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Watch a video demonstration of adjusting the scanning parameters:
A) Adjust the scanning parameters:
After starting the manualWSI software and selecting the camera, the first step is setting up the scanning parameters.
00. Place the microscope slide on the stage.
01. engage the desired objective lens for scanning.
02. if your microscope is using halogen illumination, ensure to engage the light balancing filter (daylight filter). Additionally, the light source should have been switched on for at least 5 minutes (warm up time) to stabilize color and intensity of the illumination.
03. locate the sample and bring it into focus using the eyepieces.
04. check if the sample can be seen on the live camera frame already.
05. Set the microscope illumination to the max and direct as much light as possible to the camera. Some trinocular photo tubes allow directing different amounts of light to the camera and the eyepieces (e.g. 100%/0% 80%/20% 0%/100%).
06. if the camera images is white, reduce Gain to 0dB and then reduce Brightness (= camera shutter time) until the sample can be seen on the camera.
07. refocus the sample (focus of camera and focus of the eyepieces do not match exactly).
08. move to the edge of the sample until 90% of the camera image is empty/white background and 10% is sample.
08.a. Make sure that the illumination is centered. Read more information on centering illumination.
09. Adjust brightness and gain until the three peaks of the histograms can be seen completely without being cut off at the right edge.
09.a. For hematology scanning, make sure to adjust the cameras color settings accordingly.
10. For some cameras, the green color gain is fixed. Adjust the brightness and Gain of the camera to bring the green peak in the histogram as close as possible to the right edge without cutting it off.
11. Higher Gain and Brightness values both increase the brightness of the image. Higher Gain values introduce more noise into the images. Higher Brightness values introduce more motion blur into the images. Try to keep Gain below 10dB and Brightness below 250 micro seconds.
12. The brightness can be varied by adjusting the sub stage condensers numerical aperture (N.A.), too.
13. Then adjust red and blue gain until the red and blue peaks match the location of the green peak in the histogram as close as possible.
14. Store the settings (see section C below; currently not shown in the video)
B) Save the presets
In newer software versions, it is possible to save configurations for each objective lens magnification (and even for different stains).
00. Adjust the scanning parameters as described in section B above.
01. Press the "+" button to add a new preset. Now the dropdown list turns into a text box.
02. Enter a name for the setting (e.g. "10x 0.25N.A. HE")
03. Press the ENTER or RETURN key on the keyboard to save the setting
C) Recall the presets
00. Engage the desired objective lens in the light path. (e.g. 10x)
01. Select the corresponding preset from the list of stored settings.
02. Check that the brightness of the image is OK
03. If the image does not look as expected, check that
- the sub stage condenser aperture is set correctly
- the brightness of the microscope is set correctly
- the maximum of the light is sent to the camera on trinocular heads
- check that the halogen lamp has been warmed up for a few minutes
D) Update the presets (in the case that microscope conditions changed)
00. When halogen bulb is getting old or other scanning conditions changed, existing settings should be updated.
01. Select the desired preset
02. Fine tune the settings
03. Press the update button to update the existing preset setting
E) Delete presets
00. Select a preset that is not used anymore
01. Press "-" button
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