Compound & Phase-Contrast Microscopy and Micrometry
Direct measurement of microscopic specimens requires optical micrometry because mechanical rules cannot be placed in the focal plane of an objective lens. An ocular micrometer (eyepiece graticule) is a circular glass disc engraved with an arbitrary ruled scale (typically 100 equidistant divisions of unknown physical distance). A stage micrometer is a precision glass slide featuring an etched 1.0 mm scale ruled into 100 equal divisions; therefore, exactly 1 stage division represents $10,mu ext{m}$ ($0.01, ext{mm}$).
Because the magnification power of the objective lens alters the real magnification of the specimen image projected onto the ocular diaphragm, the ocular micrometer must be individually calibrated for every objective lens. By superimposing the ocular and stage scales and identifying two points of exact coincidence, the calibration constant is computed:
In Phase-Contrast Microscopy, transparent unpigmented living cells with minimal light absorption are visualized without destructive staining. Variations in cellular refractive index ($n$) and specimen thickness shift the phase of transmitted light by $pprox lambda/4$. An annular condenser diaphragm and a phase-shifting plate inside the objective retard or advance the direct light rays by an additional $lambda/4$, converting destructive/constructive phase shifts into intensity (amplitude) variations perceptible to the human eye.
- Fresh red onion bulb (Allium cepa)
- Human cheek epithelial swab in 0.9% physiological saline
- 0.1% Methylene blue aqueous solution
- Glycerol mounting medium (50% v/v in distilled water)
- Distilled water & lens cleaning fluid (isopropanol/ether 7:3)
- Compound binocular research microscope (10×, 40×, 100× oil)
- Ocular micrometer disc (10× eyepiece insertion)
- Calibrated stage micrometer (1 mm ruled into 100 units = $10,mu ext{m}$/div)
- Fine dissection forceps, sterile cotton swabs, coverslips ($18 imes 18, ext{mm}$)
- Ocular Reticle Insertion: Unscrew the 10× ocular lens, unscrew the field stop diaphragm, insert the ocular micrometer disc with the ruled scale facing downwards, and reassemble the eyepiece.
- Stage Micrometer Alignment: Place the stage micrometer slide securely onto the mechanical stage. Focus the etched scale using low power (10× objective).
- Superimposition of Scales: Rotate the ocular lens until the ocular micrometer scale lies exactly parallel to and superimposed over the stage micrometer scale. Align the zero (0) line of both scales precisely.
- Coincidence Determination: Scan along the superimposed scales to identify a second point where a line of the ocular micrometer perfectly coincides with a line of the stage micrometer. Note both readings.
- High-Power Calibration: Switch to the high-power (40×) objective, refocus using the fine adjustment knob only, and repeat the zero-alignment and coincidence determination. Calculate calibration factors for both 10× and 40×.
- Specimen Mounting: Peel a transparent monocellular epidermal layer from the concave scale leaf of Allium cepa. Mount on a clean slide in a drop of water/saline, stain with 0.1% methylene blue for 2 minutes, wash excess stain, mount under coverslip in 50% glycerol.
- Cell Dimension Measurement: Replace stage micrometer with the specimen slide. Align the ocular scale across 10 randomly selected epidermal cells. Record length and width in ocular units, then convert to microns ($mu ext{m}$).
| Objective Lens | Coincident Stage Divs (a) | Actual Length ($a imes 10,mu ext{m}$) | Coincident Ocular Divs (b) | Calibration Factor ($mu ext{m}$/div) |
|---|---|---|---|---|
| Low Power (10×) | 10 divisions | $100,mu ext{m}$ | 10 divisions | $10.0,mu ext{m}$ |
| High Power (40×) | 6 divisions | $60,mu ext{m}$ | 24 divisions | $2.5,mu ext{m}$ |
| Oil Immersion (100×) | 4 divisions | $40,mu ext{m}$ | 40 divisions | $1.0,mu ext{m}$ |
- Never use coarse adjustment when focusing under 40× or 100× to prevent crushing the expensive stage micrometer slide.
- Recalibrate the ocular micrometer whenever an objective lens, tube length, or microscope frame is changed.
- Ensure air bubbles are eliminated beneath the coverslip by lowering it at a 45° angle with a dissection needle.
