Freezing in sealed capillaries for preparation of frozen hydrated sections

Abstract

Summary We have investigated the freezing of specimens in a confined volume for preparation of vitreous samples for cryosectioning. With 15% dextran as a cryoprotectant, a sample sealed in a copper tube begins to freeze into crystalline ice when plunged into liquid ethane. Crystallization rapidly causes an increase in the pressure to the point that much of the sample freezes in a vitreous state. We used synchrotron X‐ray diffraction of samples frozen with various amounts of dextran to characterize the ice phases and crystal orientation, providing insights on the freezing process. We have characterized cryosections obtained from these samples to explore the optimum amount of cryoprotectant. Images of cryosectioned bacteria frozen with various levels of cryoprotectant illustrate effects of cryoprotectant concentration.

Self-pressurized rapid freezing (SPRF) as a simple fixation method for cryo-electron microscopy of vitreous sections

“…After submission of this article, CEMOVIS using self-pressurized rapid freezing has been reported using copper tubes and Caulobacter crescentus as model organism (Yakovlev and Downing, 2011).…”

Macromolecular and cytological changes in fission yeast G0 nuclei

“…Self-pressurized freezing was adapted from a published method 65 as follows. Proliferating and G0 cells were pelleted by centrifuging at 1,000 × g for 5 minutes.…”

Meiotic budding yeast assemble bundled triple helices but not ladders

“…Cells were self-pressurized frozen using a modified method from (Yakovlev and Downing, 2011). Sporulating cells were pelleted by centrifugation at 3,000 × g for 5 minutes at 23°C.…”