Cryo-electron microscopy of vitrified specimens

Abstract

Cryo-electron microscopy of vitrified specimens was just emerging as a practical method when Richard Henderson proposed that we should teach an EMBO course on the new technique. The request seemed to come too early because at that moment the method looked more like a laboratory game than a useful tool. However, during the months which ellapsed before the start of the course, several of the major difficulties associated with electron microscopy of vitrified specimens found surprisingly elegant solutions or simply became non-existent. The course could therefore take place under favourable circumstances in the summer of 1983. It was repeated the following years and cryo-electron microscopy spread rapidly. Since that time, water, which was once the arch enemy of all electronmicroscopists, became what it always was in nature – an integral part of biological matter and a beautiful substance.

Transmission Electron Microscopy as a Tool for the Characterization of Soft Materials: Application and Interpretation

“…The sample is vitrified in a thin layer of solvent and imaged at very low temperatures, such that the medium neither changes phase nor evaporates in the high vacuum 41. The thin layer is made by applying and subsequently blotting a drop of sample on a lacy or holy carbon coated grid.…”

Label-Free Visualization of Ultrastructural Features of Artificial Synapses via Cryo-EM

“…Cryo-EM is a transmission electron microscopy (TEM) based technique that allows biological samples to be observed in a fully hydrated physiological environment without fixing or staining. 9 Although the approach presented here requires some alternative methods in cell culture, the observation is direct and does not require further sample processing steps that can be both tedious and experimentally biasing. This is especially true in the case of artificial synapses formed on silica beads, where thin sectioning of samples using an ultramicrotome would be difficult to achieve.…”

Magnification calibration and the determination of spherical virus diameters using cryo-microscopy

“…The high particle concentration favors the alignment of the particles into near-parallel arrays. Once in the microscope, the anticontamination shield on the specimen cold holder was retracted and the specimen left exposed to the microscope vacuum for a period of time (~ 1 h) to allow water molecules in the column to condense on the specimen in the form of cubic ice [29]. Electron diffraction patterns were obtained with the first condenser lens fully excited, with a 12.5 µm second condenser lens aperture in place, and with the objective lens aperture removed.…”

“…One might expect such expansion to create forces which would act to compress the biological sample [43]. Apparent resistance of the sample to structural change caused by vitrification could indicate that water redistributes during freezing or that differences exist between the density of vitreous water in the free solvent compared with the hydration shell surrounding the biological molecule [29].…”

“…Cryo-microscopy of biological specimens has emerged as one technique capable of minimizing or eliminating many of the problems and artifacts associated with negativestaining and other conventional electron microscopy preparative procedures [28][29][30][31][32]. The crystalline structure of a variety of frozen-hydrated biological specimens is preserved to near atomic resolution.…”