Skip to main content

Cytogenetics

Types

The light microscope is the fundamental instrument of the cytogenetics laboratory. While molecular techniques are expanding, the visual analysis of chromosomes remains the core of the discipline. Because chromosomes and nuclei are dynamic structures that require different visualization methods depending on their preparation state (unstained vs. stained vs. labeled), the cytogeneticist must be proficient in three distinct microscopy modalities: Brightfield, Phase-Contrast, and Fluorescence. Each type exploits different properties of light - absorption, refraction, and emission - to render the invisible genome visible

Brightfield Microscopy (Transmitted Light)

Brightfield is the standard mode of microscopy used for routine chromosome analysis, specifically G-banding. In this system, light from a tungsten-halogen lamp passes directly through the specimen. The image is formed based on Light Absorption: the stained chromosomes (purple/black) absorb the light, appearing dark, while the clear glass slide transmits the light, appearing bright white. This high-contrast image is essential for the resolution of the delicate light and dark bands required for karyotyping

  • Key Components & Configuration
    • Kohler Illumination: This is the critical alignment technique required for brightfield cytogenetics. It ensures that the light path is perfectly centered and focused, providing even illumination without glare. Without proper Kohler alignment, the fine grey bands (G-bands) will be washed out or obscured by artifacts
    • Objectives: A dry 10x objective is used for scanning the slide to locate metaphases. An oil-immersion 100x objective (Total Magnification: 1000x) is used for the detailed analysis and capture of the metaphase spread. The oil is necessary to match the refractive index of the glass, preventing light scattering and maximizing resolution
    • Filters: A Green Filter (540–560 nm) is almost always placed in the light path when analyzing G-banded slides. Because Giemsa stain is purple/magenta, using a green filter (the complementary color) darkens the appearance of the chromosomes, significantly enhancing the contrast of the bands against the background

Phase-Contrast Microscopy

Phase-contrast is the primary tool for Quality Control (QC) and harvest evaluation. Unstained chromosomes are transparent and have a refractive index very similar to the surrounding glass and cytoplasm; they are essentially invisible under brightfield. Phase-contrast optical systems manipulate the light path to convert slight differences in density (refractive index) into visible differences in brightness (contrast). This allows the laboratory scientist to visualize “wet” (unstained) slides immediately after dropping

  • Mechanism of Action
    • Phase Annulus: A ring-shaped aperture in the condenser allows only a hollow cone of light to illuminate the specimen
    • Phase Plate: A special ring inside the objective lens shifts the phase of the light that passes through the specimen by 1/4 wavelength relative to the background light
    • Interference: When these shifted light waves recombine, they interfere constructively or destructively. Denser structures (chromosomes) appear dark, while less dense areas appear light
  • Clinical Application
    • Harvest Evaluation: Before wasting time and reagents on staining, a test slide is checked under phase contrast to assess the Mitotic Index (are there enough cells?) and Spreading (are chromosomes overlapping?)
    • Slide Aging: Laboratory scientistsuse phase contrast to determine if chromosomes look “refractile” (shiny) or “flat” (grey). Refractile chromosomes indicate the slide is not dry enough for banding
    • Unstained Screening: Locating metaphases on a slide before sequential staining procedures (e.g., destaining a solid stain to perform FISH later)

Fluorescence Microscopy (Epifluorescence)

Fluorescence microscopy is the engine of Molecular Cytogenetics (FISH). Unlike brightfield (where light passes through), fluorescence uses Epifluorescence (light reflects off). The specimen is illuminated with high-energy light (Excitation), which is absorbed by fluorophores bound to the DNA. These fluorophores then emit light at a lower energy level (Emission). The background remains black because the excitation light is filtered out, and only the glowing signal reaches the eye

  • Key Components
    • Light Source: Traditionally high-pressure Mercury Vapor or Xenon arc lamps, which emit a broad spectrum of intense light. Modern labs are transitioning to LED (Light Emitting Diode) sources, which are cooler, longer-lasting, and can be toggled on/off instantly without a warm-up period
    • The Filter Cube (The “Heart”): This block contains three optical elements tailored to specific dyes (e.g., DAPI, FITC, Texas Red):
      • Excitation Filter: Selects the specific wavelength to hit the sample (e.g., Blue light for FITC)
      • Dichroic Mirror: A beam splitter that reflects the excitation light down to the sample but allows the emission light to pass upward to the eyepieces
      • Emission (Barrier) Filter: Blocks any stray excitation light and allows only the specific color of the glowing signal (e.g., Green) to pass
  • Filters and Fluorophores
    • DAPI (Blue): Used as a counterstain for DNA. It excites in the UV range and emits blue
    • FITC / SpectrumGreen (Green): Standard probe color. Excites in Blue, emits Green
    • TRITC / Texas Red / SpectrumOrange (Red): Standard probe color. Excites in Green, emits Red
    • Triple Bandpass Filter: A specialized filter set that allows DAPI, Green, and Red to be viewed simultaneously. This is crucial for analyzing fusion probes (Yellow) or visualizing the probe signal context within the nucleus

Summary of Applications

  • Brightfield: Routine G-banding, C-banding, NOR staining. (High resolution, permanent record)
  • Phase-Contrast: Slide preparation QC, uncultured specimen checks (amniotic fluid), harvest troubleshooting. (Low resolution, transient check)
  • Fluorescence: FISH, SKY (Spectral Karyotyping), Q-banding (historical). (Specific DNA targeting, requires dark room)