AxioVision 4.5 Brightfield Image Capture Procedure 1. STARTING-UP PROCEDURE: Remove blue dust cover and place on shelf under microscope. Turn on the halogen lamp by pushing the switch at the back right of the microscope. 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 Cardiology network 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 camera path. Select Position #5 on the filter cube turret (empty). 3. SETTING UP KOEHLER ILLUMINATION: Set the halogen lamp to 12 volts by turning up to the top value, "10, or push the black button beneath the voltage setting to achieve 3200 deg K (best). On the lower right side of microscope, set the neutral density filters: 0-1.5 Set to 1.5. (% transmission) 0-6 Set to 6. (% transmission) 0-25 Set to 0. You may use 25, but it is not necessary. Diffuser: Push to the back to swing the diffuser in. Be sure that the blue swing-in filter under the stage is in place. Make sure the moveable lens under the specimen stage is swung in when using all objectives higher than 5X. Place a high contrast specimen on the stage (H&E is good), and focus with the chosen objective. Raise the condenser (under the stage) to the upper stop position. Close the luminous field diaphragm (the large round lens at the base of the microscope) by turning the outer ring until the diaphragm is visible in the field of view. Lower the condenser until the luminous field diaphragm becomes a 12-sided figure with clear edges in focus. 1
Open the luminous field diaphragm until it almost fills the field of view but the edges are still visible. Center the luminous field diaphragm by using the two metal centering screws on the condenser carrier. Open the luminous field diaphragm until the edges exactly disappear beyond the field of view. Set the aperture diaphragm: Remove one eyepiece from the binocular tube. While looking down the eyepiece opening, slide the knob on the front of the condenser until the aperture diaphragm fills 2/3 to 4/5 of the diameter of the objective exit pupils. Because each objective has a different field size and objective aperture, you will have to set the Koehler Illumination for each change of objective. 4. STARTING THE SOFTWARE: Start AxioVision Rel. 4.5 on the computer and activate the Live image. Select the Speed of the camera. The Slow Speed gives the best detail and the Fast is most pixilated. At the bottom of the Live-AxioCam HR screen, these display icons are shown after the Snap, Camera Speed, Exposure, 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 preset display for color closest to what you see when you look through the microscope. Use the Gamma pre-set display to perform the Shading Correction and the White Balance. You may change to other display options later. 5. SETTING THE SHADING CORRECTION: Activate the Properties window from the Live-AxioCam HR window menu, from Acquisition in the menu bar at the top of the screen or from the Workflow bar on the left of the screen Select Adjust tab and set the exposure time to 20 to 50 ms. Increase as needed. Select Frame, choose RGB (or B/W). Select Resolution, usually 1300x1030 scanned, from drop-down menu. 2
Select General, set the Gain factor to "1," Index to "0. Perform the Shading Correction first, each time you change objectives. Focus on a clean, clear area of the slide. If there is no clean area, you may remove the slide completely. On Live-AxioCam HR/General, click on the Shading Correction to smooth out the background. Some color may be seen at this point, but it is temporary. A message will appear if it is too dark or too bright. Increase or decrease exposure time under Live Properties/Adjust. Click on the Shading Correction box again. Repeat until the background is even with no spots or artifacts. 6. SETTING THE WHITE BALANCE: Focus on the specimen and select Adjust again. Now do a White Balance to attain the best color representation of the specimen by one of two methods: (a) Select Interactive and hold the crosshairs on a white area. This will give a readout of the values for red, green, and blue. Choose the best spot and release the mouse. (b) If there is no white area on the specimen at the region of interest, click on 3200 K first, wait about 20 sec. for color change, then click Automatic. You may also click on the White Balance icon at the bottom of the Live window. 7. ADJUSTING THE EXPOSURE: After the White Balance is done, increase the exposure as needed by increasing the exposure setting under the Adjust tab until the background is an appropriate shade of white for your specimen. You may also do an automatic exposure measurement by checking Automatic and then click Measure. Change the exposure time if necessary until the background is white enough for your application. Disable (uncheck) the automatic to use your own settings or the software will use automatic. You must repeat the SHADING CORRECTION and WHITE BALANCE every time you change objectives if you are capturing images, even if you are going back to one done previously. 3
