Chapter 4

Microscopy

Magnification makes an image bigger. Resolution decides whether there is anything in it. Nearly every question on this chapter turns on knowing which of the two is being asked about.

Work it out

Ocular lens

Almost always 10× on a teaching scope.

Objective lens

The one currently clicked into the light path.

Oil immersion

Immersion oil has a refractive index close to glass, so light that would refract away at the slide–air boundary stays in the light path. That raises the numerical aperture to 1.25, which is what actually improves the image. Oil goes on the 100× objective only.

Total magnification

1000×

Useful ceiling for this lens

1250×

About 1000 × NA. Past this the image grows but no new detail appears.

Working

01Total magnification is the ocular multiplied by the objective.
0210× × 100×1000×

The instrument

Hover any part. The light path is drawn because the order matters: light is focused before it reaches the specimen, and magnified twice after it.

Fig. 1A compound light microscope in elevation, with the light path drawn from the illuminator up to the eye.

Nothing selected

Hover a part of the drawing, or a row below.

The four objectives

Numerical aperture is what limits resolution, and it is the reason the 100× lens needs oil.

Objective	Power	NA	With a 10× ocular	Resolves to	Useful ceiling	Oil
Scanning	4×	0.10	40×	3355 nm	100×	—
Low power	10×	0.25	100×	1342 nm	250×	—
High dry	40×	0.65	400×	516 nm	650×	—
Oil immersion	100×	1.25	1000×	268 nm	1250×	Yes

Resolving power here uses 550 nm light, the average of white light. Notice that the oil immersion lens is the only one that resolves below about 400 nm — and that even it cannot reach a virus.

How big is it, really?

Sizes worth carrying in your head, because they set what any given instrument can show you.

Thing	Size	Note
Prion	≈ 0.01 µm	Protein only
Poliovirus	0.03 µm	One of the smallest viruses
Influenza virus	0.1 µm	Below the resolution of a light microscope
Mycoplasma	0.2–0.3 µm	The smallest bacterium, and it has no cell wall
Staphylococcus	≈ 1 µm	A typical coccus
Escherichia coli	1 × 3 µm	A typical rod
Red blood cell	7 µm	A useful yardstick on a slide
Yeast cell	5–10 µm	Eukaryotic, so much larger
Protozoan	10–100 µm	Some are visible without a microscope

The 200 nm wall

A light microscope cannot resolve below roughly 0.2 µm, because visible light itself is 400–700 nm long. Everything above that line in the table is visible; everything below it needs electrons.

Units

1 mm = 1000 µm = 1 000 000 nm. Bacteria are quoted in micrometres, viruses in nanometres, and mixing the two is the fastest way to lose a mark.

Empty magnification

Blow an image up past about 1000 × NA and it gets larger and blurrier at the same time. Nothing new appears, because the resolution never changed.

Care and use

Lab 2. These are the rules a practical exam actually marks.

01Carry it with one hand on the arm and the other under the base, always.
02Start on the lowest power objective and work up.
03Use the coarse adjustment only on low power; on 40× and 100× use fine adjustment alone.
04Immersion oil goes on the 100× objective only. Oil on a 40× lens ruins it.
05Clean lenses with lens paper only — anything else scratches the glass.
06Return to the lowest power, lower the stage, and remove the slide before putting it away.