A precision cryostat design for manual and semi-automated cryo-plunge instruments.

Christopher J Russo,Steve Scotcher,Martin Kyte

doi:10.1063/1.4967864

Christopher J Russo, Steve Scotcher + Show 1 more

Open Access

https://doi.org/10.1063/1.4967864

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Abstract

Here we describe a bench-top cryostat system to control the temperature of liquid ethane in a cryo-plunge apparatus designed for biological specimen preparation for electron cryomi-croscopy. It comprises a foam insulated Dewar containing a copper cryostat cup, whose temperature is controlled via an active feedback system to within 0.1 K. The device can easily be incorporated into existing manual and semi-automatic cryo-plunge instruments that are not equipped with cryogenic temperature control. Over the course of normal use, we find that using a cryostat is convenient, fast, and does not require special mixtures of cryogens like ethane/propane. This simple cryostat improves the reliability and reproducibility of biological specimen preparation for electron cryomicroscopy.

Highlights

We describe a bench-top cryostat system to control the temperature of liquid ethane in a cryo-plunge apparatus designed for biological specimen preparation for electron cryomicroscopy
Over the course of normal use, we find that using a cryostat is convenient, fast, and does not require special mixtures of cryogens like ethane/propane
This is usually achieved using the method developed by Dubochet and colleagues, by rapidly plunging a thin biological specimen into a liquid cryogen near its melting point, at a temperatures near that of liquid nitrogen at its boiling point (77.4 K)

Summary

INTRODUCTION

Electron cryomicroscopy (cryo-EM) depends on the ability to freeze water quickly enough to ensure it enters an amorphous solid phase. The recent advances in the field have vastly increased the number of specimen being prepared for high-resolution structural analysis and the number of laboratories using cryo-preparation methods to vitrify specimens for imaging with electrons. This is usually achieved using the method developed by Dubochet and colleagues, by rapidly plunging a thin (typically less than one micrometer thick, limited by the thermal conductivity of water) biological specimen into a liquid cryogen near its melting point, at a temperatures near that of liquid nitrogen at its boiling point (77.4 K). In the intervening years, several different instruments have been developed to perform this rapid freezing process, and each incorporates varying degrees of automation and offers some control over the parameters of the process like plunge speed, humidity surrounding the specimen, blot time, and force. Other more complicated techniques like freeze-fracture deep etching, high-pressure freezing, and slam freezing are used for the preparation of specimen for electron microscopy but require more complicated instruments and are beyond the present scope. The recent advances in the field have vastly increased the number of specimen being prepared for high-resolution structural analysis and the number of laboratories using cryo-preparation methods to vitrify specimens for imaging with electrons.2,3 This is usually achieved using the method developed by Dubochet and colleagues, by rapidly plunging a thin (typically less than one micrometer thick, limited by the thermal conductivity of water) biological specimen into a liquid cryogen near its melting point, at a temperatures near that of liquid nitrogen at its boiling point (77.4 K).. It is desirable to keep the ethane temperature close to its melting point as this ensures proper vitrification of the specimen and prevents the formation of crystalline ice.1 This presents a practical problem during cryo-preparation since the ethane will freeze solid if it comes into contact with liquid nitrogen or is indirectly cooled to near liquid nitrogen temperatures with a container which is in thermal contact with liquid nitrogen. While the design is made for the FEI Vitrobot and the custom-built manual plungers of Talmon type, the principles of the design can be adapted to other freezing instruments and cryostats in general

INSTRUMENT DESIGN

RESULTS AND DISCUSSION

CONCLUSIONS