8. FOCUSING ON THE SPECIMEN: While looking at the computer screen, adjust the fine focus knob slowly, then pause a couple seconds before continuing to adjust the focus. Right click on the live image to obtain the Spot meter/focus bar. When the green color comes to the red line, the software thinks best focus is achieved. Trust your eyes, not the focus bar. When the Spot meter is activated, a green frame appears around the live window. You may use this frame to enclose the region of interest (ROI) on which you want to focus by dragging the handles of the frame. 9. SELECTING THE OBJECTIVE AND RESOLUTION: On the Live-AxioCam HR window menu, select the Objective and Resolution from the dropdown menu. This will be saved with your image, and you will be able to add a scale bar by just selecting the scale bar icon. 10. CAPTURING THE IMAGE: Capture the image by using the "Snap" at the bottom of the Live- AxioCam HR image window, or by clicking any Camera Icon. 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. Then, after processing, save it with a new name or add "edited" to the name. Do not save images on the C Drive. Save on the D Drive or local network. 11. SAVING THE IMAGE: Save the image using File/Save As from the File menu. You may select from these four formats to store the images:.zvi This format allows you to store all data microscope, user, date, annotations and measurement together in a single file. You should save your original best image as a.zvi and export as a.tif. Keep an archival copy of both..tif With these external formats, AxioVision now stores all additional.jpg information separately from the actual image data. To do this, an.bmp XML file is created for every image file that contains other information such as additional data, annotations, and interactive measurement data. 4
12. USING THE SPLITTER SCREEN: Compare saved images side by side in the Splitter display: Select from the menu at the top of the screen View/Windows/ Splitter. All the opened images will be in the Gallery. In the Splitter display choose from two to eight screens. Click on Gallery. Select from the small gallery window at the top of each screen by clicking the image. Use the small red frame in the lower right corner of the Splitter display to change the size and region of the images in the Splitter Display. Right click to zoom up or back to change the size of the images. All the images in the Splitter display may be saved as a combined single image by clicking on "Create Image. 13. ADDING AND EDITING SCALE BARS: You may add a Scale Bar before or after the merged image is saved. If you selected the appropriate objective in the 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. 14. EXPORTING IMAGES: Export by going to File/Export. An export window will appear. Export as a.tif image to a local folder, network folder, CD, or memory stick. Check "Burn in annotations" to save scalebars, etc. You will not be able to remove these annotations once they have been exported. Export as a batch by clicking on Batch, then click Add Files. Select files to be exported. The file names will appear in the window on the right. Click on Run Batch. As the files are exported, an "OK" message or an error message appears. Error messages are usually from incorrect syntax in the file name. Exported files should be saved to your folder in drive D or in a local network folder. 5
Set up the path before you export! 15. POST-PROCESSING YOUR IMAGES: Post-processing can be done on the saved images by selecting Show Properties and Display or by activating Processing from the menu at the top of the screen, then using its drop-down menu. The editing done with this dropdown menu takes some time to appear. There are several measuring tools available in this software that you may want to use. You may also measure your images with Image Pro 5.2 on the computer in WMB 306. You must get the USB dongle from Debby Martinson in WMB 303 or Lu Hilenski in WMB 329 to use the Image Pro software. 6
16. SHUTTING-DOWN PROCEDURE: Exit the AxioVision Rel. 4.5 from the File menu. Check to see if the next person is signed up to do fluorescence imaging. If so, then leave the lamp turned on and the camera plugged in. 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. Turn off the computer now a one step process. From the start menu, choose SHUTDOWN. The new monitor can be left on. Disconnect the camera computer cable. Swing a low power objective (5X or 10X) into the light path. Turn off the power supply for the mercury lamp by pushing the on/off switch. Put the blue dust cover back on the microscope, but not over the hot lamp. CLEAN UP THE AREA. You may log on the local Cardiology network and save directly to your own local shared folder. Please delete your files from the computer! 7