Microscopy and Biological Techniques
Microscopy and Biological Techniques tests whether you can choose an instrument, prepare a specimen, and interpret an image quantitatively.
Core rules
- Magnification enlarges; resolution separates two points. Resolution depends mainly on wavelength and numerical aperture, not eyepiece power.
- Brightfield is routine; phase contrast shows unstained transparent cells; fluorescence detects tagged molecules; confocal rejects out-of-focus light and gives optical sections.
- TEM transmits electrons through thin sections and reveals internal ultrastructure; SEM scans surfaces and gives topographic images.
- Fixation preserves structure, dehydration and embedding support sectioning, and staining creates contrast. A scale bar is essential for measurement.
- Aseptic technique matters in live-cell imaging; evaporation, photobleaching, and autofluorescence are common artifacts.
Worked example
A specimen is viewed with a 10x eyepiece and a 100x oil-immersion objective. Total magnification is 10 x 100 = 1000x. The objective has NA 1.25 and light of 550 nm. Using d = 0.61 lambda / NA, d = 0.61 x 550 / 1.25 = 268 nm. Increasing the eyepiece to 15x raises magnification to 1500x but does not improve resolution. The reasoning separates magnification from resolving power.
Traps
Do not claim empty magnification improves detail. Do not use oil with a dry objective or omit oil with an oil-immersion lens. Do not compare TEM and SEM as if both show internal structure. Do not measure a cell without a calibrated scale; a printed magnification is not a scale bar.