Maintenance & Troubleshooting
The imaging system is a hybrid of optical hardware (camera/microscope) and computer software. Failures can occur at the interface between the two. Regular maintenance ensures data integrity and image consistency
Routine Maintenance
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Calibration (Scaling)
- The software must know the relationship between pixels and micrometers (e.g., 100 pixels = 1 \(\mu\text{m}\))
- Frequency: Annually or after any hardware change (new camera or adapter)
- Tool: A Stage Micrometer (a slide with a certified ruler etched into it) is captured, and the software is calibrated to this known distance. This ensures that genome length measurements are accurate
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Shading Correction (Blank Field)
- Frequency: Daily
- Procedure: Capture an image of a blank area of the slide. The software saves this as the “background map” and subtracts it from all subsequent captures to remove vignetting and dust shadows
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Data Backup
- Cytogenetic images are large files. The database must be backed up daily to a secure server. Loss of patient images is a critical regulatory failure
Troubleshooting Common Issues
Problem 1: Image is grainy or “noisy”
- Diagnosis: Low Signal-to-Noise Ratio
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Cause 1: Microscope light is too dim. The camera is boosting electronic Gain (ISO) to compensate, which amplifies noise
- Fix: Turn up the microscope voltage (brightness) to maximum. Reduce the camera’s Gain setting
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Cause 2: Exposure time is too short
- Fix: Increase exposure time (e.g., from 10ms to 50ms)
Problem 2: “Hot Spots” or Bright Center
- Diagnosis: Uneven Illumination
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Cause: Condenser is not centered or Field Diaphragm is closed too much
- Fix: Perform Kohler illumination
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Cause: Shading correction is old or invalid
- Fix: Recalibrate the background (Blank Field)
Problem 3: Chromosomes look “washed out” (Low Contrast)
- Diagnosis: Poor Histogram
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Cause 1: Green Filter is missing.
- Fix: Insert green filter
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Cause 2: Condenser Iris is too open (glare)
- Fix: Close iris to 75%
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Cause 3: Gamma setting in software is incorrect
- Fix: Reset software display defaults
Problem 4: Image on screen is blurry, but sharp in eyepieces
- Diagnosis: Parfocality Error
- Cause: The camera’s focal plane is different from the eyepiece focal plane
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Fix:
- Focus the microscope sharply while looking at the Computer Monitor
- Without touching the focus knob, adjust the diopter rings on the Eyepieces: until the image is sharp for your eyes. Now they are synchronized