Abstract
Transmission electron microscopy (TEM) is an invaluable technique used for imaging the ultrastructure of samples, and it is particularly useful when determining virus–host interactions at a cellular level. The environment inside a TEM is not favorable for biological material (high vacuum and high energy electrons). Also biological samples have little or no intrinsic electron contrast and rarely do they naturally exist in very thin sheets, as is required for optimum resolution in the TEM. To prepare these samples for imaging in the TEM therefore requires extensive processing which can alter the ultrastructure of the material. Here we describe a method which aims to minimize preparation artifacts by freezing the samples at high pressure to instantaneously preserve ultrastructural detail, then rapidly substituting the ice with resin to provide a firm matrix which can be cut into thin sections for imaging. Thicker sections of this material can also be imaged and reconstructed into 3D volumes using electron tomography.
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References
Maier H, Hawes PC, Cottam EM et al (2013) Infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes. MBio 4(5):e00801-13
Zierold K (1991) Cryofixation methods for ion localisation in cells by electron probe microanalysis: a review. J Microsc 161(2):357–366
Maneta-Peyret L, Compere P, Moreau P et al (1999) Immunocytochemistry of lipids: chemical fixatives have dramatic effects on the preservation of tissue lipids. Histochem J 31(8):541–547
Lee RMKW (1984) A critical appraisal of the effects of fixation, dehydration and embedding on cell volume. In: Revel JP, Barnard T et al (eds) The science of biological specimen preparation, SEM. AMF O’Hare, Chicago, IL, pp 61–70
Studer D, Humbel BM, Chiquet M (2008) Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution. Histochem Cell Biol 130(5):877–889
Studer D, Michel M, Muller M (1989) High pressure freezing comes of age. Scanning Microsc Suppl 3:253–268
Moor H, Riehle U (1968) Snap-freezing under high pressure: a new fixation technique for freeze-etching. Proc Fourth Euro Reg Conf Electr Microsc 2:33–34
Studer D, Graber W, Al-Amoudi A et al (2001) A new approach for cryofixation by high-pressure freezing. J Microsc 203(3):285–294
Vanhecke D, Graber W, Studer D (2008) Close-to-native ultrastructural preservation by high pressure freezing. Methods Cell Biol 88:151–164
Sartori N, Richter K, Dubochet J (1993) Vitrification depth can be increased more than 10-fold by high pressure freezing. J Microsc 172:55–61
McDonald KL (2014) Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material. Protoplasma 251(2):429–448
Studer D, Chiquet M, Hunzkier EB (1996) Evidence for a distinct water-rich layer surrounding collagen fibrils in articular cartilage extracellular matrix. J Struct Biol 117(2):81–85
McDonald KL, Webb RI (2011) Freeze substitution in 3 hours or less. J Microsc 243(3):227–233
Hawes PC, Netherton CL, Mueller M et al (2007) Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells. J Microsc 226(2):182–189
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Doyle, N., Hawes, P.C. (2020). Preparation of Cultured Cells Using High-Pressure Freezing and Freeze Substitution for Subsequent 2D or 3D Visualization in the Transmission Electron Microscope. In: Maier, H., Bickerton, E. (eds) Coronaviruses. Methods in Molecular Biology, vol 2203. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0900-2_19
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DOI: https://doi.org/10.1007/978-1-0716-0900-2_19
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Publisher Name: Humana, New York, NY
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