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Cytogenetics

Components & Functions

The clinical microscope is a precision optical instrument composed of mechanical and optical systems designed to magnify, resolve, and illuminate the specimen. Understanding the anatomy of the microscope is essential for troubleshooting image quality issues (“Why is my image blurry?”) and ensuring the longevity of the equipment. The components can be broadly categorized into the Support System, the Illumination System, and the Optical System

Support System (The Frame)

  • Stand/Arm: The heavy, rigid backbone that supports all components. It provides stability to minimize vibration, which is critical at 1000x magnification
  • Stage: The flat platform where the slide is placed
    • Mechanical Stage Controls: Knobs that move the slide in the X (left/right) and Y (forward/backward) axes. In cytogenetics, these controls are used systematically to scan the slide in a “battlement” pattern (up-over-down-over) to ensure no metaphase is missed
    • Vernier Scale: A ruler on the stage axes that allows the laboratory scientist to record the specific coordinates of a cell (e.g., 115.4 / 22.1) so it can be relocated later or by another person
  • Focus Knobs: Move the stage up and down relative to the objective lens
    • Coarse Focus: Large movements. Used only with low power (10x) objectives to find the focal plane. Using coarse focus with the 100x oil objective risks crashing the lens through the glass slide
    • Fine Focus: Microscopic movements. Used for sharpening the image at high power and navigating through the “depth” of the chromosome spread

Illumination System (The Light Path)

Proper lighting is the foundation of resolution

  • Light Source (Lamp House): Typically a Tungsten-Halogen bulb located at the base. It emits continuous white light
  • Field Diaphragm: A shutter located near the light exit at the base. It controls the width of the light beam entering the condenser. It acts as a “frame” for the image; opening it too wide creates glare (stray light), reducing contrast
  • Condenser: A system of lenses located under the stage. It gathers the diffuse light from the lamp and concentrates it into a sharp cone of light that illuminates the specimen
    • Aperture Diaphragm (Iris): Located inside the condenser. This is the most critical control for resolution vs. contrast
      • Too Open: High resolution but the image looks “washed out” (low contrast)
      • Too Closed: High contrast but the image shows diffraction artifacts (resolution loss)
      • Optimal Setting: The iris should match the Numerical Aperture (NA) of the objective lens being used (typically 70–80% open)
  • Filter Holder: Located above the field diaphragm. In cytogenetics, a Green Filter is placed here to enhance the contrast of purple Giemsa-stained chromosomes

Optical System (Magnification)

Magnification is a two-step process: The Objective lens creates a magnified “Real Image,” and the Eyepiece magnifies that image further to create the “Virtual Image” seen by the eye

  • Objective Lenses (The Nosepiece): The primary lenses mounted on a rotating turret. They determine the image quality
    • 10x (Low Power): Used for scanning. Large Field of View (FOV)
    • 100x (Oil Immersion): Used for analysis
      • Numerical Aperture (NA): The most important spec (typically 1.30 or 1.40). NA measures the lens’s ability to gather light. A higher NA means better resolution (ability to distinguish two close points)
      • Immersion Oil: Because the 100x lens has a very short focal length, light refracts (bends) as it leaves the glass slide and enters the air, causing signal loss. Immersion oil has the same refractive index as glass (\(n=1.515\)). It bridges the gap between the slide and the lens, preventing refraction and capturing more light rays
  • Eyepieces (Oculars): Typically 10x magnification
    • Total Magnification: \(100\text{x Objective} \times 10\text{x Eyepiece} = 1000\text{x}\)
    • Diopter Adjustment: A ring on one or both eyepieces that allows the user to correct for differences in vision between their left and right eyes
    • Interpupillary Distance: The adjustment that spreads the eyepieces apart to match the distance between the user’s eyes, ensuring a single binocular image circle

The “Tube” (Beam Splitter)

Located between the objectives and the eyepieces

  • Trinocular Head: Most clinical microscopes have a third port on top. A prism inside allows the user to divert light from the eyepieces to a Camera mounted on top. This allows for digital imaging and karyotyping. The pull-rod usually splits the light (e.g., 100% to eyes OR 100% to camera, or a 50/50 split)

Key Concept: Resolution vs. Magnification

  • Magnification: is simply making something look bigger. You can magnify a blurry image infinitely, but it will just be a big blur (“Empty Magnification”)
  • Resolution: is the ability to distinguish detail. It is limited by the wavelength of light (\(\lambda\)) and the Numerical Aperture (NA)
    • \(Resolution (d) = \lambda / (2 \times NA)\)
    • To get better resolution (smaller \(d\)), you need a shorter wavelength (Blue/Green filter) or a higher NA (Oil Immersion). This is why the condenser alignment and oil are non-negotiable in cytogenetics