| Electron Microscopy |
What is electron microscopy (EM)? The resolution of the light microscope is limited to about 0.5 µm as visible wavelengths are relatively long (c. 300 - 500 nm). (NB. 1 µm = 1 millionth of a metre = 1 x 10-6 m, and 1 nm = 1 billionth of a metre = 10-9 m). This means, for example, that although individual cells can be viewed, relatively little of their internal structure (eg. organelles) can be discerned in any detail. In contrast, EM utilises a high energy beam of electrons which have a considerably smaller wavelength than light (c. 0.1 nm), and hence have a much greater resolution (< 1 nm). In addition to greater resolution, EMs also have much greater depth of field and focus than light microscopes at equivalent magnifications. Why use EM? As stated above, EM is excellent for viewing the fine structure of cells, and any other samples that are too small to be resolved under the light microscope. Further examples of biological material that benefit greatly from EM are bacteria (at c. 1 – 5 [m these can be seen under the light microscope but no details can be resolved eg. cell walls, flagellae) and viruses (at only a few nm these are impossible to see under the light microscope). What types of EM are there? There are two basic types of EM: 1. Transmission EM (TEM). As the name implies, this involves the transmission of the electron beam through a sample so that detail can be discerned of areas that are more or less opaque to the electrons. This is analogous to a compound light microscope, in which light is transmitted through a section (1 to several [m in thickness) of a specimen mounted on a glass slide, and the specimen, which is often stained with a dye, is then viewed as an image with regions of greater or lesser colour intensity, depending on it’s nature and density and/or how it has absorbed the applied stain. In the case of TEM, very thin sections (c. 70 nm) are prepared from a specimen using an ultramicrotome (LINK) and the tissues in the section are differentially stained using solutions of heavy metal salts (eg. those of lead and uranium) which, because they are so dense, are opaque to the electrons transmitted through it. Therefore, unstained or lightly stained regions appear on the viewing screen as clear areas, whereas parts of the section that have absorbed the heavy metal stains appear as darker regions.TEM tends to be used for the analysis of internal structure eg. of cells, and therefore samples need to be sectioned on an ultramicrotome. However, it can also be used to examine very small whole, unsectioned, specimens, such as bacteria (particularly their flagellae) and virus particles. 2. Scanning EM (SEM). As with TEM, specimens are subjected to a beam of electrons, but instead of the electrons being transmitted through the specimen, the beam is "scanned" across it creating a 3-dimensional image in a TV screen. This image is achieved via the detection of "secondary" electrons that are released from the specimen as a result of it being scanned by very high energy "primary" electrons (ie. those emitted from the electron "gun" in the SEM). As most biological specimens are made up of non-dense material the amount of secondary electrons produced is too low to be of much use in creating an image and therefore they are usually coated with a very fine layer of a metal which readily produces secondary electrons (eg. Au/Pd, Cr). Therefore, in summary, a 3-D image of the metal-coated specimen is produced as the electron beam scans across it, resulting in the release of secondary electrons that are detected and then amplified for viewing on a screen. Contrast in the specimen is seen whenever the secondary electron signal changes as the electron beam scans across it. SEM is generally used for analysing surface morphology and topography, and therefore sectioning is not usually necessary. Fixation "Fixation" is essential for virtually all biological EM samples. The purpose of fixing a sample is to firstly prevent it from deteriorating (eg. once it has been excised from its parent organism) and secondly to stabilise it so that it can be subjected to further microscopical analysis (and the various steps leading up to this) without its structure changing significantly from its "natural" state (ie. that before it was fixed). |
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