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15.1 Why does the use of electrons allow cellular organelles to be visualized in great detail?
The limit to resolution in an optical system is the wavelength of the illumination used. Using visible light, the wavelength is around 400 - 700 nm and the limit to resolution around 200 nm. Electrons have dual wave/particle characteristics. Their wavelengths are thousands of times shorter than that of visible light, hence the resolution that can be achieved is much better, around 0.2 nm in typical EMs, allowing much finer detail to be seen.
15.2 The passage of electrons is blocked very easily. What impact does this have on the design of the EM?
Air molecules inside the microscope would block the electron beam. Hence, the inside of the microscope must contain a vacuum. This imposes major technical requirements – vacuum pumps that can maintain the vacuum permanently, valves that are operated by compressed air that control the flow of air as it is pumped from the microscope and seals between components that can withstand the vacuum. In addition, to introduce samples without losing the vacuum requires a special airlock to enable rapid sample insertion and removal.
15.3 Why are formaldehyde-based fixatives a second choice for TEM? Think about chemical composition and what happens when formaldehyde solutions are stored.
The formaldehyde molecule contains a single aldehyde group that binds to proteins, cross- linking them. The better alternative, glutaraldehyde, contains two aldehyde groups and is therefore better at cross-linking, improving the preservation. Formaldehyde needs to be made fresh because after a relatively short period of time, polymerisation occurs and it is no longer as effective.
15.4 Why are tissue blocks for EM impregnated with resin and not wax?
To cut sections for microscopy requires giving support to the tissue, otherwise it would deform or break up when it touches the cutting blade, especially when the sections needed have to be thin (about 4 – 10 m) to allow light through, or ultrathin (70 – 100 nm) to allow electrons to pass through. Wax is not hard enough to allow ultrathin sections to be cut, so resin is used that is much harder than wax, once polymerised.
15.5 Why are tissues fixed in glutaraldehyde and processed into araldite resin generally not suitable for immunoelectron microscopy?
The extra cross-linking achieved by glutaraldehyde modifies proteins to a greater extent than formaldehyde which is essentially a weaker fixative. Thus the antigenic site recognised by the antibody in immunoelectron microscopy may be modified to an extent that the antibody, normally made against the native (unfixed) protein or an amino acid sequence from it, no longer binds to it. Araldite resins are not miscible with water so the small amount of antibody penetration needed to bind to the sample is harder to achieve and the aqueous solutions used in labelling the sections are repelled by the section surface.
15.6 At what thickness are ultrathin sections cut, and why is this important?
Around 90 nm (70 nm – 100 nm) typically (although thinner and thicker sections can be used but with increasing difficulty in handling and observation). This is necessary to allow electrons to penetrate through the section. However, it also means that the apparatus needed to cut the sections is technically more complicated than for light microscope wax sections.
15.7 Why do we not pick up ultrathin sections on a glass slide?
The EM beam will not penetrate through the thickness of a glass slide, hence sections are collected onto grids which have a fine mesh. Different sizes of mesh are available, but all obscure the section to some extent because the beam will not penetrate through the bars of the grid that form the mesh. Imaging is done through the gaps between the bars. It is possible to use a very thin plastic support that can be made quite easily to cover grids that lack a mesh (slot or hole grids). This plastic support allows the full section to be observed, but has other problems associated with it such as it tends to pick up stain deposits and it is also very delicate so is easily ruptured.
15.8 Why are heavy metals used as stains for EM?
Heavy metal atoms tend to scatter electrons more easily than the typical components of biological tissue, which are generally relatively small atoms. This scattering increases the contrast in the EM by scattering more electrons out of the beam path, making the areas stained more heavily darker on the image.