Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Freeze-fracture electron microscopy


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 options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Photograph of the Balzers BAF 400 T machine.
Figure 2: Cutaway diagram of the apparatus for freeze fracture in the vacuum chamber of the Balzers BAF 400T machine.
Figure 3: View of the apparatus in the vacuum chamber with the door opened.
Figure 4: Diagram illustrating the two principal methods for fracturing frozen specimens, knife fracture and tensile fracture.
Figure 5: The principal types of specimen carrier designed for mounting specimens for knife fracture and tensile fracture.
Figure 6: Detachable tables for holding specimen carriers for knife fracture in the Balzers BAF 400.
Figure 7: Detachable cold table for tensile fracture (original type), designed for use with specimen sandwiches mounted between pairs of gold-stub carriers.
Figure 8: Detachable cold table for tensile fracture, with spring-loaded hinged partners, designed for use with specimen sandwiches mounted between pairs of copper hat carriers.
Figure 9: Liquid nitrogen bench dewar with stainless steel basket for holding frozen specimens.
Figure 10: Electron beam evaporation gun (type EK 552) as used in the Balzers BAF 400 machine.
Figure 11: To set up the guns, a filament centering rod is used to position the filament.
Figure 12: Loading gold carrier-mounted frozen specimens onto a standard specimen table.
Figure 13: Loading sandwich-mounted frozen specimens onto a tensile fracture specimen table.
Figure 14: Inserting the loaded specimen table into the vacuum chamber and onto the cold stage.
Figure 15: Replica mounted on an electron microscope grid.
Figure 16: Low-power survey freeze-fracture image of a capillary in heart muscle.
Figure 17: High-magnification freeze-fracture micrograph illustrating plasma membrane structure of adjacent endothelial cells.
Figure 18: Freeze-fracture view of the interior of a Chlorella cell.
Figure 19: Nomenclature for describing the aspects of the plasma membrane revealed by freeze fracture and etching.
Figure 20: Diagram illustrating the three basic fracture paths through frozen spheroidal cells, with corresponding examples from freeze-fractured yeast cells.
Figure 21: Freeze-fracture views of human airway epithelial cells.
Figure 22: Freeze-fracture imaging of the intercalated disk plasma membrane of cardiac muscle cells.
Figure 23: A gap junction, fractured in a similar manner to that in Figure 22, depicted at higher magnification.
Figure 24: Freeze-fracture view of the luminal part of the plasma membrane of rat urinary bladder, seen in E-face view.
Figure 25: E surface of the bladder luminal membrane revealed by deep etching to expose the surface.
Figure 26: Examples of the application of ultrarapid freezing.
Figure 27: Apart from the study of membrane and organelle structure in cells and tissues, freeze fracture has wide application to model systems, for example cell cultures (a), isolated membranes (b) and artificial liposomes (c).


  1. 1

    Steere, R.L. Electron microscopy of structural detail in frozen biological specimens. J. Biophys. Biochem. Cytol. 3, 45–60 (1957).

    CAS  Article  Google Scholar 

  2. 2

    Moor, H. & Mühlethaler, K. Fine structure of frozen-etched yeast cells. J. Cell Biol. 17, 609–628 (1963).

    CAS  Article  Google Scholar 

  3. 3

    Bullivant, S. & Ames, A. A simple freeze-fracture replication method for electron microscopy. J. Cell Biol. 29, 435–447 (1966).

    CAS  Article  Google Scholar 

  4. 4

    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).

    CAS  Article  Google Scholar 

  5. 5

    Branton, D. et al. Freeze-etching nomenclature. Science 190, 54–56 (1975).

    CAS  Article  Google Scholar 

  6. 6

    Moor, H., Mühlethaler, K., Waldner, H. & Frey-Wyssling, A. A new freezing-ultramicrotome. J. Biophys. Biochem. Cytol. 10, 1–13 (1961).

    CAS  Article  Google Scholar 

  7. 7

    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).

    CAS  Article  Google Scholar 

  8. 8

    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).

    CAS  Article  Google Scholar 

  9. 9

    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).

    CAS  Article  Google Scholar 

  10. 10

    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).

    CAS  Article  Google Scholar 

  11. 11

    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).

    CAS  Article  Google Scholar 

  12. 12

    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).

