Microscopy
Introduction
This domain covers the light microscope, magnification, resolution, contrast methods, and correct use of oil immersion. The exam tests whether you can separate useful magnification from empty magnification and choose a microscopy method for a specimen.
Core rules
- Total magnification = ocular lens magnification × objective lens magnification.
- Resolution is the smallest distance at which two points remain distinct; it is more important than magnification alone. For a light microscope, d = 0.61λ/NA, where λ is wavelength and NA is numerical aperture.
- Immersion oil has the same refractive index as glass and prevents light loss between the slide and the 100× oil objective.
- Brightfield microscopy suits stained smears; darkfield suits unstained live organisms such as Treponema pallidum; phase contrast suits transparent live cells; fluorescence uses fluorochromes and specific excitation.
- Clean lenses with lens paper only, and never use oil with a dry objective.
Worked example
A microscope has a 10× ocular and a 100× oil objective with NA 1.25. Using green light at 550 nm, d = 0.61 × 550 / 1.25 = 268.4 nm, approximately 0.27 µm. Increasing the ocular to 15× raises total magnification to 1500× but does not improve this resolution, because resolution depends on wavelength and objective NA, not ocular magnification.
Traps
Do not report greater detail merely because the image is larger. Do not apply oil to the 40× objective unless it is specifically an oil objective. Do not use xylene or rough paper on lenses; residual oil can damage lenses and distort images.