A versatile high‐vacuum cryo transfer system for cryo microscopy and analytics

Abstract

Cryogenic microscopy methods have gained increasing popularity, as they offer an unaltered view on the architecture of biological specimens. As a prerequisite, samples must be handled under cryogenic conditions below their recrystallization temperature, and contamination during sample transfer and handling must be prevented. We present a high-vacuum cryo-transfer system that streamlines the entire handling of frozen-hydrated samples from the vitrification process to low temperature imaging for scanning transmission electron microscopy and transmission electron microscopy. A template for cryo-electron microscopy and multimodal cryo-imaging approaches with numerous sample transfer steps is presented.

A streamlined workflow for automated cryo focused ion beam milling

“…To avoid contamination and devitrification during sample transfer from the plunge freezer to the glove box and from there to the cryo-FIB/SEM, we implemented a previously developed high vacuum cryo transfer system [Tacke2016] (Figure 2c), which consists of a transfer rod, a liquid nitrogen reservoir, a vacuum chamber, a valve and a plug & play adapter for the glove box or cryo-FIB/SEM. To avoid devitrification during transfer, the cryo-FIB shuttle is actively cooled by the liquid nitrogen reservoir as soon as the transfer rod is retracted in its parking position.…”

“…To avoid devitrification during transfer, the cryo-FIB shuttle is actively cooled by the liquid nitrogen reservoir as soon as the transfer rod is retracted in its parking position. Moreover, because the cooled surfaces of the nitrogen reservoir act as a cryo-pump, the vacuum level is kept constant as long as the reservoir is filled [Tacke2016].…”

Non-contact luminescence lifetime cryothermometry for macromolecular crystallography

“…This feature is essential for cryogenic experiments where accuracy of temperature determination can be significantly affected by heat load through contact wires and thermal resistance of interfaces [7], [8], [9], [10] which are difficult to account for in a reliable manner. Hence, the real temperature at the point of interest can be quite different from the sensor reading [11], [12], [13]. If instead the luminescence response of the sensor is measured in the experiment, the accuracy of the temperature measurement is far less influenced by heat leaks, detail of heat transfer at the interfaces or other effects, giving more reliable estimations of the true sample temperature.…”