click on the linebetween two nodes.

Hold down the Shift-key andclick on the line between twonodes.

Delete node

Hold down the Ctrl-keyand double-click with theleft mouse button on thenode.

Hold down the Command-keyand double-click on the node.

Deletepolygon

Hold down the Ctrl-keyand double-click with theleft mouse button on a linebetween two nodes.

Hold down the Command-keyand double-click on a linebetween two nodes.

Change size

In the drawing mode, thesize of the vertices as wellas the connecting linescan be increased ordecreased by repeatedlypressing the + key or keyon the numerical keypad.

In the drawing mode, the sizeof the vertices as well as theconnecting lines can beincreased or decreased byrepeatedly pressing the + keyor key on the numericalkeypad.

The process of drawing the contour can be interrupted and continuedlater. To change the position of the object while drawing the contour,switch the pointer to Navigate mode, move to the required location, andswitch the pointer back to Select.

Tip: You can easily switch between the two pointer modes using theESC-Key. The effect is directly visible on screen by the altered mousepointer display.



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10.6Calculate Contour Surface

In the next step you calculate the new Contour Surface. Click on thebutton Calculate Surface.

You can change to the pointer mode Navigate (press the ESC-key) andnavigate the Contour Surface to any desired position.



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10.7Create Surface Object

In the next step you create new Surfaces.

Click on the button Create Surfaces.

In the SurpassTree the new item Surfaces1_of_ContourSurface1appears.

You can change to the pointer mode Navigate (press the ESC-key) andnavigate the new Surfaces to any desired position.



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10.8Mask Channel

Sometimes it is useful to apply a certain function not on the entireimage but only in a region of interest (ROI). As region of interest youcan take the Surface object. Be sure that in the Object List the Surfacesobject is highlighted and click on the tab Edit.

Click in the Mask Properties on the button Mask All ... .

Window Mask Channel

The Surfaces object is your "cookie-cutter", the selected channel your"cake mixture". You duplicate the original red channel and then you usethe "cookie-cutter" to mask this channel. In the following you will cut outthe red area to see only the inside of the the Surfaces object. To getthis result you will exclude all voxels outside the "cookie-cutter" (set thevoxels outside the surface to zero).


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Select the channel (destination channel) you want to mask with theSurface object (ROI). In this case you select the red channel,Channel 1 - Ch1. Check the box Duplicate Channel before applying Mask and an

additional masking channel displays automatically in the DisplayAdjustments window. Select Constant inside/outside and check the box Set voxels

Outside Surface to: and use the default value 0.000. This means,that no voxels outside the region of interest are displayed. Leave the next box un-checked (Set voxels Inside Surface to:). The

original channel intensities inside the region of interest are displayed. Check the box Apply to all time points. Click on the button OK to apply the mask.

See also: Design Mixed Model Rendering - Display Adjustment 44


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10.9Display Masked Channel

To display only the masked channel in the viewing area you have toadjust the channel display in the Display Adjustment window.Additionally you have to set up the visibility of the Object List items inthe viewing area.

Display Adjustment Window

To open the Display Adjustment window select the menu Edit - ShowDisplay Adjustment.

In the Display Adjustment window you can find the additional channelmasked Ch1.

Un-ckeck the original red and green channel. Adjust the upperthreshold for the new channel masked Ch1 (move the right arrow thatrepresents the upper threshold to the left).

Adjust Object List

In the Object List check the item Volume and un-check the ContourSurface and the Surfaces. As the result of the masking everythingoutside the surface is cut away and only the inside voxels are visible.


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The initial object for the masking is the Contour object. Based on thisContour object you create a Surface object. Then you use this Surfaceobject as the region of interest ("cookie-cutter") for the masking.

New, Modified Red Channel


See also: Design Mixed Model Rendering - Surpass View andDisplay Adjustment

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11 - Volume over Time

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11 Volume over Time

In this tutorial you analyze the chromosome volume over time in amitotic cell. The demo data set is a time series of a HeLa cell goingthrough all mitotic phases in the cell cycle. In the green channel, themitotic spindle is visualized by EGFP tagged alpha-tubulin. In the redchannel, chromatin is visualized by tagged core histone 2B. You willstart with the visualization of the data set. First you Add a Volume reconstruction for both channels, then yougenerate an Surfaces reconstruction of the chromosomes. In thechapter Time Concept you will learn how-to-handle time series inImaris. Then you Calculate and export statistical data for each timepoint. The focus of the last chapter is the Presentation of the Results

in MS Excel.

Statistical Data, HeLa Cell

User Level: AdvancedModule: Imaris, MeasurementPro, Tracking

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Volume over Time - 11

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11.1Visualization


Open the demo image HeLa cell in the Slice view.PC: \ProgramFiles\Bitplane\Imaris\images\HeLaCell.imsMac:/Applications/Imaris.app/Contents/SharedSupport/images/HeLaCell.ims

To select the Surpass mode click on the icon Surpass in the maintoolbar of Imaris. The HeLa cell displayed. To open the Display Adjustment window select in the menu Edit -Show Display Adjustment. Click on Advanced to open the advancedsettings. Set the threshold Range for for Ch1 (red) and Ch2 (green) toMin:45 and Max:200. The HeLa cell displays in the viewing area.

See also: Design Mixed Model Rendering - Add Volume , DesignMixed Model Rendering - Display Adjustment , Analyze Neuron - MIPDisplay

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11.2Add Surfaces

In the Objects toolbar of the Surpass view click on the icon to adda new Surfaces item.


For algorithm setting select

quick start tutorials - emory university department of...

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