Electron Microscopy of Structural Detail in Frozen Biological Specimens

Abstract

A procedure is described whereby preshadowed replicas can be obtained from frozen biological specimens which have been cut and then etched by sublimation of the ice from their surfaces. Electron micrographs showing details of the internal structure of plant virus crystals are presented to demonstrate the values of the procedure. Crystals of purified tobacco ringspot virus and squash mosaic virus and some portions of turnip yellow mosaic virus crystals have been shown to exhibit hexagonal packing. Sections through in situ crystals of tobacco mosaic virus show the rods to be parallel within each layer and arranged in a square net as viewed end on. Individual rods in each layer of the latter measure 300 mµ in length and are somewhat tilted with respect to the rods of adjacent layers. This results in the formation of a herring-bone appearance when a crystal is cut perpendicular to its hexagonal face. It is suggested that the procedure outlined here might well serve to supplement other procedures for the preparation of many cytological specimens for electron microscopy.

Collecting, shipping, storing, and imaging snow crystals and ice grains with low‐temperature scanning electron microscopy

“…Similarly, cryofixation and coating, which consist of plunging a sample into a cryogen, and coating with a heavy metal, are not new procedures. Nearly 50 years ago, Steere (1957) demonstrated the use of these techniques to prepare replicas of virus particles for observation in a transmission electron microscope. Through the years, this technique has also been widely applied to biological studies.…”

In focus in HCB

“…Given the above introduction, it is perhaps fitting and appropriate that the first issue of HCB in 2018 includes a review of the technique of freeze fracture for the ultrastructural analysis of cellular membranes, which was reported about 60 years ago (Steere 1957;Moor et al 1961). In this technique, aldehyde-fixed cells or pieces of tissues are cryoprotected followed by rapid freezing and fracturing by a microtome in a liquid nitrogen cooled apparatus under a high vacuum.…”

Catching sight of lysosomes

“…With this modest claim, Steere et al (1957) introduced a method that has played "an absolutely critical role in the elucidation of membrane structure," according to L. Andrew Staehelin (University of Colorado, Boulder, CO). Steere started with frozen samples and then combined a series of known methodsusing an ultramicrotome for cutting, freeze drying to etch and expose surface features, and finally creating a replica with heavy metals.…”