January 11, 2013

The Basics

A Very Rough Guide to Microscopy

The Cell Imaging Centre is primarily equipped to benefit research scientists within the faculty of medicine and dentistry at the University of Alberta, and is also available to users from other academic institutes and commercial companies.

There are 2 main types of microscope – LIGHT microscopes and ELECTRON microscopes.

Both these technologies are available in the facility.

PDF versions of popular light microscopy guides:

Zeiss' practical introductory manual for microscopy Olympus' introductory manual for microscopy

PDF versions of helpful publications:

 

Rossner and Yamada. What's in a picture? The temptation of image manipulation. The J. Cell Biol. (2004) vol 166(1) pp. 11-15North. Seeing is believing? A beginner's guide to practical pitfalls in image acquisition. J. Cell Biol. (2006) vol. 172 (1) pp. 9-18

 

 

 

LIGHT MICROSCOPY

The most common type of light microscopy involves shining a regular tungsten filament bulb, a bit like you have in your light fittings at home, onto the sample. This can be shone through the sample (this is called transmitted light microscopy) or bounced off the surface (reflected light microscopy). Because the sample is made up of different structures, light travelling through it is absorbed in different ways (in much the same way that when your windows are covered in gunk you see darker and lighter patches depending on how much sunlight is absorbed and how much gets through). By using a magnifying lens, we can use this approach to see structures in fairly small samples, such as cells.

Sometimes, the sample is so thin or translucent that not enough light is absorbed to see a difference (imagine covering your window with pieces of selotape – it would be difficult to see the seloptape on a sunny day). Although we could magnify the image to make it look bigger, we wouldn’t actually be able to see anything much at all because there is no contrast. Fluorescence microscopy is a light microscopy technique that dramatically improves contrast. This is done by putting a dye onto the sample, which becomes attached to a specific part of the cell (e.g. the dye DAPI only stains a cell’s nucleus). By adding several dyes, which stain different parts of the cell, we can increase the contrast in the sample and see more of the biological structures we’re interested in by seeing less of the structures surrounding them.

Light microscopy is a very useful tool in biology because the light used to image the sample does not tend to kill it. On the down side, we are limited in how much we can magnify a sample because the wavelength of light is quite long, as compared to the wavelength of the electron beam used in electron microscopy. This is a bit like the difference between trying to pick up something while wearing a thick pair of gloves, and then trying the same thing with a pair of tweezers. If we need to look at very small structures and we don’t mind killing the cell in the process, we can learn much more about fine structure with the electron microscope.

 

ELECTRON MICROSCOPY

As we have already seen, the light microscope can be used to look at single cells and we can even see something of their internal structure. By comparison, an electron microscope can be used to see much smaller structures within biological and material specimens.

Just like with light microscopy, where we have transmitted and reflected light microscopy, we also have two types of electron microscopy. These are called Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM).

TEM is very similar to transmitted light microscopy, only in this case it is a beam of electrons, rather than a beam of light, which is passed through the sample. The other difference is to do with size. On a light microscope we might use a 20 micrometer thick sample with some success, on a TEM the sample is usually about 60 nanometres thick (a micrometer is 1 millionth of a meter, a nanometer is one billionth of a meter). Because the sample is so thin, it needs to be stained to improve contrast. This is done with a solution of heavy metal salts such as Uranium or Lead. The end results is that electrons are able to pass through the clear and lightly stained regions of the sample, whereas stained parts of the sample scatter the electrons and appear dark on the image.

Scanning electron microscopy is a little bit like reflected light microscopy, but in this case it is not electrons from the original beam that return to the detector to make up an image. Instead, electrons scanned across the sample are absorbed, and secondary electrons are emitted from the sample. It is these secondary electrons that are captured in the detector. The denser a material is, the more secondary electrons it tends to produce. Unfortunately, most biological specimens are not very dense and therefore don’t emit many electrons. To get round this problem, samples are coated with a very fine layer of a dense substance (such as gold) to increase the number of secondary electrons and improve the quality of the image. Because SEM images are a record of electrons that have been emitted from the surface of the sample we tend to use it to look at surface structures, therefore sectioning is not usually necessary.

 

Pro’s and Con’s

Although the resolution of images acquired with an electron microscope is significantly better than those acquired with a light microscope, it is helpful to think of these as complimentary techniques. The main reason is that –

1. Understanding the structure of a biological specimen in fine detail is fundamental to understanding its function. Because electron microscopy can provide information at a higher resolution than light microscopy, it will always be an important technique in life sciences research.

2. It is often important to look at events in living cells to improve our understanding of them. Understanding the dynamics of bacterial chemotaxis, or organellar translocation from one part of the cell to another may reveal functionally important aspects of biology. Due to its sample imaging requirements – samples are imaged in a vacuum and at short wavelengths with high energy electrons – electron microscopy is incompatible with living cell imaging, and therefore light microscopy will excel at revealing the dynamic nature of living cells.