axiovision 4.8.1 image capture for fluorescent...

Hilenski 2013 Microscopy in Medicine Core (MiM) website: http://medicine.emory.edu/MIMCore

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AxioVision 4.8.1 Image Capture for Fluorescent Dyes

Images of Axioskop 2 microscope are from the following website: http://www.einstein.yu.edu/aif/instructions/zeiss/axioskop2/index.htm

1. STARTING-UP PROCEDURE:

• Remove blue dust cover and place on shelf under microscope. • Turn on the X-Cite lamp FIRST—ALWAYS! • Turn on the computer by pushing the button on the top front right side. • Connect the camera computer cable to the back of the camera. • Log on to the Emory network (EMORYUNIVAD) using your login name

and password. 2. USING A CLEAN, DRY SLIDE:

• Clean your slide with 70% ethanol using Q-tips and Kimwipes if it has fingerprints or dust on it.

• Make sure AxioCam cable has been hooked up to camera for at least 15 minutes.

• Open the light path to the eyepieces (push in Beam Splitter--see below) to find an area on your slide you wish to image.

http://www.einstein.yu.edu/aif/instructions/zeiss/axioskop2/index.htm 3. STARTING THE SOFTWARE:

• Start AxioVision Rel. 4.8 on the computer desktop and activate the AxioCamHR:Live image by clicking on Live at the top of the window. Open the light path to the camera (pull out Beam Splitter--see above)

http://medicine.emory.edu/MIMCore

http://www.einstein.yu.edu/aif/instructions/zeiss/axioskop2/index.htm

http://www.einstein.yu.edu/aif/instructions/zeiss/axioskop2/index.htm


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• Select the Speed of the camera. The Slow Speed gives the best detail and the Fast is most pixilated. Right click on the image to show the green focusing bar. Maximum focus results when the bar contacts the red line.

At the bottom of the Live Image screen, these display icons are shown after the Snap, Camera Speed, Exposure and White balance analysis icons:

The first icon on the left is the Best Fit (usually the most vivid). The next one is the Min/Max. Next is Linear (full range of possible values). 4th icon is Gamma display for color closest to what you see when

you look through the microscope. At the bottom of the AxioCamHR:Live window, click on the Linear

icon. You cannot get a black reference that is truly black unless Linear is activated.

In the AxioCamHR:Live window, activate Properties to show the AxioCamHR window.

4. BLACK REFERENCE:

• Perform a Black Reference approximately 15 minutes after attaching the cable to the camera, as the camera will then be warmed up. The Black Reference ensures that the image background retains a uniform darkness, even with exposure times of several seconds.

1. Close the light path to the camera (Beam Splitter IN). 2. Close the shutter for the X-Cite lamp (Fluorescence Slider

IN). 3. Turn off the halogen lamp (if on). 4. Turn off the room lights, except for red lights.

• Click on Black Reference from the AxioCamHR/General tab and a

window will show you the progress. Make sure the Shading Correction is



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not checked unless you plan to merge brightfield images with fluorescent images. In that case you should now do a Shading Correction and a White balance with the halogen lamp on by following the AxioVision 4.8 Brightfield directions.

• Open the light path to the camera (Beam Splitter OUT).

5. SETTING SCANNING PARAMETERS:

• Go to Frame tab in AxioCamHR, select RGB (or B/W for monochrome) and select the Resolution, usually 1300x1030 standard color.



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• In the AxioCamHR:Live window, select Objective and Resolution in current use in dropdown menu. The objective and resolution chosen here will be saved as a text file with your image.

• In AxioCamHR:Live window, choose Frame Capture Speed. The Slow speed is the easiest to focus.

• Select the right filter set by turning the turret wheel above the objectives to these positions: #1 is for Rhodamine Red X with excitation at 570 nm and emission

at 620 nm. #2 is for FITC with excitation at 480 nm and emission at 535 nm. #3 is for DAPI or Hoechst with excitation at 360 nm and emission at

all wavelengths. #4 is for Quantum dots with excitation at 460 nm and a long pass

emission >= 500 nm. #5 is usually empty for use with transmitted light. Other filter sets

can be installed: Cy5 with excitation at 620 nm and emission at 700 nm TRITC (rhodamine) with excitation at 545 and emission at

610 nm. 6. SETTING UP NEW EXPERIMENT:

• Go to the tab marked "C" on the Multidimensional Acquisition window.

