ANATOMY AND HUMAN BIOLOGY
Electron Microscopy for Biological Samples
Luis Filgueira
General Principals
The principal of microscopy is to illuminate the sample to get information about it. In electron microscopy (EM), the specimen is illuminated with an electron beam and the illumination source is an electron gun. Similar to light microscopy, there is a sequence of lenses to manipulate the beam which in the case of EM are electromagnetic. A high vacuum is needed to sustain and control the electron beam. Consequently, the samples have to be processed specifically for that purpose that the vacuum is not disturbed
Roughly, two different types of electron microscopes are available: A) transmission electron microscope, B) scanning microscope. The following paragraphs will describe both types and the corresponding most common biological applications.
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The Centre for Microscopy and Microanalysis (CMM) at The University of Western Australia
What are Electron Microscopes?
Transmission Electron Microscopy (TEM)
The principals of TEM is very similar to that of a light microscopy (see Figure 1). The illumination source at the beginning generates a beam. This beam is focused on the specimen by the condenser lens. The resulting image of the specimen is modified through the objective lens and the projector lens. The electron image is not visible for human eyes. Therefore, the image is transformed to a visible one on a fluorescent screen. Finally, the image of the specimen is somehow a shadow-graph of it, i.e. black-and-white. For light microscopy, the specimen has to be transparent for visible light. For TEM, the specimen has to be transparent for the electron beam. Electrons are very easily absorbed. Consequently, the specimen has to be thin enough (around 100nm) to get an image due to the transmission and scattering of the electron beam trough it. Image contrast is achieved by strongly scattered electrons, failing to reach the viewing screen. The higher the atomic number of the elements in the specimen, the more scattering and the more contrast results for the image. Most biological materials are composed of low atomic number elements such as H, N, O and C, resulting in a very low image contrast. However, the contrast can be enhanced by staining the specimen with salt solutions of metals with high atomic number, e.g. Os, Pb and U.
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Figure 1: Comparison between a light microscope and a transmission electron microscope (courtesy of Martin Saunders, CMM)
Applications: The maximal resolution of TEM is in the range of 1nm (light microscopy: 500nm). Due to this high resolution and the fact that the specimen has to be sliced to 100nm thickness, TEM is very well suited for most human and animal tissues, where ultra-structural information is required. Ultra-structural information can be obtained with TEM from the following cellular and extracellular structures: Golgi-complex, mitochondria, endoplasmic reticulum, lysosomes, vesicles, membranes, nucleus, cytoskeleton, extra-cellular matrix (collagen) and inter-cellular junctions. It is possible to include additional information using specific labeled antibodies. This technique is called immune electron microscopy.
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Processing of the specimen for conventional TEM: Different sample processing methods are available for TEM. The biological material has to be fixed. Usually, for conventional TEM, the samples are fixed with glutaraldehyde (1% to 3%) and/or paraformaldehyde (1% to 4%) in buffered salt solutions. Both, the fixative and the buffer solution have to be adequate and optimized for the specific tissue. There are protocols available for most animal tissues. After the first fixation step, often the sample is post-fixed with osmium (1% OsO4), which stabilizes and already stains the lipid layers of biological membranes.
The tissue has to be cut into 100nm thick slices. This is done with a sharp knife made of glass, semi-diamond or diamond using an ultramicrotome. The specimen must not be too soft; otherwise it is not possible to obtain thin slices. To increase the hardness, the tissue is embedded into a resin (e.g. epon). For this purpose, the tissue is passed through an alcohol series (dehydration) into the liquid resin, which polymerizes afterwards to a hard piece containing the tissue.
Usually the slices (~ 500m in diameter) are placed on copper grids for easy handling. To enhance the contrast, the specimen is often stained with lead citrate and uranyl acetate.
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Sample preparation:
Technical tips:
Reagents for TEM:
Knives:
For immune electron microscopy (IEM), beside the optimal conservation of the tissue for a good ultra-structural morphology, the antigenicity of the target molecules for the labeling with the specific antibodies has high priority. It is important to fix the tissue in a way that the antibody binding side is not denaturated. Usually, glutaraldehyde concentration has to be kept low (less than 0.5%), which has to be compensated with a higher concentration of paraformaldehyde (2% to 4%). It is recommended to test the antibodies with light microscopy methods first (immune fluorescence or immune cytochemistry), using the tissue processed with different IEM protocols (variation of fixatives, buffers or resins).
Resins normally used for conventional TEM, such as epon, can not be used for IEM. Different resins are on the market for IEM. LR White is a resin recommended and widely used for IEM. The only disadvantage of most of the resins for IEM is the poor contrast for membrane structures. If membrane structures are of interest, the tissue has to be process for cryo-IEM, which means, that the tissue is cut frozen. For this procedure the technical equipment has to be more sophisticated, as the tissue has to be cut in liquid nitrogen. The most common difficulty for cryo IEM is formation of ice crystals during freezing and thawing of the specimen which disrupts the ultra-structure.
The specimen slices on the grid can be treated like other specimens for any immune staining. The only difference is that the secondary antibody is labeled with gold particles sized 1 to 30nm in diameter. Thus, it is finally the localization of the gold particles which indicates the location of the immunogenic structure.
IEM Methods:
Surface Scanning Electron Microscopy (SEM)
SEM is used to get information about the surface of the specimen. It is very well suited for single cells or epithelial surfaces, as well as for smaller organisms, such as insects, parasites and even bacteria. Usually, SEM is not useful for tissue biopsies. The technical principals of SEM are similar to TEM: there are the electron gun, and the electromagnetic lenses. The big difference is that the beam is scanned over the specimen. Detection of the secondary electrons produced through the beam-specimen interaction generates the image on a screen.
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For SEM sample preparation, usually, the specimen has to be fixed with glutaraldehyde. Thereafter, the specimen has to be dried in a way that the surface is not altered. Critical point drying is an easy and widely used method. The dry specimen is usually mounted on an aluminium stub. Usually, the specimen is coated with a thin carbon layer (20nm) and/or sputtered with gold (10nm) to increase conductivity and contrast.
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