    CAS  Article  Google Scholar 

  13. 13

    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).

    Google Scholar 

  14. 14

    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).

    Google Scholar 

  15. 15

    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).

    CAS  Article  Google Scholar 

  16. 16

    Müller, M., Meister, N. & Moor, H. Freezing in a propane jet and its application in freeze-fracturing. Mikroskopie (Wien) 36, 129–140 (1980).

    Google Scholar 

  17. 17

    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).

    Google Scholar 

  18. 18

    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).

    CAS  Article  Google Scholar 

  19. 19

    Escaig, J. New instruments which facilitate rapid freezing at 83K and 6K. J. Microsc. 126, 221–230 (1982).

    Article  Google Scholar 

  20. 20

    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).

    Google Scholar 

  21. 21

    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).

    CAS  Article  Google Scholar 

  22. 22

    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).

    CAS  Article  Google Scholar 

  23. 23

    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).

    Google Scholar 

  24. 24

    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).

    Google Scholar 

  25. 25

    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).

    CAS  Article  Google Scholar 

  26. 26

    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).

    CAS  Article  Google Scholar 

  27. 27

    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).

    CAS  PubMed  Google Scholar 

  28. 28

    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).

    CAS  Article  Google Scholar 

  29. 29

    Robenek, H. et al. Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis. J. Cell Sci. 119, 4215–4224 (2006).

    CAS  Article  Google Scholar 

  30. 30

    Robenek, H. et al. Butyrophilin controls milk fat globule secretion. Proc. Natl. Acad. Sci. USA 103, 10385–10390 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Robenek, H. et al. Lipid droplets gain PAT family proteins by interaction with specialized plasma membrane domains. J. Biol. Chem. 280, 26330–26338 (2005).

    CAS  Article  Google Scholar 

  32. 32

    Severs, N.J. & Shotton, D.M. (Rapid Freezing, Freeze Fracture, and Deep Etching . (Wiley-Liss Inc., New York, 1995)).

  33. 33

    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).

    Google Scholar 

  34. 34

    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).

    Google Scholar 

  35. 35

    Rash, J.E. & Hudson, C.S. Freeze-fracture. Methods, artifacts and interpretation (Raven Press, New York, 1979).

    Google Scholar 

  36. 36

    Hui, S.W. Freeze Fracture Studies of Membranes (CRC Press, Boca Raton, Florida, 1989).

    Google Scholar 

  37. 37

    Roberts, K.L., Kessel, R.G. & Tung, N.-H. Freeze Fracture Images of Cells and Tissues (Oxford University Press, Oxford, 1991).

    Google Scholar 

  38. 38

    Orci, L. & Perrelet, A. Freeze-Etch Histology: A Comparison between Thin Sections an Freeze-Etch Replicas (Springer-Verlag, Berlin, 1975).

    Book  Google Scholar 

  39. 39

    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).

    Google Scholar 

  40. 40

    Steinbrecht, R.A. & Zierold, K. Cryotechniques in Biological Electron Microscopy (Springer-Verlag, Berlin, Heidelberg, 1987).

    Book  Google Scholar 

  41. 41

    Echlin, P. Low-Temperature Microscopy and Analysis (Plenum Pub. Corp., New York, 1992).

    Book  Google Scholar 

  42. 42

    Pauli, B.U., Weinstein, R.S., Soble, L.W. & Alroy, J. Freeze-fracture of monolayer cultures. J. Cell Biol. 72, 763–769 (1977).

    CAS  Article  Google Scholar 

  43. 43

    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).

    Google Scholar 

  44. 46

    Severs, N.J. Inverted logic. Nature (Scientific Correspondence) 308, 776 (1984).

    CAS  Google Scholar 

  45. 44

    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).

    CAS  Article  Google Scholar 

  46. 45

    Severs, N.J. & Green, C.R. Rapid freezing of unpretreated tissues for freeze-fracture electron microscopy. Biol. Cell 47, 193–204 (1983).

    Google Scholar 

  47. 47

    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).

    Google Scholar 

  48. 48

    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).

    CAS  Article  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Nicholas J Severs.

Ethics declarations

Competing interests

The author declares no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Severs, N. Freeze-fracture electron microscopy. Nat Protoc 2, 547–576 (2007).

Download citation

Further reading


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.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing