For ultrastructural analysis of biological specimens such as various tissues, cells (suspension and cell monolayers), bacteria or plants, the biological sample is stabilized by immersion in a mixture of chemicals containing Glutaraldehyde and formaldehyde. Glutaraldehyde reacts with many nucleophiles in the cell and produces irreversible cross-linking networks throughout the cytoplasm in seconds to minutes. Formaldehyde cross-links amino groups of proteins, and the reaction is much slower. The fixation reaction results in a drop in pH from a significant release of protons, making adequate buffering important.

Osmium tetroxide is used as a secondary fixative and it reacts with unsaturated acyl chains of membrane lipids and nucleophiles like amino and sulphhydryl groups. While its primary purpose is contrasting, it also increases the retention of lipids in the tissue. Uranyl acetate (UA) is then used to enhance the contrast by interaction with proteins and lipids with sialic acid carboxyl groups such as glycoproteins and ganglioside and to nucleic acid phosphate groups of DNA and RNA. UA delivers good contrasting results of membranes, nucleic acids and nucleic acids containing protein complexes such as ribosomes.

Following post-fixation with Osmium and UA, the sample is then completely dehydrated in solvents like propylene oxide or acetone, before infiltration with a plastic resin and polymerization at 50-60° C temperatures. Ultrathin sections (~70 nM) are cut from the sample blocks with a diamond knife using an ultra-microtome and transferred to a TEM grid. Grids are then stained with heavy metals such as uranyl acetate and lead citrate and imaged in the electron microscope. The image is formed by a beam of electrons passing through the sections, and contrast in the image arises from differential absorption of electrons by native or stained structures within the specimen.

Transmission electron micrograph of a section through a goblet cell and secretory cells of a mouse small intestine. Courtesy of Prof. Shalev Itzkovitz's group.