The plastic-embedded tissue is sectioned on specially designed microtomes using diamond knives.
Because of the limited penetrating power of electrons, sections for routine transmission electron microscopy range from 50 nm to no more than 150 nm. Also, for the reason that abrasives used to sharpen steel knives leave unacceptable scratches on sections viewed in the TEM, diamond knives with a nearly perfect cutting edge are used. Sections cut by the diamond knife are much too thin to handle; they are floated away from the knife edge on the surface of a fluid-filled trough and picked up from the surface onto plastic-coated copper mesh grids. The grids have 50 to 400 holes/inch or special slots for viewing serial sections. The beam passes through the holes in the copper grid and then through the specimen, and the image is then focused on the viewing screen, CCD, or photographic film.
Routine staining of transmission electron microscopy sections is necessary to increase the inherent contrast so that the details of cell structure are readily visible and photographable.
In general, transmission electron microscopy sections are stained by adding materials of great density, such as ions of heavy metals, to the specimen. Heavy-metal ions may be bound to the tissues during fixation or dehydration or by soaking the sections in solutions of such ions after sectioning. Osmium tetroxide, routinely used in the fixative, binds to the phospholipid components of membranes, imparting additional density to the membranes.
Uranyl nitrate is often added to the alcohol solutions used in dehydration to increase the density of components of cell junctions and other sites. Sequential soaking in solutions of uranyl acetate and lead citrate is routinely used to stain sections before viewing with the TEM to provide high-resolution, high-contrast electron micrographs.
Sometimes, special staining is required to visualize results of histocytochemical or immunocytochemical reactions with the TEM. The phosphatase and esterase procedures are used for this purpose (see Fig. 1.3). Substitution of a heavy metal–containing compound for the fluorescent dye that has been conjugated with an antibody allows the adaptation of immunocytochemical methods to transmission electron microscopy. Similarly, routine EM autoradiography techniques have been refined for use with transmission electron microscopy (see Fig. 1.8b). These methods have been particularly useful in elucidating the cellular sources and intracellular pathways of certain secretory products, the location on the cell surface of specific receptors, and the intracellular location of ingested drugs and substrates.
Freeze fracture is a special method of sample preparation for transmission electron microscopy; it is especially important in the study of membranes.
The tissue to be examined may be fixed or unfixed; if it has been fixed, then the fixative is washed out of the tissue before proceeding. A cryoprotectant such as glycerol is allowed to infiltrate the tissue, and the tissue is then rapidly frozen to about 160 C. Ice crystal formation is prevented by the use of cryoprotectants, rapid freezing, and extremely small tissue samples. The frozen tissue is then placed in a vacuum in the freeze fracture apparatus and struck with a knife edge or razor blade.
The fracture plane passes preferentially through the hydrophobic portion of the plasma membrane, exposing the interior of the plasma membrane.
The resulting fracture of the plasma membrane produces two new surfaces. The surface of the membrane that is backed by extracellular space is called the E-face; the face backed by the protoplasm (cytoplasm) is called the P-face. The specimen is then coated, typically with evaporated platinum, to create a replica of the fracture surface. The tissue is then dissolved, and the surface replica, not the tissue itself, is picked up on grids to be examined with the TEM. Such a replica displays details at the macromolecular level (see Fig. 2.5, page 30).