Skip to main content

Cytogenetics

Imaging System

The transition from optical microscopy to digital karyotyping relies on the Imaging System. This workstation captures the analog view, digitizes it, and provides tools for analysis

Capture Images

  • Hardware: A high-resolution monochrome CCD/CMOS camera mounted to the microscope. Monochrome is preferred for G-banding (better grey sensitivity)
  • Procedure (G-band)
    1. Focus: Must be sharp on the screen (not just eyes)
    2. Filter: Ensure Green Filter is used for contrast
    3. Exposure: Adjust shutter speed so background is light grey (not pure white/blown out) and chromosomes are dark
    4. Shading Correction: The system subtracts a “blank” background image to remove uneven lighting (vignetting)
  • Procedure (FISH)
    • Captures separate black-and-white images through specific filters (DAPI, Green, Red)
    • Software merges these into a composite pseudocolor image
    • Z-Stacking: Captures images at multiple focal depths to create a sharp composite of 3D nuclei

Enhance Images

Digital tools improve visibility but must be used ethically

  • Standard Enhancements
    • Contrast/Brightness: Stretching the histogram to make bands distinct
    • Background Flattening: Making the empty space uniform white
    • Sharpening: Edge-detection filters to define bands
  • Geometric Corrections
    • Straightening: Unbending curved chromosomes to facilitate homology comparison
    • Separation: Digital cutting of touching chromosomes
  • Ethical Limits: Never delete signals, add bands, or clone chromosomes. The Raw Image is always preserved for audit

Maintenance & Troubleshooting

  • Routine
    • Calibration: Using a Stage Micrometer to ensure measurement accuracy (pixels to \(\mu\text{m}\))
    • Shading Correction: Daily calibration on a blank field to remove dust shadows
    • Backup: Daily archiving of patient data
  • Troubleshooting
    • Grainy Image: Light source too dim (High Gain). Fix: Increase lamp voltage
    • Hot Spots: Uneven light. Fix: Check Kohler illumination
    • Blurry Screen/Sharp Eyes: Parfocality issue. Fix: Focus on screen first, then adjust eyepiece diopters