Human Fibroblast Intracellular Network Prepared with Digitonin for Field Emission Scanning Electron Microscopy

Peter LEA, Robert J. TEMKIN
1992 Archives of histology and cytology  
Results obtained by extracting human fibroblast cells for the study of cytoskeletal structures are compared. Cells grown in culture were treated with digitonin in contrast to other methods of detergent preparation using Triton and Saponin. The three dimensional intracellular network which resulted from digitonin treatment was found to be similar in appearance to the structures observed by high voltage transmission electron microscopy of untreated cells described as a microtrabecular lattice by
more » ... becular lattice by PORTER and TUCKER (1981) . Our results, obtained by high resolution, field emission scanning electron microscopy indicate that the microtrabecular lattice may indeed be one conformation of a dynamic cytoskeleton. Advances in the development and design of high resolution field emission scanning electron microscopes (SEM) have achieved instrumental resolution beyond 1nm and are now approaching less than 0.5nm. However, major problems in imaging at this level of instrument resolution remain in how to prepare a specimen to actually obtain similar specimen resolution. Our present specimen preparation techniques are formulated to preserve the structural integrity of proteins in an attempt to obtain as much information as possible before the effects of the electron beam, including charging, heating and placing in a vacuum combine to reduce the specimen to ashes. A most important side effect is beam-induced specimen motion which becomes increasingly more pronounced when observing biological samples at high magnifications. Beam irradiation may result in broken co-valent bonds and, to counter this effect, the specimen may be destroyed by actual mass loss. In order to prolong the survival time of the proteins under the electron beam, the specimen should be irradiated as little as possible. If only a fraction of the specimen is exposed per unit time and this beam spot is rastered over the specimen,
doi:10.1679/aohc.55.suppl_57 pmid:1290677 fatcat:cjtile5tx5d7vgqgfawqwofm3y