AEMI: Cryosectioning

Specimen preparation and Cryo-protection

The real breakthrough in cryosectioning of biological material came in 1973 when Tokuyasu introduced the use of sucrose as a cryo-protectant after fixation but prior to freezing (Tokuyasu 1973). This step meant that the material could be frozen into a vitrified (i.e. no ice crystals formed) state just by immersing it in liquid nitrogen. Tokuyasu found that immersion in 1.6 M sucrose was enough to allow this freezing to occur. Once vitrified, biological material is relatively easy to section at low temperatures.

The use of 2.3 M sucrose as a cryoprotectant, first used by Janice Griffith in Jan Slot's laboratory made the blocks easier to section and introduced the possibility of manipulating the block hardness. By varying the concentration of sucrose used it is possible to alter the hardness of the block (lower the concentration of sucrose and the harder is the block). Alternatively, if the sucrose concentration is kept constant then the block hardness can be manipulated by varying the sectioning temperature (lower the temperature to get harder blocks).

For routine sectioning, it is enough to leave the tissue or cells in 2.1 M sucrose in phosphate buffered saline (PBS) for 15-60 min, depending on the block size and cell density. For tissue pieces it is just a matter of trimming the blocks into small pyramid-shaped pieces and placing them into the sucrose. Cell pellets can be a little more difficult.

If cells are fixed in suspension, pelleted down as soon as the fixative is added and the pellet is left in the presence of the fixative then the cell pellet should hold together as one piece. This can be carefully manipulated with forceps. If the cell pellet cannot be manipulated with forceps, small drops of the concentrated cell suspension may be placed directly into a small amount of cryo-protectant. After a suitable incubation time a drop of the cell suspension, in sucrose, is placed on a specimen stub and frozen by immersion in liquid nitrogen. However, this method of preparation may result in a low concentration of cells in the sections.

An alternative way of keeping cell suspensions together is to embed them in gelatin. Cells can be pelleted down in warm 10% gelatin and, after the gelatin has cooled, the embedded pellet is cut into pieces for infiltration with cryo-protectant. (N.B.If the warm gelatin is not centrifuged prior to use then it can be difficult to section). Infiltration is done on ice to prevent the gelatin from warming. Lower concentrations of gelatin may be used and fixed prior to cryo-protection.

An improvement introduced by Tokuyasu (1989) is to use a mixture of 1.6M sucrose and 10-30% (w/v) polyvinyl pyrolidone (PVP, with a molecular weight of 10,000) instead of sucrose. The PVP appears to act as a support, entering extracellular spaces, and greatly facilitates the sectioning process. It may require that the biological material be left in the solution for longer than for sucrose alone.

The PVP-sucrose solution is not stable if left at room temperature so should be stored in a refrigerator or in a freezer (see below for information on storage).

Mounting Specimens and Freezing

Ideally the pieces of tissue or cell pellets should be trimmed to a pyramidal shape so that when mounted onto the specimen stub the base is wider than the top. If possible, the block face should be flat and resemble a square or trapezoid. The block is mounted onto the specimen stub, excess sucrose is removes with a damp filter paper and the specimen stub with block is quickly immersed in liquid nitrogen until freezing is complete.

Some sucrose should be left around the base of the block so as to anchor the block to the stub. It is also important not to allow the specimen to dry out. Not only will higher sucrose concentrations produce softer blocks but there is also a possibility of fine structure damage occurring.

If the sucrose has completely penetrated the sample then vitrification of the specimens can be achieved simply by immersion in liquid nitrogen (Griffiths et al, 1984). No improvement of the cooling can be made using faster coolants. Once frozen, the specimens can be stored indefinitely under liquid nitrogen. They can also be sectioned and stored multiple times.

Freezing the sample: Once the sample is fixed and cryoprotected by immersion in 2.3M sucrose, it is cut into small pieces and mounted onto a metal specimen pin. The sample in this drawing is shaped in the form of pyramid. Once on the pin, the sample and pin are frozen by immersion in liquid nitrogen and transferred rapidly to the cooled cryochamber of an ultramicrotome.

Next step: Cryosectioning.

References Cited

Tokuyasu, K. T. 1973.

A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol.. 57:551-565.

Tokuyasu, K. T. 1989.

Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy. Histochem. J. 21:163-171.

Griffiths, G., A. W. McDowall, R. Back and J. Dubochet. 1984.

On the preparation of cryo-sections for immunocytochemistry. J. Ultrastruct. Res. 89:65-78.

 

Ahmanson Center for
Advanced Electron Microscopy and Imaging