Virtual Tissue (slide scanning): Troubleshooting imaging issues (Brightfield)

Problem 1:

Dark image 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:

Bright specks consistently spaced throughout the image

Background image contains 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.
  • Click the Acquire Image icon

Problem:

Over-saturated or “washed out" image

Light level is too high

Solution:

• Turn down the light level and/or exposure and perform the Clip Detect process again.
• Ensure that the background image and live image are acquired at the same light level.

Problem:

Distinct horizontal and vertical seam lines at every image tile

No background image or background image with uneven lighting

Solution:

• Try enabling background correction (Acquisition>Enable Background Correction) or acquire another background image.
• Verify that lighting is uniform, especially around the edges of the image.

Problem:

“Doubling” or blurring along horizontal or vertical seam lines

Insufficient microscope calibration/lack of sufficient trim.

Solution:

• Verify that the microscope is properly calibrated (i.e., camera is aligned, lenses are grid-tuned, stage movements are functioning properly) - Virtual Tissue (slide scanning): Fine-tuning calibration
• Turn up the trim on the appropriate edges under the Virtual Tissue Trim/Blend Options dialog box.
• If trimming does not work, a fine-tune manual calibration may be needed.

Problem:

Large out of focus areas in the image

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:

Very distinct tile lines. Individual tiles are not lined up.

Mismatched objective and software lens

Solution:

• Verify that the lens selected in the software’s drop-down menu matches the objective selected on the microscope .