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Simple procedures for evaluating the cryofixation of biological samples
Author(s) -
Costello M. J.,
Fetter R.,
Höchli M.
Publication year - 1982
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1982.tb00330.x
Subject(s) - materials science , lamellar structure , thermocouple , fracture (geology) , phase (matter) , scanning electron microscope , composite material , analytical chemistry (journal) , jet (fluid) , coolant , freezing point , chemistry , chromatography , thermodynamics , physics , organic chemistry
SUMMARY Ultra‐rapid cooling of biological material can be achieved in the absence of cryoprotectants by using thin samples. Three methods now employed to prepare thin samples for freeze‐fracture electron microscopy are compared: contacting the sample against a liquid helium‐cooled copper surface (Heuser et al. , 1979), spraying the sample with a jet of propane (Mueller et al. , 1980), and plunging a streamlined copper ‘sandwich’ into liquid propane (Costello, 1980). In the first method a thin surface layer of the sample is ultra‐rapidly cooled while in the other methods the entire sample sandwiched between sheets of conducting metals is cooled. The morphology of fracture‐faces of dilauryllecithin‐water systems is used to evaluate the effectiveness of cooling methods. At optimum cooling rates the initial disordered arrangement of lipid in the lamellar (L α ) phase is preserved, giving smooth fracture faces. At slower cooling rates a worm‐like texture appears which signals the formation of molecular ordering characteristic of the P β , phase. All three methods are capable of cooling these lipid‐water phases as well as other more dilute aqueous suspensions without evidence of ice crystal growth or damage. Measurement of cooling rates employing miniature thermocouples embedded in samples indicates that rates for all three methods are in excess of 10,000 K/s. The propane jet (32 times 10 3 K/s, slope at 273 K) exposes the sample to coolant more rapidly than the sandwich plunging method (10 times 10 3 K/s, slope at 273 K) and therefore produces slightly higher cooling rates for samples of equivalent mass and thickness. Each method has its advantages. The contact method is well suited for tissues; the sandwich method is simple and inexpensive; the jet method can potentially produce the highest cooling rates. The last two methods yield complementary replicas.