Microscope and Microscopy (Advanced)

How microscopes see the very small: total magnification, real size, field of view, resolution, and choosing between light, phase-contrast, fluorescence, TEM and SEM.

Key ideas

The objective forms a real, inverted, magnified image; the eyepiece magnifies it again into a virtual image.
More magnification is not always better: without enough resolution the bigger image is just blurred.
The eye resolves about 0.1 mm, a light microscope about 0.2 µm and an electron microscope about 0.2 nm.
Phase-contrast shows living cells without stains; fluorescence makes dyed structures glow against a dark background.
TEM passes electrons through ultra-thin sections for internal details in 2D; SEM scans a metal-coated surface for 3D-like images.
Electron microscopes need a vacuum and chemical preparation, so they cannot show living cells.
Temporary mounts dry out; permanent slides are fixed, stained, dehydrated and sealed in Canada balsam or DPX.

Remember

Total magnification	M = objective × eyepiece
Real size	real size = image size ÷ M; image size = real size × M
Field of view	inversely proportional to M; cell size ≈ field width ÷ number of cells across
Units	1 cm = 10 mm; 1 mm = 1,000 µm; 1 µm = 1,000 nm
Resolving power	1 ÷ resolution (the smaller the resolution, the greater the power)

Watch out

Adding the eyepiece and objective powers instead of multiplying them.
Forgetting to change mm to µm before dividing by the magnification.
Thinking the field of view gets bigger at higher magnification.

Worked example

A TEM photograph at 5,000X shows a structure 42 mm long. Find its real length (a) in µm and (b) in nm. (c) Could a light microscope, which resolves about 0.2 µm, show its two ends as separate points? (Yes or No) (Give the answer to 2 decimal places where needed.)

Working: (a) 42 mm = 42,000 µm, ÷ 5,000; (b) × 1,000; (c) compare with 0.2 µm

Answer: (a) 8.4 µm; (b) 8,400 nm; (c) Yes