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Cytogenetics

Microscope & Imaging Equipment

The generation of a karyotype or FISH result relies on a chain of optical and digital technologies. The light microscope magnifies the specimen, while the imaging system captures and processes the visual data. The quality of the final diagnosis is constrained by the weakest link in this chain, making proper configuration, maintenance, and technique essential

Microscope

The clinical microscope is a precision instrument designed to resolve fine genomic details. It utilizes different optical modalities depending on the specimen type

  • Types & Applications
    • Brightfield: Uses transmitted light and a Green Filter to visualize G-banded chromosomes (absorption). Standard for karyotyping
    • Phase-Contrast: Converts refractive index differences into contrast. Essential for Quality Control of unstained/wet slides (checking mitotic index)
    • Fluorescence: Uses epifluorescence (reflected light). High-energy excitation causes fluorophores to emit colored light against a dark background. Used for FISH
  • Components
    • Illumination: The Condenser focuses light onto the specimen. The Aperture Diaphragm (Iris) controls the balance between contrast and resolution
    • Optics: Objectives (10x, 100x Oil) determine magnification and resolution. Immersion Oil (\(n=1.515\)) is mandatory for 100x to prevent light refraction
  • Achieving Resolution
    • Resolution (\(d\)) is limited by wavelength (\(\lambda\)) and Numerical Aperture (\(NA\))
    • To optimize: Use Oil: (High NA), a Green Filter (Short \(\lambda\)), and perform Kohler Illumination (perfect alignment of the light path)
  • Maintenance
    • Daily: Clean oil off lenses to prevent cement dissolution. Cover to block dust
    • Troubleshooting: A blurry image is usually a dirty objective. An uneven image is usually a misaligned condenser

Imaging System

The imaging system digitizes the microscopic view, allowing for computer-assisted analysis. It consists of a high-resolution camera and specialized software

  • Capture Images
    • G-Banding: Uses a monochrome camera for high greyscale sensitivity. Requires correct exposure (not too bright/dark) and Shading Correction (removing background shadows/vignetting). Focus must be set on the monitor, not the eyepieces
    • FISH: Uses multi-channel capture (DAPI, Green, Red) merged into a composite. Z-Stacking captures multiple focal planes to resolve 3D signals
  • Enhance Images
    • Tools include Contrast Adjustment: (histogram stretching), Sharpening, and Background Flattening
    • Geometric Correction: Allows straightening curved chromosomes or separating touching ones
    • Ethics: Enhancement clarifies existing data; Manipulation (adding/deleting signals) is fraudulent. The raw image is always preserved
  • Maintenance
    • Calibration: Scaling pixels to microns using a stage micrometer
    • Troubleshooting: “Grainy” images are caused by low light and high camera gain. “Hot spots” are caused by poor Kohler alignment