Transmission Electron Microscopy

Fig. 50.1

Transmission electron microscope, working at intermediate voltages. It consists of a tungsten filament as an emission source, electromagnetic lenses for beam convergence, a specimen stage to hold the grids, and a fluorescent screen to observe the image

Fig. 50.2

Flat embedding molds. After fixation, postfixation, dehydration, and embedding, the specimen is placed in flat molds. Epoxy resin (Spurr, Epon 812) is poured over the tissue and the molds containing specimens are placed in an oven at 70 °C for polymerization for 2 days. It must be ensured that no air bubbles are trapped within the resin

Fig. 50.3

(a) Knife preparation; knife maker used to prepare glass knives. Plastic troughs are fixed to the knives with dental wax. (b) Block trimming. The block should be trimmed (removing excess resin) to a trapezoid-shaped pyramid, with the section area being as small as possible, to include the part of the specimen of interest

Fig. 50.4

(a) Ultramicrotome used to obtain semithin and ultrathin sections. (b) Floating semithin sections (0.5–1 μm) are collected on slides and stained with toluidine or Richardson’s methylene blue

Fig. 50.5

Light microscope used for observation of semithin sections

Fig. 50.6

The quality of fixation can be appreciated in semithin sections. (a) Well-fixed and postfixed retina semithin section (×1,000). (b) In poorly postfixed samples, semithin sections show areas of different staining intensity, which indicates that the osmium tetroxide did not penetrate across the tissue (×400)

Fig. 50.7

Ultrathin sections (50–70 nm) are cut using a diamond knife and mounted on grids

Fig. 50.8

Staining of ultrathin sections using the Hiraoka Staining Kit. (a) The grids with the ultrathin sections are inserted into the slits in the plastic plate. (b) The plate with the grids is inverted over the dish containing uranyl acetate 1 % and Reynolds’ lead citrate successively. (c) The grids are stored in the grid box