Glutaraldehyde, which is used as a fixative, is efficient in cross-linking proteins and maintaining cell ultrastructure, but penetrates tissues relatively slowly. Formaldehyde penetrates tissues rapidly, apparently due to its low molecular weight. Osmium tetroxide, most commonly used for postfixation, acts both as a fixative and as an electron stain. It preserves many lipids and it is able to stabilize some proteins by transforming them into clear gels without destroying many of their structural features (12).

Sample fixation is followed either by en bloc staining to enhance contrast or by OTOTO (osmium tetroxide/thiocarbohydrazide/osmium—tetroxide/thiocarbohydrazide/osmium—tetroxide) staining for conductivity. In the OTOTO conductive staining technique, thiocarbohydrazide (T) is used as a ligand. It is bidentate, binding the osmium (O) tetroxide molecules together giving T–O–T. In this manner the natural affinity of osmium for unsaturated lipids is enhanced or amplified. The electrical conductivity imparted to the tissue by this increased deposition of osmium reduces specimen charging during the SEM operation (13).

Critical point drying (CPD) is an automated process that requires approximately 40 min to be completed. It relies on the following physical principle: the liquid in the biological tissue is replaced with a suitable inert fluid whose critical temperature at a realizable pressure is just above the ambient temperature. The choice of fluids is severely limited and despite early work with Freon 13 and N₂O, CO₂ is now commonly used. With CO₂, a critical point of approximately 35°C can be achieved at a pressure of about 1,200 psi. Samples are transferred in 100% acetone to the pressure chamber of a critical point dryer. When the acetone is replaced with liquid CO₂ and the temperature is raised to above the critical temperature, the liquid CO₂ vaporizes with no change in density. As a consequence, there are no surface tension effects that can distort morphology and ultrastructure. However, another method using hexamethyldisilazane can be used for drying. In our experience, the two drying methods are found to produce a similar appearance of cellular ultrastructure. When the two protocols are compared, we find that HMDS drying takes less effort, has lower costs for chemicals, requires no specialized equipment and has a high rate of success. In our laboratory and elsewhere (14), CPD- and HMDS-dried samples have typically shown equally good results (Fig. 2) (15). The high magnifications in Fig. 2a, b are unusual for SEM investigation of biological samples prepared by drying. This is evidence of satisfactory sample stabilization and conductivity, as well as, of successful FIB operation.

For drying with Hexamethyldisilazane:

Dried samples are mounted on standard SEM microscope stubs prior to FIB/SEM investigation. Double-stick adhesive tape, conductive silver paint, or both can be used to attach specimens to stubs. Due to the small size of specimens and because sometimes samples are mechanically opened in order to expose their subsurface structure, a stereo microscope with magnification up to 10× is indispensible.

Samples are sputter coated with a layer of carbon or platinum/palladium, or other metals (thickness of ∼10–30 nm). Metal atoms ejected from the target by the ionized gas cross the plasma and are deposited onto any surface within the coating unit, including the specimen. A low vacuum is used (0.1–0.05 mbar), which with a modern low voltage sputter coater, enables metal to be deposited at a rate of 1 nm/s. For SEM imaging, when high magnifications are required, sputtering is a very important step that is needed to ensure the thickness and evenness of the cover layer. In Fig. 3a, b we provide examples of different qualities of sample coating.

Fixation is the same as described above (Subheading 3.1.1), using a mix of paraformaldehyde and glutaraldehyde in sodium cacodylate buffer.

Following fixation, the samples are stained en bloc for enhancement of contrast. Uranyl acetate, usually used for staining thin sections for transmission electron microscopy, is used here for en bloc staining before dehydration. It also acts as both an electron stain and as a fixative. As a fixative, uranyl acetate stabilizes membranous and nucleic acid-containing structures but also reacts with proteins (12).

The dehydration steps are the same as described above (Subheading 3.1.3), using an increasing series of ethanol concentration.

The embedding steps are similar to those commonly used for preparing samples for TEM. To embed our samples we use Spurr’s-based resin.