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Cytogenetics

Microscope

The microscope is the primary instrument of the cytogeneticist. Mastery of its optical principles is required to transform faint chromosomal structures into analyzable data

Microscope Types

  • Brightfield
    • Use: Standard routine analysis (G-banding)
    • Principle: Light passes through the specimen. Stained chromosomes absorb light (appear dark) against a bright background
    • Requirement: Requires a Green Filter to enhance the contrast of purple Giemsa stain
  • Phase-Contrast
    • Use: Quality Control (QC) of unstained slides, checking mitotic index, and harvest troubleshooting
    • Principle: Converts invisible differences in refractive index (density) into visible differences in brightness. Allows visualization of “wet” transparent cells
  • Fluorescence
    • Use: FISH and molecular cytogenetics
    • Principle: Epifluorescence. High-energy light (Excitation) hits the sample; fluorophores emit lower-energy light (Emission). The background is black. Requires specific filter cubes (DAPI, FITC, Texas Red)

Components & Functions

  • Support: The heavy Stand and Stage provide stability
  • Illumination
    • Lamp: Source of white light
    • Condenser: Concentrates light into a cone to illuminate the specimen
    • Diaphragms: Field Diaphragm controls beam width (glare); Aperture Diaphragm (Iris) controls contrast vs. resolution
  • Optics
    • Objectives: The primary lenses (10x Scan, 100x Oil)
    • Eyepieces: Further magnify the image (10x). Total Mag = \(100 \times 10 = 1000\text{x}\)

Achieve Optimal Resolution

Resolution (\(d\)) is the ability to distinguish two close points. \(d = \lambda / (2 \cdot NA)\). To minimize \(d\) (improve resolution):

  1. Decrease Wavelength (\(\lambda\)): Use a Green Filter (shorter wavelength than red)
  2. Increase Numerical Aperture (\(NA\)): Use Immersion Oil (\(n=1.515\)) with the 100x objective. Oil bridges the air gap, preventing light refraction/loss
  3. Align Light: Perform Kohler Illumination to center and focus the condenser
  4. Optimize Iris: Set the Condenser Iris to ~75% open to balance resolution and contrast

Maintenance & Troubleshooting

  • Daily: Clean oil off the 100x lens after use (oil dissolves cement). Cover to prevent dust
  • Troubleshooting
    • Blurry Image: Usually a dirty objective or slide. Clean thoroughly
    • Black Field: Check light path (camera pull-rod engaged? Field diaphragm closed?)
    • Drifting Focus: Tighten the tension collar on the focus knob
    • Uneven Light: Condenser is off-center (Fix: Kohler)