Skip to main content
SpringerLink
Log in

Freeze Fracture and Freeze Etching

  • Protocol
  • First Online:
Electron Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1117))

Abstract

Freeze fracture depends on the property of frozen tissues or cells, when cracked open, to split along the hydrophobic interior of membranes, thus revealing broad panoramas of membrane interior. These large panoramas reveal the three-dimensional contours of membranes making the methods well suited to studying changes in membrane architecture. Freshly split membrane faces are visualized by platinum or tungsten shadowing and carbon backing to form a replica that is then cleaned of tissue and imaged by TEM. Etching, i.e., removal of ice from the frozen fractured specimen by sublimation prior to shadowing, can also reveal the true surfaces of the membrane as well as the extracellular matrix and cytoskeletal networks that contact the membranes. Since the resolution of detail in the metal replicas formed is 1–2 nm, these methods can also be used to visualize macromolecules or macromolecular assemblies either in situ or displayed on a mica surface. These methods are available for either specimens that have been chemically fixed or specimens that have been rapidly frozen without chemical intervention.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 279.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Williams RC, Wyckoff RWC (1944) The thickness of electron microscopic objects. J Appl Physiol 15:712–716

    Article  CAS  Google Scholar 

  2. Williams RC, Wyckoff RWC (1964) Applications of metal shadow-casting to microscopy. J Appl Physiol 17:23–33

    Article  Google Scholar 

  3. Williams RC, Wyckoff RW (1945) Electron shadow micrography of the tobacco mosaic virus protein. Science 101:594–596

    Article  CAS  PubMed  Google Scholar 

  4. Price WC, Williams RC, Wyckoff RW (1945) The electron micrography of crystalline plant viruses. Science 102:277–278

    Article  CAS  PubMed  Google Scholar 

  5. Chandler DE (1986) Rotary shadowing with platinum-carbon in biological electron microscopy: a review of methods and applications. J Electron Microsc Tech 3:305–335

    Article  Google Scholar 

  6. Steere RL (1957) Electron microscopy of structural detail in frozen biological specimens. J Biophys Biochem Cytol 3:45–60

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Steere RL (1989) Freeze-fracture: a personal history. J Electron Microsc Tech 13:159–164

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  9. Weinstein RS, Bullivant S (1967) The application of freeze cleaving technics to studies on red blood cell fine structure. Blood 29:780–789

    CAS  PubMed  Google Scholar 

  10. Moor H, Muhlethaler K, Waldner H et al (1961) A new freezing-ultramicrotome. J Biophys Biochem Cytol 10:1–13

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  12. Moor H (1966) Use of freeze-etching in the study of biological ultrastructure. Int Rev Exp Pathol 5:179–216

    CAS  PubMed  Google Scholar 

  13. Moor H (1969) Freeze-etching. Int Rev Cytol 25:391–412

    Article  CAS  PubMed  Google Scholar 

  14. Newman TM (1995) A guide to equipment for production of freeze-fracture replicas. In: Severs NJ, Shotton DM (eds) Rapid freezing, freeze fracture and deep etching. Wiley-Liss Inc., New York, pp 51–67

    Google Scholar 

  15. Branton D (1966) Fracture faces of frozen membranes. Proc Natl Acad Sci U S A 55:1048–1056

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Branton D (1967) Fracture faces of frozen myelin. Exp Cell Res 45:703–707

    Article  CAS  PubMed  Google Scholar 

  17. Pinto da Silva P, Branton D (1970) Membrane splitting in freeze-etching. Covalently bound ferritin as a membrane marker. J Cell Biol 45:598–605

    Article  CAS  PubMed  Google Scholar 

  18. Branton D, Bullivant S, Gilula NB et al (1975) Freeze-etching nomenclature. Science 190:54–56

    Article  CAS  PubMed  Google Scholar 

  19. Gilula NB, Branton D, Satir P (1970) The septate junction: a structural basis for intercellular coupling. Proc Natl Acad Sci U S A 67:213–220

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Yu J, Branton D (1976) Reconstitution of intramembrane particles in recombinants of erythrocyte protein band 3 and lipid: effects of spectrin-actin association. Proc Natl Acad Sci U S A 73:3891–3895

