Measurement of cooling rate during plunge freezing of sample preparation in cryo electron microscopy

Abstract

Plunge freezing is used as a sample preparation method to freeze biological samples in a vitrified thin water layer of <500nm. Typically sample is loaded on thin (<10nm) carbon membranes of a copper mesh grid. The vitrified biological sample is imaged using high resolution cryogenic transmission electron microscopy (Cryo-EM).The common practice of rapidly plunge freezing biological material prepares it to be placed in the vacuum chamber of the Cryo-EM by cooling it to a vitrified state (causing vitrification). In the present sample preparation methods, 90% of the biological materials used experience some form of contamination or damage during the transfer process and end up unusable. While improvements have been made, such as the automation of sample loading into the Cyro-EM, these improvements have brought on their own set of complications. In want of a better solution for the issues surrounding the cryo-sample preparation and loading, the research and experiments of this thesis focused on the question: is it possible for biological material to be vitrified on an Autogrid with plunge freezing? To answer this question experiments were carried out to measure the cooling rate of EM-grid and Autogrids during plunge freezing with a Vitrobot. A small thermocouple was embedded into an EM standard-grid and Autogrid glued in place with cryo-varnish. The EM-grid was then mounted onto Vitrobot tweezers and plunged into liquid ethane (cryogen). The set-up mimics to the best of its possibilities the same conditions and procedure of the standard Cyro-EM workflow and temperature was recorded at a rate of 32000 samples per second. Cooling rates measured with an EM-grid were as expected. The cooling rate of the Autogrid is slower than that of standard grid, which implies that the Vitrobot is not capable of vitrifying biological materials on an Autogrid. Research and experiments showed that the Vitrobot would probably not be capable of achieving vitrification on Autogrid because the measured cooling rate was slower then that of EM-grid. Cooling rates with the Vitrobot could be improved by a faster and deeper plunger.