Capturing a brightfield background image for slide scanning

Purpose

Once you've adjusted the camera settings, it is useful to capture a background correction image over an empty region of the slide to correct for uneven illumination and to reduce image-tiling unevenness.

Procedure

  1. Navigate to an area on the tissue where some white area is visible. In the Camera Histogram window, check Clip Detect. Clip Detect fills any white areas that are overexposed in the field of view with dark red pixels.

    If nothing appears when it is enabled, raise the exposure or light level until the red pixels appear; keep the exposure between 10 and 100 ms.

  2. Adjust the exposure to allow for only a “light dusting” of red pixels in the white areas.
  3. Once a light dusting of red pixels has been achieved, perform another white balance.
  4. When the white balance is finished, you should still see a light dusting of red pixels in the white area.

    If more or less pixels appear after the white balance is complete, adjust the exposure and try again until the amount of pixels is roughly the same as before the white balance.

  5. When finished, uncheck Clip Detect to turn it off.
  6. Using Joy Track and the joystick, navigate completely off the tissue to a blank space on the slide. To obtain a background image that is completely white, make sure that the field of view is free of tissue or debris.
  7. Click Acquire>BG Acquire.
  8. Select the Type of background correction, check Enable background correction, and click OK.

    If you see a message indicating that the image is saturated, lower the light level or exposure. Make some slight adjustments and try again. The most recent background image captured is automatically the one used for the acquisition, even if multiple background images appear in the image organizer.

  9. Once the background image has been captured, it is displayed in the image organizer.
    • Ensure that it appears completely white and free of debris (sometimes debris that were not visible in the field of view can appear in the image).
    • If there is debris, slightly adjust the location of the background image to away from the debris and acquire the image again.
  10. When the background image is satisfactory, return to the live image.

 

If all the acquisitions are performed at the same magnification and with the same tissue, a single background image can often be used for all images. Keep the background image “open” for each new image. A new background image needs to be acquired when there are changes to the tissue, slide, magnification, or light level.