Light microscope use and care

Principle

• A compound brightfield microscope magnifies in two stages: total magnification = objective × eyepiece (e.g. 100× oil × 10× = 1,000×).
• Resolution depends on wavelength and numerical aperture (NA); with oil immersion the limit is about 0.2 µm.
• Immersion oil (refractive index about 1.515, similar to glass) reduces light loss by refraction at high magnification.

Reagents and equipment

• Eyepieces (usually 10×), objectives (commonly 4×, 10×, 40× and 100× oil), condenser with aperture diaphragm, field diaphragm, fine and coarse focus, stage, light source; lens paper, lens cleaner, immersion oil.

Procedure

• Carry with one hand on the arm and one under the base.
• Start with the lowest-power objective; focus with coarse, then fine adjustment.
• Köhler illumination: focus the specimen, close the field diaphragm, focus its edges with the condenser height, center it, then open until it just clears the field.
• Adjust the aperture diaphragm for contrast (commonly about 70–80% of the objective NA); control brightness with the lamp intensity, not the condenser.
• Most objectives are parfocal, so only fine focus is needed after changing magnification.
• For oil immersion, place a drop of oil on the slide, rotate the 100× objective into it and use fine focus only; do not swing the 40× (dry) objective through oil.

Quality control and pitfalls

• Clean oil from the 100× objective and stage after each use with lens paper and recommended cleaner; avoid excess solvents that can loosen lens cement.
• Hazy image: oil or dirt on a dry objective, slide upside down, or coverslip too thick.
• Uneven lighting: condenser not centered or misadjusted.
• Turn the lamp down before switching off, cover when not in use, and have routine preventive maintenance.
• Calibrate the ocular micrometer for each objective when measuring organisms or cells.

Clinical use

• Essential for blood films, urine sediment, Gram stains, parasites, fungi, histology and cytology slides.