Biological Electron Microscopy: Theory, Techniques, and by Michael J. Dykstra, Laura E. Reuss

By Michael J. Dykstra, Laura E. Reuss

Electron microscopy is often portrayed as a self-discipline that stands by myself, separated from molecular biology, gentle microscopy, body structure, and biochemistry, between different disciplines. it's also awarded as a technically not easy self-discipline working principally within the sphere of "black bins" and ruled via many absolute legislation of technique. on the introductory point, this portrayal does the self-discipline and the scholar a disservice. The instrumentation we use is complicated, yet finally comprehensible and, extra importantly, repairable. The strategies we hire for getting ready tissues and cells aren't completely understood, yet sufficient details is accessible to permit investigators to make moderate offerings in regards to the most sensible suggestions to use to their parti­ cular difficulties. There are numerous really good strategies within the box of electron and light-weight microscopy that require the purchase of specialised wisdom, rather for interpretation of effects (electron tomography and effort dispersive spectroscopy instantly come to mind), yet such a lot laboratories owning the gear to impression those methods have experts to aid the informal consumer. the appearance of machine operated electron microscopes has additionally broadened entry to those tools, permitting clients with little technical wisdom approximately electron microscope layout to quick develop into operators. This has been a welcome strengthen, simply because previous instru­ ments required a degree of information approximately electron optics and vacuum platforms to supply optimum photos and to prevent "crashing" the tools that sometimes made it tough for newcomers.

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Extra resources for Biological Electron Microscopy: Theory, Techniques, and Troubleshooting

Example text

This kind of change is important to consider when designing protocols and evaluating resulting photographs. This whole section on fixation should make it clear that a variety of approaches can usually be taken to solve the same problem. If the initial simple attempts with general protocols fail, move on to more esoteric formulations to stabilize the samples in question. The approach in our laboratory is to try something like 4F: 1G fixative first; we rarely have to move on to the more complicated procedures.

Specimen Preparation for Electron Microscopy 29 5. 5 and thus finds most use in cytochemical incubation solutions for demonstrating enzymes with activity under alkaline conditions. It may react with glutaraldehyde, so washing after primary fixation must be thorough. 6. Sodium Bicarbonate Sodium bicarbonate is frequently encountered in buffers prepared for physiological studies but is not commonly recommended for electron microscopy fixative preparation. Salema and Brandao (1973) suggested that it was superior to phosphate buffers for the preservation of plant tissues.

When the specimen has sunk to the bottom, the capsule is rapidly immersed in liquid nitrogen. Ultrathin sections are then cut (though the blocks are extremely hydrophilic and must be blotted dry frequently during sectioning) and placed on Formvar and polY-L-lysine-coated grids (the latter to keep the sections attached to the plastic film). They are next dehydrated again to remove the PEG and critical-point dried. This results in critical-point dried sections on grids without any surrounding medium, which thus have sufficient contrast to eliminate the need for poststaining.

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