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Heuser J (2011) The origins and evolution of freeze-etch electron microscopy. J Electron Microsc (Tokyo) 60:S3–S29

    Article  CAS  Google Scholar 

  22. Severs NJ (2007) Freeze fracture electron microscopy. Nat Protoc 2:547–576

    Article  CAS  PubMed  Google Scholar 

  23. McIntyre JA, Gilula NB, Karnovsky MJ (1974) Cryoprotectant-induced redistribution of intramembranous particles in mouse lymphocytes. J Cell Biol 60:192–203

    Article  CAS  PubMed  Google Scholar 

  24. Chandler DE, Heuser J (1979) Membrane fusion during secretion: cortical granule exocytosis in sea urchin eggs as studied by quick-freezing and freeze fracture. J Cell Biol 83:91–108

    Article  CAS  PubMed  Google Scholar 

  25. Dubochet J (2007) The physics of rapid cooling and its implications for cryoimmobilization of cells. Methods Cell Biol 79:7–21

    Article  CAS  PubMed  Google Scholar 

  26. Gilkey J, Staehelin LA (1986) Advances in ultrarapid freezing for the preservation of cellular ultrastructure. J Electron Microsc Tech 3:177–210

    Article  Google Scholar 

  27. Heuser JE, Reese TS, Landis DM (1976) Preservation of synaptic structure by rapid freezing. Cold Spring Harb Symp Quant Biol 40:17–24

    Article  CAS  PubMed  Google Scholar 

  28. Walther P (2003) Recent progress in freeze-fracturing of high-pressure frozen samples. J Microsc 212:34–43

    Article  CAS  PubMed  Google Scholar 

  29. McDonald KL (2009) A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues. J Microsc 235:273–281

    Article  CAS  PubMed  Google Scholar 

  30. Rash JE, Hudson CS (1979) Freeze-fracture. Methods artifacts and interpretation. Raven, New York

    Google Scholar 

  31. Abeysekera RM, Robards AW (1995) Freeze fracture artifacts: how to recognize and avoid them. In: Severs NJ, Shotton DM (eds) Rapid freezing, freeze fracture and deep etching. Wiley-Liss Inc., New York, pp 69–88

    Google Scholar 

  32. Heuser JE, Reese TS, Dennis MJ et al (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol 81:275–300

    Article  CAS  PubMed  Google Scholar 

  33. Chandler DE, Heuser J (1980) Arrest of membrane fusion events in mast cells by quick-freezing. J Cell Biol 86:666–674

    Article  CAS  PubMed  Google Scholar 

  34. Plattner H, Hentschel J (2006) Sub-second cellular dynamics: time-resolved electron microscopy and functional correlation. Int Rev Cytol 255:133–176

    Article  CAS  PubMed  Google Scholar 

  35. Chandler DE, Heuser J (1980) The vitelline layer of the sea urchin egg and its modification during fertilization: a freeze fracture study using quick freezing and deep etching. J Cell Biol 84:618–632

    Article  CAS  PubMed  Google Scholar 

  36. Larabell CA, Chandler DE (1988) The extracellular matrix of Xenopus laevis eggs: a quick-freeze, deep-etch analysis of its modification at fertilization. J Cell Biol 107:731–741

    Article  CAS  PubMed  Google Scholar 

  37. Larabell C, Chandler DE (1991) Fertilization-induced changes in the vitelline envelope of echinoderm and amphibian eggs: self assembly of an extracellular matrix. J Electron Microsc Tech 17:294–318

    Article  CAS  PubMed  Google Scholar 

  38. Hirokawa N, Heuser JE (1981) Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J Cell Biol 91:399–409

    Article  CAS  PubMed  Google Scholar 

  39. Hirokawa N (1986) Quick freeze, deep etch of the cytoskeleton. Methods Enzymol 134:598–612

    Article  CAS  PubMed  Google Scholar 

  40. Heuser J (2000) The production of ‘cell cortices’ for light and electron microscopy. Traffic 1:545–552

    Article  CAS  PubMed  Google Scholar 

  41. Nermut MV (1995) Manipulation of cell monolayers to reveal plasma membrane surfaces for freeze-drying and surface replication. In: Severs NJ, Shotton DM (eds) Rapid freezing, freeze fracture and deep etching. Wiley-Liss Inc., New York, pp 151–172

    Google Scholar 

  42. Heuser JE, Kirschner MW (1980) Filament organization revealed in platinum replicas of freeze-dried cytoskeletons. J Cell Biol 86:212–234

    Article  CAS  PubMed  Google Scholar 

  43. Heuser J (1980) Three-dimensional visualization of coated vesicle formation in fibroblasts. J Cell Biol 84:560–583

    Article  CAS  PubMed  Google Scholar 

  44. Hanson PI, Roth R, Lin Y et al (2008) Plasma membrane deformation by circular arrays of ESCRT-III protein filaments. J Cell Biol 180:389–402

    Article  CAS  PubMed  Google Scholar 

  45. Heuser JE (1983) Procedure for freeze-drying molecules adsorbed to mica flakes. J Mol Biol 169:155–195

    Article  CAS  PubMed  Google Scholar 

  46. Heuser J (1989) Protocol for 3-D visualization of molecules on mica via the quick freeze, deep etch technique. J Electron Microsc Tech 13:244–263

    Article  CAS  PubMed  Google Scholar 

  47. Szajner P, Weisberg AS, Lebowitz J et al (2005) External scaffold of spherical immature poxvirus particles is made of protein trimers, forming a honeycomb lattice. J Cell Biol 170:971–981

    Article  CAS  PubMed  Google Scholar 

  48. Matsuoka K, Schekman R, Orci L et al (2001) Surface structure of the COPII-coated vesicle. Proc Natl Acad Sci U S A 98:13705–13709

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  49. Takizawa T, Saito T (1996) Freeze fracture enzyme cytochemistry: application of enzyme cytochemistry to freeze-fracture cytochemistry. J Electron Microsc (Tokyo) 45:242–246

    Article  CAS  Google Scholar 

  50. Elias PM, Goerke J, Friend DS et al (1978) Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes. J Cell Biol 78:577–596

    Article  CAS  PubMed  Google Scholar 

  51. Elias PM, Friend DS, Goerke J (1979) Membrane sterol heterogeneity. Freeze-fracture detection with saponins and filipin. J Histochem Cytochem 27:1247–1260

    Article  CAS  PubMed  Google Scholar 

  52. Skepper JN (1989) Membrane segregation in atrioventricular nodal myocytes of the golden hamster (Mesocricetus auratus). A cytochemical study using filipin and tomatine. J Anat 163:143–154

    CAS  PubMed  Google Scholar 

  53. Pinto da Silva P, Torrisi MR, Kachar B (1981) Freeze-fracture cytochemistry: localization of wheat-germ agglutinin and concanavalin A binding sites on freeze-fractured pancreatic cells. J Cell Biol 91:361–372

    Article  CAS  PubMed  Google Scholar 

  54. Pinto da Silva P, Parkison C, Dwyer N (1981) Fracture-label: cytochemistry of freeze-fracture faces in the erythrocyte membrane. Proc Natl Acad Sci U S A 78:343–347

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  55. Pinto da Silva P, Kan FWK (1984) Label-fracture: a method for high resolution labeling of cell surfaces. J Cell Biol 99:1156–1161

    Article  CAS  PubMed  Google Scholar 

  56. Pavan A, Mancini P, Cirone M et al (1989) Capping of HLA antigens in human lymphocytes as followed by immunogold label-fracture. J Histochem Cytochem 37:1489–1496

    Article  CAS  PubMed  Google Scholar 

  57. Andersson Forsman C, Pinto da Silva P (1988) Fracture-flip: new high-resolution images of cell surfaces after carbon stabilization of freeze-fractured membranes. J Cell Sci 90:531–541

    PubMed  Google Scholar 

  58. Pavan A, Mancini P, Lucania G et al (1990) High-resolution surface views of human lymphocytes during capping of CD4 and HLA antigens as revealed by immunogold fracture-flip. J Cell Sci 96:151–157