• A number "1" appears in the Settings section indicating Channel 1. • Go to the Dye dropdown menu and select dye. • The color will appear. The dropdown menu allows a color change.



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• The Name box contains the dye name. You may type a more accurate name or choose a more accurate color from the dropdown menus.

• To select another dye, go to the Channel Actions section at the bottom of the window and click on Duplicate. The next number appears at the top of the Settings section with the same color as the last dye but changes when you select the next dye. Select Remove to eliminate a dye.

• Repeat above steps for each channel selected. You may have up to eight channels per image. A Brightfield channel is available. When you take a brightfield image, Koehler illumination, Shading Correction and White balance must be performed for each objective used before capturing the brightfield image. See AxioVision 4.8 Brightfield instructions.

7. ADJUSTING THE EXPOSURE:

• Go to the Exposure section of tab "C" and determine exposure type and time. For each dye to be used, show the camera the sample and adjust the exposure time under the Adjust tab exposure setting in the AxioCamHR window. When you think you have the right exposure, you may enter it and check "Fixed" in the Exposure section of the Multidimensional Acquisition window or click Measure to see what the software chooses as the exposure time.



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• There are THREE ways to adjust exposure:

Camera: The exposure time entered on the AxioCamHR page. Adjust tab is used. The time entered in the Time field of tab "C" is ignored.

Fixed: The time entered in the Time field is used. Any value

entered on the AxioCamHR adjust tab page is ignored. For each set of slides from one experiment, choose a good positive to use to set the exposure times for each dye and enter times as fixed. All positive and negative controls and experiment images must be captured with the same exposure settings. Always use the Fixed option for each experiment set.

Auto: The camera's automatic exposure time is used. See

"Interactive Exposure." This exposure time will vary for each slide. Interactive Exposure Determination

Select dye and set correct filter on the turret wheel, then proceed. 1. Click on Measure button under Exposure section under tab "C." 2. A live image box appears with various icons at the bottom. 3. Increase the window size to your liking. 4. Use the ROI icon (a frame) to get a region of interest frame to choose the area or cell on which you want to base your exposure time. 5. Click on the Measure button in the auto measure window. If you want to use this exposure time, click OK in the Measure box and it will be entered in the time field of the Exposure section in tab "C" as Fixed. You may change the exposure time by adjusting the time up or down from that auto value and then click OK. Make sure that the Fixed button is selected.



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Close the shutter after exposure time is selected! Activate the Extended Parameters box to verify your settings. NOTE: The exposure times will be different for each channel!

• Go back to the Multidimensional Acquisition tab. Under the Experiment section, click on the Save dropdown menu. Choose Save As.

A window appears called "Save Experiment As" with a folder that the software will use named "Experiment." The name should include exposure type, channels, and objective (Fixed_FITC_Rho_DAPI_40x).



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This will be a .zvx file. Save the .zvx file in My Computer>Desktop>My Documents>Carl Zeiss>Data>Experiment.

8. CAPTURING THE IMAGE

• If you have saved the experiment, load Experiment from Experiment tab, then go to tab C. You may change exposure settings for a new experiment and Save As with a new name under Experiment tab.

• Move filter wheel to desired filter for channel chosen to use for focus. • Open the shutter to the X-Cite lamp light path. • Focus image of the dye of primary interest while looking at the monitor.

This dye is the only one on which you can set the focus! Make focus adjustments slowly and wait two or three seconds before turning focus wheel again. The Spot Meter or focus bar is available from the menu that comes up when you right click on the live image. When the green bar contacts the red line, maximum focus is achieved. The focus must remain the same for all channels so that the images can be merged. Focus on the most important dye selected only.

• Snap the first image using only the Snap button in the Multidimensional window.

• Activate the next channel and move turret wheel to the correct filter cube.

• Snap the image. Repeat above steps for all channels for this experiment. The merged image will be generated as you snap each image. There are three windows showing the process.

• Close the shutter after the last snap. • The image below shows three channels: FITC/Rhodamine/DAPI.



