You will need to know about the difference between light , transmission electron and scanning electron microscopes – LM, TEM and SEM. Both the latter (as the name suggests) use a beam of electrons, rather than light, to produce an image of the sample. TEM uses electrons which pass through the sample, so the resulting micrograph (image) shows everything within the sample in black and white , for example organelles in a cell. SEM uses electrons which scan the sample in 3D , resulting in a coloured micrograph with 3D detail, but no components from within the sample. In light microscopy, light does go through the sample, but the outcome depends on the thickness of the sample. For example, the plant root slice in the diagram (LM) is thin enough to be able to see through the thickness of the sample. Light would also travel freely through air but not various materials of high opacity.
When talking about microscopes, differentiating between resolution and magnification is important. In principle, it’s not hard to understand. Imagine zooming in a photo to try to see a detail. That is magnification . Now imagine the photo has a low resolution, and if you magnify it, you can only see annoying pixels. If the image had a high resolution, you would be able to see the detail clearly after zooming in. So magnifying is zooming in, while resolution is the focus power. You will need to be able to calculate actual sizes and magnifications of various drawings. The equation for that is Image size on paper = Magnification x Actual size . This gives magnification = image size on paper / actual size. “I AM” summarises it nicely in a triangle.
The theory is all good, but in practice, someone’s gotta mash those cells, separate organelles and what not. The technique used is called cell fractionation and it involves ultracentrifugation . These are the steps:
1. Take some tissue such as liver, and blend it in a blender; this is called homogenisation. This must be done in a special solution – an isotonic buffer . Isotonic means of the same water potential as the sample. This ensures enzyme reactions are minimised, organelles don’t get distorted by water gain or loss, and changes in pH are resisted. Learn this (examiners have the hots for isotonic buffers).
2. The resulting mixture is filtered to remove any debris, and then spun in an ultracentrifuge very fast. The increased gravitational field produced will separate the organelles according to their density and even shape.
3. The big ol’ nuclei are the first to separate. The other organelles, still in the liquid at the top called supernatant , are poured into a different test tube and centrifuged further.
4. The organelles are separated in the order: mitochondria and lysosomes, rough ER, plasma membranes, smooth ER, ribosomes.