Achieve Optimal Resolution
Resolution (Resolving Power) is the single most important performance metric of a microscope in cytogenetics. It is defined as the minimum distance (\(d\)) between two points at which they can still be distinguished as separate entities. If the bands on a chromosome are closer together than this distance, they will blur into a single blob, making accurate diagnosis impossible. Achieving optimal resolution is not automatic; it requires the laboratory scientist to actively manage the physics of the optical system through alignment and technique
The Physics of Resolution (Abbe’s Equation)
The limit of resolution is governed by Ernst Abbe’s formula:
\[d = \frac{\lambda}{2 \cdot NA}\]
Where:
- \(d\): = Minimum resolvable distance (Smaller is Better)
- \(\lambda\) (Lambda): = Wavelength of light used
- \(NA\): = Numerical Aperture of the objective lens
To improve resolution (minimize \(d\)), the laboratory scientist has two levers to pull: Decrease \(\lambda\) or Increase \(NA\)
Decreasing Wavelength (\(\lambda\))
White light is a mixture of all colors (Red = 700nm, Blue = 400nm)
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The Green Filter: By inserting a green filter (approx. 550nm) into the light path, we remove the longer red wavelengths. This physically sharpens the image because shorter wavelengths diffract less
- Bonus: Green is also the complementary color to the purple Giemsa stain, increasing Contrast: simultaneously
- Fluorescence: In fluorescence microscopy, we use even shorter wavelengths (UV/Blue excitation), which inherently supports high resolution
Increasing Numerical Aperture (\(NA\))
\(NA\) is a measure of the light-gathering cone of the lens. \(NA = n \cdot \sin(\mu)\), where \(n\) is the refractive index of the medium
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Immersion Oil: Air has a refractive index (\(n\)) of 1.0. Glass has an \(n\) of 1.515. When light passes from the glass slide into the air gap, it refracts (bends) away from the lens, causing data loss
- Action: Placing immersion oil (\(n = 1.515\)) between the slide and the lens creates a “homogeneous immersion system.” The light travels straight from the specimen into the lens without bending. This increases the \(NA\) from ~0.95 (Dry) to 1.40 (Oil), drastically improving resolution
- Technique Tip: Use only high-quality synthetic oil. Do not introduce air bubbles. A single bubble in the oil acts as a lens, scattering light and destroying resolution
Kohler Illumination (The Critical Alignment)
Even with the best lens and oil, resolution is lost if the light path is misaligned. Kohler Illumination is the standard procedure to ensure the light source is perfectly centered and focused at the specimen plane
- Why it matters: It ensures the condenser’s cone of light perfectly matches the objective’s cone of acceptance
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The Procedure
- Focus on the specimen (10x)
- Close the Field Diaphragm: (at the base) until you see a small octagon of light
- Adjust the Condenser Height: knob until the edges of the octagon are sharp/crisp (focusing the light)
- Adjust the Condenser Centering Screws: to move the octagon to the exact center of the field of view
- Open the Field Diaphragm until it just disappears beyond the edge of the view (reducing glare)
The Aperture Diaphragm (The Balancing Act)
Located inside the condenser, the Aperture Iris controls the angle of the light cone. It controls the trade-off between Resolution and Contrast
- Too Closed (Stopped Down): The image becomes very high contrast (dark chromosomes), but diffraction artifacts appear (“ringing” around edges), and fine resolution is lost
- Too Open: The image is flooded with light. Resolution is theoretically maximized, but glare washes out the detail (low contrast), making the bands invisible to the eye
- Optimal Setting: The iris should be open to approximately 70–80% of the objective’s NA. To set this, remove one eyepiece and look down the tube. Close the iris until the edges of the octagon block the outer 20% of the light circle
Slide Corrections
- Coverslip Thickness High-NA objectives are corrected for a specific coverslip thickness (usually 0.17 mm, or a #1.5 coverslip). Using a #1 (too thin) or #2 (too thick) coverslip introduces spherical aberration, causing a “hazy” image that cannot be focused
- Mounting Medium: The mounting media must have a refractive index close to glass. If the media shrinks or crystallizes (old slides), resolution is lost