Microscopy
Senior 4 · Biology
Microscopy
Lesson 4 of 4
25 min
Under the best light microscope a cell shows a nucleus, perhaps some dots in the cytoplasm, and in plants chloroplasts and a vacuole. Yet a cell contains a whole system of membranes, thousands of ribosomes each about 25 nm across, and mitochondria folded inside like crumpled paper. None of these details can be resolved with light, because their spacing is far smaller than about 200 nm, the limit set by the wavelength of light (Lesson 1). The electron microscope, with a resolution hundreds of times better, revealed them in the 1950s and 1960s. What you will learn in Unit 4 about the ultrastructure of cells comes almost entirely from electron micrographs.
Definition
Transmission electron microscope (TEM): a microscope that passes a beam of electrons through an ultrathin section of a specimen and forms a two-dimensional image of its internal structure.
Scanning electron microscope (SEM): a microscope that sweeps a fine beam of electrons across the surface of a specimen and detects the electrons given off, forming a three-dimensional-looking image of the surface.
Electromagnetic lens: a coil of wire carrying a current, whose magnetic field focuses a beam of electrons as a glass lens focuses light.
Electron micrograph: a photograph taken with an electron microscope. It is black and white; any colour is added later and is called false colour.
Artefact: a structure seen in a prepared specimen that was not present in the living cell, caused by the preparation itself.
An electron gun at the top of a tall column fires a beam of electrons downward. Because electrons are scattered by air molecules, the whole column is kept under a vacuum. Electromagnets focus the beam onto the specimen, which must be an ultrathin section, typically less than 100 nm thick, so that electrons can pass through it. The specimen is stained with salts of heavy metals such as lead and uranium, which bind to membranes and other structures and scatter electrons strongly. Where electrons are scattered, few reach the screen, and the area appears dark (electron-dense); where they pass through, it appears bright. Further electromagnetic lenses magnify the image onto a fluorescent screen or a digital camera.
In an SEM the specimen is not sliced. It is dried and coated with a layer of metal, often gold, a few nanometres thick, so that it conducts electricity. A fine beam of electrons is swept back and forth across the surface, line by line. At each point the beam knocks secondary electrons out of the surface; a detector counts them, and a computer builds up the image point by point. Surfaces facing the detector send more electrons and look brighter, which gives the image its striking depth. An SEM shows surfaces, such as the sculpture of a pollen grain or the hooks on an insect's leg, but not the inside of cells.
Because the specimen sits in a vacuum and is bombarded by electrons, it must be dead and completely dry. A typical TEM preparation: fix the tissue with chemicals (such as glutaraldehyde) that cross-link proteins and hold everything in place; dehydrate it with alcohol; embed it in hard resin; cut ultrathin sections with a diamond or glass knife; and stain with heavy metals.
Each step can change the cell, creating artefacts. A famous case: in the 1960s, folds of the membrane called "mesosomes" were seen in electron micrographs of chemically fixed bacteria and were thought to be organelles. By the late 1970s evidence showed that they were damage caused by chemical fixation, and by the 1980s, when cells were frozen extremely fast instead (cryofixation), most biologists concluded that mesosomes do not exist in living cells. Modern cryo-electron microscopy freezes specimens in a fraction of a second without chemicals, so that they are seen close to their living state.
| Feature | Light microscope | TEM | SEM |
|---|---|---|---|
| Radiation | Light | Electrons passing through the specimen | Electrons scanning the surface |
| Lenses | Glass | Electromagnets | Electromagnets |
| Best resolution (approximate) | 200 nm | 0.5 nm or better | 3–10 nm |
| Useful magnification (approximate) | up to ×1500 | ×500 000 or more | up to about ×100 000 |
| Specimen | Living or dead; thin sections or smears | Dead; ultrathin sections | Dead; whole, coated in metal |
| Image | Natural colour or stains; 2-D | Black and white; 2-D internal detail | Black and white; 3-D-looking surface |
| Cost, size and skill | Cheap, portable, quick | Very expensive, large, needs trained staff and a stable power supply | Very expensive, large, needs trained staff |
Worked example 1: how much better is the TEM?
Taking the resolution of a light microscope as 200 nm and that of a TEM as 0.5 nm, (a) how many times better is the TEM's resolution? (b) Explain why the light microscope cannot simply be given stronger lenses to match it.
Worked example 2: magnification of an electron micrograph
In a TEM image, a ribosome 25 nm across appears 10 mm across. Calculate the magnification.
Worked example 3: choosing the right microscope
Choose a microscope for each task, and give a reason: (a) watching Paramecium swim; (b) the sculpted surface of a pollen grain; (c) the folded inner membrane of a mitochondrion.
Cognito: "Electron Microscopes: Transmission Electron & Scanning Electron". Note how each microscope forms its image, and list the advantages and limitations it gives for each.
Warning
Common mistakes
Tip
Exam technique. For "compare" questions, write in matched pairs: "TEM uses electrons, whereas the light microscope uses light; TEM has a resolution of about 0.5 nm, whereas…". Always link the shorter wavelength of electrons to the higher resolution.
Practice: electron microscopes
Key points
Further reading for this unit
Sources and credits
This lesson adapts CC BY-SA material and is shared under CC BY-SA 4.0.
Quick check
Why can a TEM resolve much finer detail than a light microscope?
