Capture Images

In modern clinical cytogenetics, the analysis is rarely performed solely through the eyepieces. The Computer-Assisted Karyotyping System is the industry standard. The imaging system captures the analog view from the microscope, digitizes it, and provides software tools to arrange the chromosomes. The quality of the final karyotype is entirely dependent on the quality of the initial image capture. “Garbage in, garbage out” is the rule: software cannot fix a blurry, poorly illuminated image

The Hardware Interface

• The Camera
  • CCD (Charge-Coupled Device) / CMOS Sensors: High-resolution monochrome cameras are preferred for G-banding. Monochrome sensors are more sensitive to light intensity (greyscale resolution) than color sensors, providing sharper band definition
  • Resolution: Typically 1.4 to 5 Megapixels. Excessive resolution slows down processing without adding diagnostic value (optical resolution is limited by the microscope lens, not the camera pixels)
• The Adapter (C-Mount)
  • Connects the camera to the microscope trinocular head. Ideally, it includes a reduction lens (e.g., 0.7x) to match the camera’s field of view to what is seen in the eyepieces

Steps for Optimal Capture (G-banding)

1. Optical Perfection First
   • Before touching the computer, the image must be perfect in the eyepieces
   • Focus: Use the fine focus knob. The camera focus is often slightly different from the eye focus (parfocality issue). Laboratory scientistsmust learn to focus on the screen, not through the oculars, during capture
   • Green Filter: Ensure the green filter is in the light path to maximize contrast
2. Exposure Time (Brightness)
   • The software controls how long the camera shutter stays open
   • Too Bright (Overexposed): The background is pure white (“blown out”). Chromosomes look thin or eroded. Faint grey bands disappear
   • Too Dark (Underexposed): The background is grey. The image is “grainy” (electronic noise)
   • Goal: The background should be just below saturation (light grey/white), and the chromosomes should be distinct dark objects
3. Shading Correction (Flat-Fielding)
   • Microscope illumination is naturally uneven (brighter in the center, darker at the edges/vignetting)
   • Correction: The software subtracts a “blank” background image from the captured image to create a perfectly even, white background. This must be calibrated daily on a blank area of the slide
4. Contrast Enhancement
   • The software applies a “histogram stretch” (adjusting the Black and White points) to make the darkest band black and the background white

Fluorescence Capture (FISH)

Capturing FISH images is more complex because it involves multiple colors and low light levels

• Multi-Channel Acquisition
  • The camera takes separate black-and-white photos through different filter blocks:
    • Exposure 1: DAPI filter (Blue channel)
    • Exposure 2: FITC filter (Green channel)
    • Exposure 3: Texas Red filter (Red channel)
  • The software merges these into a single composite image 2. Integration Time
  • Because fluorescence is faint, exposure times are long (milliseconds to seconds)
  • Binning: A technique where pixels are grouped (e.g., 2x2) to increase sensitivity, though this lowers resolution 3. Z-Stacking (Extended Focus)
  • In a 3D nucleus, signals are at different depths. A single focal plane might miss a signal
  • Technique: The system takes a series of images at different focal depths (like a CT scan) and collapses them into a single sharp 2D image

Common Capture Artifacts

• Halos: Bright white rings around chromosomes
  • Cause: Condenser aperture is closed too much (diffraction) or Phase Contrast condenser was left in
• Vignetting: Dark corners
  • Cause: Camera adapter mismatch or misaligned condenser
• Grainy Image
  • Cause: Light source is too dim, forcing the camera to boost “Gain” (electronic noise)
  • Fix: Turn up the microscope lamp voltage and reduce camera gain