Slide scanning: Troubleshooting for brightfield
Seven common problems and their solutions are outlined in the slideshow below.
Problem 1: The image is dark which is probably due to poor lighting.
Solution:
- Adjust light level and/or exposure level (see camera settings).
- Acquire a new background image.
Ensure that live image and background image are acquired at the same light level.
Problem 2: There are bright specks consistently spaced throughout the image which suggests the presence of debris.
Solution:
- Acquire another background image free of debris.
- Acquire a quick test image in the current field of view to ensure that the background image is suitable.
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 Detect again.
- 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:
- Try enabling background correction (Acquire>Background Correction>Apply) or capture another background image.
- 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: trim/blend options.
- If trimming does not work, a fine-tune manual calibration may be needed.
Problem: There are large out-of-focus areas in the image, most likely because there were not enough focus sites.
Solution:
- Try adding more focus sites to the 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.
Problem: There are out-of-focus tiles dispersed throughout an otherwise in-focus image. In our experience, this occurs with extremely “wavy” tissue with drastic differences in z-depth between focus sites. The focus map interpolation cannot account for these large differences resulting in out-of-focus tiles.
Solution: Try increasing the number of focus sites in the focus map to create a tighter triangulation grid.