Microscopy and cell fractionationAQA A-Level Biology: Revision notes

Magnification is how many times larger the image is than the real object. Resolution is the ability to distinguish between two points that are close together as separate points; it determines the detail in an image.

magnification = size of image ÷ size of real object

actual size = size of image ÷ magnification

Image and object must be in the same units: 1 mm = 1000 μm and 1 μm = 1000 nm.

Worked example: an image of a mitochondrion is 45 mm long at ×30 000. Actual size = 45 mm ÷ 30 000 = 0.0015 mm = 1.5 μm.

An optical (light) microscope uses light and glass lenses. Light passes through a thin specimen and the lenses magnify the image.

• Advantages: cheap, easy to use, specimens can be living, shows colour.
• Limitations: low resolution (about 0.2 μm), because light has a relatively long wavelength, so small organelles such as ribosomes cannot be seen in detail.

Measuring size: an eyepiece graticule (a scale in the eyepiece) is calibrated with a stage micrometer (a slide with a scale of known size). If 40 graticule divisions line up with 100 μm, one division = 100 ÷ 40 = 2.5 μm. The object is measured in graticule divisions and multiplied by this value. The graticule must be recalibrated for each objective lens.

Electron microscopes use a beam of electrons, which have a much shorter wavelength than light, so they give a much higher resolution and can show organelle detail. Electromagnets focus the beam, and the specimen is in a vacuum, so it must be dead.

Transmission electron microscope (TEM): electrons pass through a very thin specimen. Denser parts absorb more electrons. It gives the highest resolution and shows internal structure, but only a 2D image, and complex preparation can create artefacts.

Scanning electron microscope (SEM): a beam scans the surface of the specimen and electrons reflected from the surface are detected. It gives a 3D image of the surface and can use thick specimens, but its resolution is lower than the TEM.

Cell fractionation separates organelles so that their structure and function can be studied.

1. Homogenise the tissue (blend it) to break open the cells and release the organelles.
2. Use a cold, isotonic, buffered solution: cold to reduce enzyme activity (so organelles are not digested), isotonic to prevent organelles bursting or shrinking by osmosis, buffered to keep the pH constant.
3. Filter to remove cell debris and unbroken cells.
4. Ultracentrifugation: spin the filtrate in a centrifuge at low speed; the heaviest organelles (nuclei) form a pellet. Pour off the supernatant and spin at a higher speed to pellet the next heaviest (mitochondria), then chloroplasts, lysosomes, and finally ribosomes at the highest speeds.

Organelles separate in order of mass/density.

An artefact is something seen in an image that is not part of the specimen in life. Preparing specimens for electron microscopy (fixing, dehydrating, staining, slicing, viewing in a vacuum) can distort cells or create structures that are not real.

When electron microscopes were first used, there was a considerable period during which the scientific community had to distinguish artefacts from real organelles. They did this by preparing specimens in different ways and using different techniques: a structure seen consistently, and supported by evidence such as isolation by cell fractionation, is likely to be a real organelle; one seen only with a single preparation is likely to be an artefact.