Conventional TEM

Visualizing a biological specimen with an electron microscope is a complex process primarily due to the inherent interactions between electrons and matter that contribute to image formation. To enable imaging using TEM, the specimen must be extremely thin and placed within a high vacuum environment. As a result, biological specimens cannot be imaged in their natural state and require extensive processing. Therefore, sample preparation plays a critical role.

A conventional protocol for preparing samples for ultrastructure imaging typically involves the following steps:

Primary fixation with aldehydes (proteins): During this step, proteins and other cell molecules are crosslinked using formaldehyde and/or glutaraldehyde. The specimen should be dissected to a maximum thickness of 1 mm in at least one direction and fixed through immersion (using buffered 2.5% glutaraldehyde/paraformaldehyde solution).

Secondary fixation with osmium tetroxide (lipids): This step ensures the preservation of lipids, such as phospholipids that form membranes, preventing their extraction during dehydration. The fixation process results in the formation of a black insoluble precipitate on the membranes, providing membrane contrast (using 1% osmium tetroxide in water).

Dehydration series with solvent (ethanol or acetone): The fixed specimen is dehydrated using a series of ethanol or acetone solutions. The concentration of the solvent is gradually increased to gently remove water without causing artefacts such as shrinkage.

Resin infiltration and embedding: After dehydration, the solvent is replaced with liquid resin (typically epoxy) with a gradually increasing concentration. The specimen is placed in a mould filled with liquid resin and cured into a solid block using heat. Once this step is completed, the sample can be stored indefinitely.

Sectioning and mounting sections on specimen grids: The specimen embedded in hardened resin can be sectioned into extremely thin slices, typically less than 100 nm. These sections are then mounted on copper grids, which fit into the microscope sample holder.

Contrasting: Biological specimens are naturally not very electron-opaque due to their composition of low atomic number atoms, which allows the electron beam to pass through easily. To enhance sample contrast, the sections can be post-stained with lead citrate and uranyl acetate, which bind to cell components and scatter the incident beam.