Cryo-fixation by Self-Pressurized Rapid Freezing

Abstract

High-pressure freeze fixation is the method of choice to arrest instantly all dynamic and physiological processes inside cells, tissues, and small organisms. Embedded in vitreous ice, such samples can be further processed by freeze substitution or directly analyzed in their fully hydrated state by cryo-electron microscopy of vitreous sections (CEMOVIS) to explore cellular ultrastructure as close as possible to the native state. Here, we describe the procedure of self-pressurized rapid freezing as fast, easy-to-use, and low-cost freeze fixation method, avoiding the usage of a high-pressure freezing (HPF) apparatus. Cells or small organisms are placed in capillary metal tubes, which are tightly closed and plunged directly into liquid ethane cooled by liquid nitrogen. In parts of the tube, crystalline ice is formed and builds up pressure sufficient for the liquid-glass transition of the remaining specimen. The quality of samples is equivalent to preparations by conventional HPF apparatus, allowing for high-resolution cryo-EM applications or for freeze substitution and plastic embedding.

Direct Measurement of Water States in Cryopreserved Cells Reveals Tolerance toward Ice Crystallization

“…Self-pressurized rapid freezing (SPRF) was introduced as an alternative cryofixation method that included subsequent freeze substitution and resin embedding for room-temperature EM ( 34 ). An improved protocol to achieve vitrification of biological samples after SPRF using dextran was reported recently ( 35 , 36 ). Briefly, cell suspensions with 2.5 × 10 7 cells/mL were prepared using different cryoprotective media and taken up into biocompatible aluminum tubes (300 μ m inner diameter and 600 μ m outer diameter; Goodfellow, Bad Nauheim, Germany) ( 35 ).…”

Reversible Cryopreservation of Living Cells Using an Electron Microscopy Cryo-Fixation Method

“…coli from overnight cultures were centrifuged and resuspended in 200 μl PBS. Suspensions in indicated media were taken up into metal tubes as described [ 34 , 35 ]. We used aluminum tubes (300 μm inner and 600 μm outer diameter; Goodfellow GmbH, Bad Nauheim, Germany) and silver tubes (300 μm inner and 600 μm outer diameter; Heimerle & Meule GmbH, Pforzheim, Germany) cut to a length of 15 mm.…”

“…The expansion of water and hexagonal ice during cooling causes increased pressure inside the tube and thus supports vitrification of the remaining sample similar to the LeChatelier principle. In subsequent studies, it has been proven by cryo-electron microscopy, that a modified procedure indeed enables vitrification of biological material like mammalian cells, and to a certain extend their survival [ 5 , 34 , 35 ]. The combined beneficial effects of high thermal diffusivity and pressure in the confined volume might also enable an efficient rewarming process.…”

Self‐Pressurized Rapid Freezing as Cryo‐Fixation Method for Electron Microscopy and Cryopreservation of Living Cells

“…Excellent morphology and ultrastructure preservation of prokaryotes, unicellular eukaryotes, and C. elegans was shown after SPRF cryo‐fixation combined with freeze substitution (Grabenbauer et al., ; Han et al., ; Leunissen & Yi, ) (see Figure ). Recently, several mammalian cell types, prokaryotes, yeast S. cerevisiae , and plant A. thaliana cells were successfully cryo‐immobilized by SPRF and analyzed at high resolution by cryo‐electron microscopy after vitreous sectioning (CEMOVIS) (Dittmann et al., ; Han et al., ; Sarkar et al., ).…”

“…For subsequent freeze-substitution procedures, refer to already published protocols (e.g., McDonald, 2014). Recent protocols using small amounts of water in the substitution medium usually resulted in enhanced membrane contrast (Buser & Walther, 2008;Grabenbauer, Han, & Huebinger, 2014). A detailed protocol for application of cryo-electron microscopy of vitrified sections (CEMOVIS) after SPRF is beyond the scope of this protocol and can be found elsewhere (Grabenbauer et al, 2014).…”