    PubMed  Google Scholar 

  59. Fujimoto K (1995) 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

    CAS  PubMed  Google Scholar 

  60. Fujimoto K (1997) 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

    Article  CAS  PubMed  Google Scholar 

  61. Heuser JE (1980) Quick-freeze, deep-etch method of preparing samples for 3-D electron microscopy. Trends Biochem Sci 6:64–68

    Article  Google Scholar 

  62. Heuser JE (1981) Preparing biological specimens for stereo microscopy by the quick-freeze, deep-etch, rotary-replication technique. Methods Cell Biol 22:97–122

    Article  CAS  PubMed  Google Scholar 

  63. Chandler DE, Kazilek CJ (1986) Extracellular coats on the surface of Strongylocentrotus purpuratus eggs: stereo electron microscopy of quick-frozen and deep-etched specimens. Cell Tissue Res 246:153–161

    CAS  PubMed  Google Scholar 

  64. Morone N, Fujiwara T, Murase K et al (2006) Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography. J Cell Biol 174:851–862

    Article  CAS  PubMed  Google Scholar 

  65. Morone N, Nakada C, Umemura Y et al (2008) Three-dimensional molecular architecture of the plasma membrane associated cytoskeleton as reconstructed by freeze-etch electron tomography. Methods Cell Biol 88:207–236

    Article  CAS  PubMed  Google Scholar 

  66. Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137A–138A

    Google Scholar 

  67. Chandler DE, Roberson RW (2009) Bioimaging: current concepts in light and electron microscopy. Jones and Bartlett Learning, Burlington, MA

    Google Scholar 

  68. Merkle CJ, Chandler DE (1993) Visualization of exocytosis by quick freezing and freeze-fracture. Methods Enzymol 221:112–123

    Article  CAS  PubMed  Google Scholar 

  69. Severs NJ, Shotton DM (1995) Rapid freezing, freeze fracture, and deep etching. Wiley-Liss Inc., New York

    Google Scholar 

  70. Shotton DM (1998) Freeze fracture and freeze etching. In: Celis JE (ed) Cell biology: a laboratory handbook, vol 3. Academic, New York, pp 310–322

    Google Scholar 

  71. Severs NJ, Shotton DM (1998) Rapid freezing of biological specimens for freeze fracture and deep etching. In: Celis JE (ed) Cell biology: a laboratory handbook, vol 3. Academic, New York, pp 299–309

    Google Scholar 

  72. Heuser JE, Salpeter SR (1979) Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane. J Cell Biol 82:150–173

    Article  CAS  PubMed  Google Scholar 

  73. Hirokawa N (1989) Quick-freeze, deep-etch electron microscopy. J Electron Microsc (Tokyo) 38:S123–S128

    Google Scholar 

  74. Lupu F, Constantinescu E (1989) A new freeze-drying device for platinum replica studies of cell surface and cytoskeleton: an example using immunogold-labeled human erythrocytes. J Electron Microsc Tech 11:76–82

    Article  CAS  PubMed  Google Scholar 

  75. Severs NJ, Robenek H (2008) Freeze fracture cytochemistry. Methods Cell Biol 88:181–204

    Article  CAS  PubMed  Google Scholar 

  76. Severs NJ (1995) Freeze-fracture cytochemistry: an explanatory survey of methods. In: Severs NJ, Shotton DM (eds) Rapid freezing, freeze fracture and deep etching. Wiley-Liss Inc., New York, pp 173–208

    Google Scholar 

  77. Ciolofan C, Li XB, Olson C et al (2006) Association of connexin 36 and zona occludins-1 with zona occludins-2 and the transcription factor zona occudins-1-associated nucleic acid-binding protein at neuronal gap junctions in rodent retina. Neuroscience 140:433–451

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. School of Life Sciences, Arizona State University, Tempe, AZ, USA

    Douglas E. Chandler & William P. Sharp

Authors
  1. Douglas E. Chandler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. William P. Sharp
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University of Western Australia, Crawley, Australia

    John Kuo

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Chandler, D.E., Sharp, W.P. (2014). Freeze Fracture and Freeze Etching. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology, vol 1117. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-776-1_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-776-1_6

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-775-4

  • Online ISBN: 978-1-62703-776-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation