Abstract
The freeze-fracture technique consists of physically breaking apart (fracturing) a frozen biological sample; structural detail exposed by the fracture plane is then visualized by vacuum-deposition of platinum–carbon to make a replica for examination in the transmission electron microscope. The four key steps in making a freeze-fracture replica are (i) rapid freezing, (ii) fracturing, (iii) replication and (iv) replica cleaning. In routine protocols, a pretreatment step is carried out before freezing, typically comprising fixation in glutaraldehyde followed by cryoprotection with glycerol. An optional etching step, involving vacuum sublimation of ice, may be carried out after fracturing. Freeze fracture is unique among electron microscopic techniques in providing planar views of the internal organization of membranes. Deep etching of ultrarapidly frozen samples permits visualization of the surface structure of cells and their components. Images provided by freeze fracture and related techniques have profoundly shaped our understanding of the functional morphology of the cell.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Steere, R.L. Electron microscopy of structural detail in frozen biological specimens. J. Biophys. Biochem. Cytol. 3, 45–60 (1957).
Moor, H. & Mühlethaler, K. Fine structure of frozen-etched yeast cells. J. Cell Biol. 17, 609–628 (1963).
Bullivant, S. & Ames, A. A simple freeze-fracture replication method for electron microscopy. J. Cell Biol. 29, 435–447 (1966).
Pinto da Silva, P. & Branton, D. Membrane splitting in freeze-etching. Covalently bound ferritin as a membrane marker. J. Cell Biol. 45, 598–605 (1970).
Branton, D. et al. Freeze-etching nomenclature. Science 190, 54–56 (1975).
Moor, H., Mühlethaler, K., Waldner, H. & Frey-Wyssling, A. A new freezing-ultramicrotome. J. Biophys. Biochem. Cytol. 10, 1–13 (1961).
Wehrli, E., Mühlethaler, K. & Moor, H. Membrane structure as seen with a double replica method for freeze-fracturing. Exp. Cell Res. 59, 336–339 (1970).
Tillack, T.W. & Marchesi, V.T. Demonstration of the outer surface of freeze-etched red blood cell membranes. J. Cell Biol. 45, 649–653 (1970).
Pinto da Silva, P. Translational mobility of the membrane intercalated particles of human erythocyte ghosts. pH-dependent, reversible aggregation. J. Cell Biol. 53, 777–787 (1972).
Pinto da Silva, P. & Nicolson, G.L. Freeze-etch localization of concanavalin A receptors to the membrane intercalated particles of human erythrocyte ghost membranes. Biochim. Biophys. Acta 363, 311–319 (1974).
Heuser, J.E. & Salpeter, S.R. Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary replicated Torpedo postsynaptic membrane. J. Cell Biol. 82, 150–173 (1979).
Heuser, J.E. Preparing biological specimens for stereo microscopy by the quick-freeze, deep-etch, rotary-replication technique. Methods Cell Biol. 22, 97–122 (1981).
Severs, N.J., Newman, T.M. & Shotton, D.M. A practical introduction to rapid freezing techniques. in Rapid Freezing, Freeze Fracture, and Deep Etching (eds. Severs N.J. & Shotton D.M.) 31–49 (Wiley-Liss Inc., New York, 1995).
Severs, N.J. & Shotton, D.M. Rapid freezing of biological specimens for freeze fracture and deep etching. in Cell Biology: A Laboratory Handbook Vol. 3. (ed. Celis, J.E.) 299–309 (Academic Press, New York, 1998).
Galway, M.E., Heckman, M.E., Hyde, G.J. & Fowke, L.C. Advances in high-pressure and plunge-freeze fixation. Methods Cell Biol. 49, 3–19 (1995).
Müller, M., Meister, N. & Moor, H. Freezing in a propane jet and its application in freeze-fracturing. Mikroskopie (Wien) 36, 129–140 (1980).
Knoll, G. Time resolved analysis of rapid events. in Rapid Freezing, Freeze Fracture and Deep Etching (eds. Severs, N.J. & Shotton, D.M.) 105–126 (Wiley-Liss Inc., New York, 1995).
Heuser, J.E. et al. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J. Cell Biol. 81, 275–300 (1979).
Escaig, J. New instruments which facilitate rapid freezing at 83K and 6K. J. Microsc. 126, 221–230 (1982).
Kiss, J.Z. & Staehelin, L.A. High pressure freezing. in Rapid Freezing, Freeze Fracture and Deep Etching (eds. Severs, N.J. & Shotton, D.M.) 89–104 (Wiley-Liss Inc., New York, 1995).
Heuser, J. Protocol for 3-D visualization of molecules on mica via the quick freeze, deep etch technique. J. Electron Microsc. Tech. 13, 244–263 (1989).
Severs, N.J. & Warren, R.C. Analysis of membrane structure in the transitional epithelium of rat urinary bladder. 1. The luminal membrane. J. Ultrastruct. Res. 64, 124–140 (1978).