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Image of vascular smooth muscle cells labeled with FITC phalloidin (F-actin green), caveolin (Rhodamine Red X secondary antibody) and DAPI (nucleus blue). 9. ADDING A SCALEBAR:

• You may add a Scale Bar before or after the merged image is saved. If you selected the appropriate objective in the AxioCamHR:Live window dropdown menu, just select the scale bar icon and place it on the image. Otherwise, you must select from the top menu Measure/Scalings.

• To edit the Scalebar, go to the Attributes tab of Show Properties, available from the bottom menu of the image window, or from the workflow icons on the left of the screen. Highlight Scalebar. The line, color and font sizes can be edited here and saved as a default setting for all your scale bars.

• Undo is available from the Edit menu at the top of the screen.

NOTE: Images submitted for publication are being scrutinized carefully because of an increasing problem with fraudulent images made with editing! You may do post-processing of your image before you save it, but the best practice is to save your image as captured as a .zvi, not a .tif. When editing, edit only using histogram stretch, brightness/contrast, and gamma. Then save it with a new name or add "ed" or" edited" to the name. See the notebook with AxioVision instructions for articles on the editing problem. 10. SAVING AN IMAGE:

• Do not save images to the C or D Drives. Save only to your Emory server folder.



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• Save the image using File/Save As from the file menu. You may select from four formats to store the images: .zvi This format allows you to store all data belonging to an

image in a single file. Microscope, user, date, annotations and measurement data are stored together. This will save all data; saving original in any other format could cause loss of data.

.tif, .jpg, .bmp AxioVision now stores all additional information separately from the actual image data. To do this, an XML file is created for every image file that contains all additional information For multidimensional images, individual images have to be saved. The first image is always stored in the chosen folder, and an additional sub-folder is created with all the other images and the XML file. When you reload the images in AxioVision, multidimensional images are also assembled correctly.

11. EXPORTING AN IMAGE:

• File/Export as a .tif (recommended) image to a local folder, local network folder or CD/DVD.

• Several File types of export files are available in the export window. Select one (.tif is recommended).

• Select "Burn in annotations" to save scalebars, etc. You will not be able to remove these annotations once they have been exported. The exported files will be saved to your selected folder.

• Set up your path for saving exported files before you start exporting. • You may also export all files as a batch, but export one by itself when it is

open in order to set up the way you want all files to be exported. Make sure all channels are selected and the image window

channels are "ON.” Select "Batch" then click "Add Files.” Select files from your folder to be exported and these will appear in the window on the right.

Click "Run Batch.” As the files are exported, an "OK" message or an "Error" message appears. Error messages usually are

caused by incorrect syntax in the file name. 12. SHUTTING-DOWN PROCEDURE:

• ALWAYS Exit the AxioVision Rel. 4.8.1 from the File menu. • Log off the computer but do not turn the computer off. • If you are the last person to use the system today, shut down the

computer but MAKE SURE THAT NO ONE ELSE IS SIGNED UP TO USE FLUORESCENCE.

• Turn off the computer—now a one step process. From the start menu, choose SHUTDOWN. The monitor can be left on.

• Disconnect the camera computer cable. • Swing a low power objective (5X or 10X) into the light path.



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• Turn off the power supply for the X-Cite lamp by pushing the on/off switch ONLY if no one else is signed up to use fluorescence.

• Put the blue dust cover back on the microscope, but not over the hot lamp.

• CLEAN UP THE AREA. You can download a free version of the AxioVision LE software at the following website: http://microscopy.zeiss.com/microscopy/en_de/home.html Click on Downloads>AxioVision>Download Microscope Software AxioVision LE. If you do not have Administrator rights to your lab computer, you must contact the DOM IT ([email protected]) or 404-712-1443 for help with installation of the free AxioVision LE software.

REMEMBER! IMAGES SAVED ON ANY LOCAL COMPUTER DRIVES

WILL BE PERIODICALLY DELETED BY THE MiM CORE STAFF WITHOUT NOTIFICATION. YOU ARE

RESPONSIBLE FOR SAVING YOUR IMAGES.


http://microscopy.zeiss.com/microscopy/en_de/home.html