Slide scanning: Troubleshooting for brightfield

Problem: The image appears washed out; the over-saturation is probably caused by an excessive light level.

Solution:

• Turn down the light level and/or exposure and use Clip Detection again (see Step 6: Background correction).
• Ensure that the background image and live image are acquired at the same light level.

Problem: There are distinct horizontal and vertical seam lines on every image tile because either no background image was used, or the background image has uneven lighting.

Solution:

• Capture another background image (see Step 6: Background correction).
• Verify that lighting is uniform, especially around the edges of the image.

Problem: There is "doubling" or blurring along horizontal and vertical seam lines, caused by either an incorrect microscope calibration or an insufficient trim.

Solution:

• Verify that the microscope is properly calibrated (i.e., the camera is aligned, lenses are calibrated, stage movements are functioning properly) - See Slide scanning: Fine-tune calibration
• Turn up the trim on the appropriate edges using the Slide scanning settings: pixel trim images and seam blending options (see Step 7: Scan settings).
• If trimming does not work, a fine-tune manual calibration may be needed.

problem 1: Thermal drift.

Solution: Power your hardware on at least 2 hours before you set up your focus map and make sure that there is no hot or cold air blowing on it.

Problem 2: There were not enough focus sites.

Solution:

• Try adding more focus sites to the focus map (see Step 9: Define focus map).
• Verify that the focus sites are consistently dispersed throughout the contour or grid.
• If stage drift is the issue (and you allowed your microscope to warm up prior to acquisition), modify the focus adjustments settings by adding a manual focus interval. A manual focus interval enables you to adjust the focus during the imaging process, and to periodically update the focus map to compensate for drift.
  • First, try adding a manual focus 3-4 times throughout the image and see if it helps (e.g., if the image has 2000 tiles, add a manual focus every 500 sites).
  • Adjust as needed.

Problem: There are very distinct tile lines and individual tiles are not lined up. This is likely caused by using mismatched objective and software lens.

Solution: Verify that the lens selected in the software matches the objective selected on the microscope.