MicroscopyEdexcel International A Level Biology: Revision notes

Magnification is how many times larger the image is than the real object:

magnification = size of image ÷ actual size of object

Rearranged, actual size = image size ÷ magnification. Always convert to the same units first (1 mm = 1000 µm; 1 µm = 1000 nm).

Resolution is the minimum distance between two points that can still be seen as separate. A microscope with a higher resolution shows more detail. Making the image larger beyond the resolution limit only gives a bigger, blurred image: this is called empty magnification.

Worked example: a cell image is 24 mm long at ×3000, so the actual length is 24 000 µm ÷ 3000 = 8 µm.

A light microscope uses visible light and glass lenses. Its resolution is about 0.2 µm (200 nm), limited by the wavelength of light, and its useful magnification is up to about ×1500.

Advantages: specimens can be living, preparation is quick and cheap, and colour stains can be used. Disadvantage: it cannot resolve small structures such as ribosomes or the cristae of mitochondria.

An electron microscope uses a beam of electrons, which have a much shorter wavelength than light, so the resolution is far higher (about 0.1 nm in a TEM) and the magnification is much greater. Electromagnets focus the beam and the specimen must be in a vacuum, so it must be dead.

• Transmission electron microscope (TEM): electrons pass through a very thin section; internal structures (ultrastructure) are seen in a two-dimensional image.
• Scanning electron microscope (SEM): electrons scan the surface and the image shows the surface in three dimensions, with a lower resolution than a TEM.

Disadvantages: expensive, complex preparation, black and white images and possible artefacts from preparation.

Most cell structures are almost transparent and have little contrast. A stain is taken up by, or binds to, particular structures so that they absorb more light and can be seen and distinguished.

• Methylene blue or acetic orcein: stain the nucleus and DNA.
• Iodine in potassium iodide: stains starch blue-black.
• Eosin: stains cytoplasm.

For electron microscopy, heavy metals such as lead or uranium compounds are used, because they scatter electrons and give contrast.

Making a temporary mount: place a thin piece of tissue in a drop of water or stain on a slide and lower the coverslip at an angle with a mounted needle to avoid air bubbles.

Drawing: focus on low power first, then high power. Use a sharp pencil, single continuous lines, no shading or colour, a large drawing, ruled label lines, a title and the magnification.

Using an eyepiece graticule: it is a scale in the eyepiece with arbitrary units. Calibrate it against a stage micrometer (divisions of 10 µm) for each objective lens. For example, if 80 graticule divisions line up with 400 µm, one division is 5 µm; a cell 14 divisions long is 70 µm.

Magnification of a drawing = length of drawing ÷ actual length (same units).