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Microscopy

You will need to know about the difference between lighttransmission 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.




Staining is a key precursor to microscopy. Most samples would not register well under a microscope without some form of staining. This can also be critical to the experiment carried out. For example, we might need a stain for the cell nucleus as well as a stain for the cell fibres.

For microscopes with fluorescent wavelength filters, fluorescent stains are used. These can be bound to very specific antibodies to target specific cell types or cell organelles.


Muscle cells can be stained for their nuclei (blue) or one of the constituent proteins like actin (red). Stains of different colours and hence wavelengths are used to differentiate the various parts that we want to visualise. Advanced microscopes such as confocal microscopes that use lasers can focus on multiple points in the sample, and relay multiple wavelengths at the same time to create an impression of a section through the sample, add together the data through the multiple layers in the sample, and create complex images of specimens.

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