Nermut, M.V. Manipulation of cell monolayers to reveal plasma membrane surfaces for freeze-drying and surface replication. in Rapid Freezing, Freeze Fracture and Deep Etching (eds. Severs, N.J. & Shotton, D.M.) 151–172 (Wiley-Liss Inc., New York, 1995).
Severs, N.J. Freeze-fracture cytochemistry: an explanatory survey of methods. in Rapid Freezing, Freeze Fracture, and Deep Etching (eds. Severs, N.J. & Shotton, D.M.) 173–208 (Wiley-Liss Inc., New York, 1995).
Pinto da Silva, P., Parkison, C. & Dwyer, N. Fracture-label: cytochemistry of freeze-fracture faces in the erythrocyte membrane. Proc. Natl. Acad. Sci. USA 78, 343–347 (1981).
Pinto da Silva, P. & Kan, F.W.K. Label-fracture: a method for high resolution labeling of cell surfaces. J. Cell Biol. 99, 1156–1161 (1984).
Fujimoto, K. Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins—application to the immunogold labeling of intercellular junctional complexes. J. Cell Sci. 108, 3443–3449 (1995).
Fujimoto, K. SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembrane: practical procedure, interpretation and application. Histochem. Cell Biol. 107, 87–96 (1997).
Robenek, H. et al. Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis. J. Cell Sci. 119, 4215–4224 (2006).
Robenek, H. et al. Butyrophilin controls milk fat globule secretion. Proc. Natl. Acad. Sci. USA 103, 10385–10390 (2006).
Robenek, H. et al. Lipid droplets gain PAT family proteins by interaction with specialized plasma membrane domains. J. Biol. Chem. 280, 26330–26338 (2005).
Severs, N.J. & Shotton, D.M. (Rapid Freezing, Freeze Fracture, and Deep Etching . (Wiley-Liss Inc., New York, 1995)).
Robards, A.W. & Wilson, A.J. Low-temperature methods for TEM and SEM. in Procedures in Electron Microscoopy (eds. Robards, A.W. & Wilson, A.J.) (Wiley-Liss Inc., New York, 1993).
Shotton, D.M. Freeze fracture and freeze etching. in Cell Biology: A Laboratory Handbook Vol. 3. (ed. Celis, J.E.) 310–322 (Academic Press, New York, 1998).
Rash, J.E. & Hudson, C.S. Freeze-fracture. Methods, artifacts and interpretation (Raven Press, New York, 1979).
Hui, S.W. Freeze Fracture Studies of Membranes (CRC Press, Boca Raton, Florida, 1989).
Roberts, K.L., Kessel, R.G. & Tung, N.-H. Freeze Fracture Images of Cells and Tissues (Oxford University Press, Oxford, 1991).
Orci, L. & Perrelet, A. Freeze-Etch Histology: A Comparison between Thin Sections an Freeze-Etch Replicas (Springer-Verlag, Berlin, 1975).
Robards, A.W. & Sleytr, U.B. Low temperature methods in biological electron microscopy. in Practical Methods in Electron Microscopy Vol. 10. (ed. Glauert, A.M.) (Elsevier, Amsterdam, 1985).
Steinbrecht, R.A. & Zierold, K. Cryotechniques in Biological Electron Microscopy (Springer-Verlag, Berlin, Heidelberg, 1987).
Echlin, P. Low-Temperature Microscopy and Analysis (Plenum Pub. Corp., New York, 1992).
Pauli, B.U., Weinstein, R.S., Soble, L.W. & Alroy, J. Freeze-fracture of monolayer cultures. J. Cell Biol. 72, 763–769 (1977).
Newman, T.M. A guide to equipment for production of freeze-fracture replicas. in Rapid Freezing, Freeze Fracture, and Deep Etching (eds. Severs, N.J. & Shotton D.M.) 51–67 (Wiley-Liss Inc., New York, 1995).
Severs, N.J. Inverted logic. Nature (Scientific Correspondence) 308, 776 (1984).
Severs, N.J., Gourdie, R.G., Harfst, E., Peters, N.S. & Green, C.R. Review. Intercellular junctions and the application of microscopical techniques: the cardiac gap junction as a case model. J. Microsc. 169, 299–328 (1993).
Severs, N.J. & Green, C.R. Rapid freezing of unpretreated tissues for freeze-fracture electron microscopy. Biol. Cell 47, 193–204 (1983).
Rash, J.E. et al. Grid-mapped freeze fracture: correlative confocal laser scanning microscopy and freeze-fracture electron microscopy of preselected cells in tissue slices. in Rapid Freezing, Freeze Fracture, and Deep Etching (eds. Severs, N.J. & Shotton, D.M.) 127–150 (Wiley-Liss Inc., New York, 1995).
Stolinski, C., Gabriel, G. & Martin, B. Reinforcement and protection with polystyrene of freeze-fracture replicas during thawing and digestion of tissue. J. Microsc. 132, 149–152 (1983).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The author declares no competing financial interests.
Rights and permissions
About this article
Cite this article
Severs, N. Freeze-fracture electron microscopy. Nat Protoc 2, 547–576 (2007). https://doi.org/10.1038/nprot.2007.55
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2007.55
This article is cited by
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.