Controlled environment vitrification system: An improved sample preparation technique

Abstract

The controlled environment vitrification system (CEVS) permits cryofixation of hydrated biological and colloidal dispersions and aggregates from a temperature- and saturation-controlled environment. Otherwise, specimens prepared in an uncontrolled laboratory atmosphere are subject to evaporation and heat transfer, which may introduce artifacts caused by concentration, pH, ionic strength, and temperature changes. Moreover, it is difficult to fix and examine the microstructure of systems at temperatures other than ambient (e.g., biological systems at in vivo conditions and colloidal systems above room temperature). A system has been developed that ensures that a liquid or partially liquid specimen is maintained in its original state while it is being prepared before vitrification and, once prepared, is vitrified with little alteration of its microstructure. A controlled environment is provided within a chamber where temperature and chemical activity of volatile components can be controlled while the specimen is being prepared. The specimen grid is mounted on a plunger, and a synchronous shutter is opened almost simultaneously with the release of the plunger, so that the specimen is propelled abruptly through the shutter opening into a cryogenic bath. We describe the system and its use and illustrate the value of the technique with TEM micrographs of surfactant microstructures in which specimen preparation artifacts were avoided. We also discuss applications to other instruments like SEM, to other techniques like freeze-fracture, and to novel "on the grid" experiments that make it possible to freeze successive instants of dynamic processes such as membrane fusion, chemical reactions, and phase transitions.

Mechanisms behind the Faceting of Catanionic Vesicles by Polycations: Chain Crystallization and Segregation

“…Cryogenic transmission electron microscopy (cryo-TEM) studies were performed at 9, 25, and 30°C, following well-established procedures. 36 Before film preparation, Figure 1. Polymer-vesicle system investigated in this work: (a) structure of the two polymers; (b) phase maps of the LM200-vesicle system and JR400-vesicle system (adapted from ref 21).…”

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

“…Vitrification of water is achieved by very rapid cooling, usually by plunging the sample into liquid ethane cooled to its melting point 42. There is no dehydration, because all procedures are carried out in a humidity controlled environment,43, 44 which also allows the study of structures at different temperatures. Since the vitrification process is very fast, the sample is physically fixed in its current state 45.…”

Physicochemical and toxicological properties of novel amino acid-based amphiphiles and their spontaneously formed catanionic vesicles

“…A copper grid with a thin film of the sample was quickly vitrified by immersion in liquid ethane from the temperature of 25 • C [42]. The sample was then carefully transferred to a Philips CM 120 Bio-Twin transmission electron microscope under liquid nitrogen environment.…”