Vitrification of thick samples for soft X-ray cryo-tomography by high pressure freezing.
Soft X-ray cryo-microscopy (cryo-XT) offers an ideal complement to electron cryo-microscopy (cryo-EM). Cryo-XT is applicable to samples more than an order of magnitude thicker than cryo-EM, albeit at a more modest resolution of tens of nanometers. Furthermore, the natural contrast obtained in the "water-window" by differential absorption by organic matter vs water yields detailed images of organelles, membranes, protein complexes, and other cellular components. Cryo-XT is thus ideally suited for tomography of eukaryotic cells. The increase in sample thickness places more stringent demands on sample preparation, however. The standard method for cryo-EM, i.e., plunging to a cryogenic fluid such as liquid ethane, is no longer ideally suited to obtain vitrification of thick samples for cryo-XT. High pressure freezing is an alternative approach, most closely associated with freeze-substitution and embedding, or with electron cryo-microscopy of vitreous sections (CEMOVIS). We show here that high pressure freezing can be adapted to soft X-ray tomography of whole vitrified samples, yielding a highly reliable method that avoids crystallization artifacts and potentially offers improved imaging conditions in samples not amenable to plunge-freezing.
Dept. of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel.
Allon
A
Weiner
Sergey
S
Kapishnikov
Eyal
E
Shimoni
Sandra
S
Cordes
Peter
P
Guttmann
Gerd
G
Schneider
Michael
M
Elbaum
D015925:Cryopreservation / Q000379:methods; D003460:Crystallization; D004747:Entamoeba / Q000648:ultrastructure; D004912:Erythrocytes / Q000469:parasitology / Q000473:pathology; D004980:Ethane / Q000737:chemistry; D005615:Freezing; D006801:Humans; D010963:Plasmodium falciparum; D011312:Pressure; D014056:Tomography, X-Ray / Q000379:methods*; D058989:Vitrification
D004980:Ethane