Freeze-Fracture (-Etch) Electron Microscopy

  • Russell L. Chapman
  • L. Andrew Staehelin


Freeze-fracture electron microscopy (FEM) is a unique and powerful research technique that provides a triad of major advantages for the microbiologist interested in ultrastructure. First, because FEM is a replicating technique, it can provide both face-on as well as cross-sectional views of cellular components exposed by the fracturing of frozen cells. Thus, the investigator can visualize the morphology and distribution of specific components both within the plane and on both surfaces of membranes. In addition, it is often possible to perceive three-dimensional relationships that would require tedious reconstruction if studied in thin-section transmission electron microscopy. Second, even when chemical pretreatment is used during processing of samples, FEM provides an alternate method of subsequent processing and thus allows comparative analysis of thin- section electron microscopy data and FEM data. Third, when coupled with a purely physical specimen fixation method (i.e., cryofixation), FEM can completely eliminate the need for chemical fixation and is thus extremely valuable for the identification of artifactual structures in electron microscopy (EM) images. Furthermore, because of the rapid rate at which all cellular activities can be halted by cryofixation, FEM can be used to investigate rapid cellular processes that cannot be preserved in chemically fixed samples. From the start, FEM has offered the promise of an “artifact-free” glimpse at the ultrastructure of cells. Even if the ultimate goal is not a routine achievement, FEM has been and remains an important research tool in biology in general, and in microbiology in particular.


Fracture Face Cell Wall Layer Freeze Fracture High Pressure Freezing Specimen Support 
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  1. Bachmann, L., Schmitt, W. W., 1971, Improved cryofixation applicable to freeze-etching, Proc. Natl. Acad. Sci. USA 68: 2149–2152.PubMedCrossRefGoogle Scholar
  2. Bôhler, S., 1979, Artifacts and defects of preparation in freeze-etch technique, in: Freeze Fracture: Methods, Artifacts, and Interpretations ( J. E. Rash and C. S. Hudson, eds.), pp. 19 - 29, Raven Press, New York.Google Scholar
  3. Branton, D., 1966, Fracture faces of frozen membranes, Proc. Natl. Acad. Sci. USA 55: 1048–1056.PubMedCrossRefGoogle Scholar
  4. Branton, D., Deamer, D. W., 1972, Membrane structure, Protoplasmatologia II/E/1.Google Scholar
  5. Branton, D., Southworth, D., 1967, Fracture faces of frozen Chlorella and Saccharomyces cells, Exp. Cell Res. 47: 648–653.PubMedCrossRefGoogle Scholar
  6. Branton, D., Bullivant, S., Gilula, N. B., Karnovsky, M. J., Moor, H., Muehlethaler, K., Northcote, D. H., Packer, L., Satir, B., Satir, P., Speth, V., Staehelin, L. A., Steere, R. L., Weinstein, R. S., 1975, Freeze-etching nomenclature, Science 190: 54–56.PubMedCrossRefGoogle Scholar
  7. Bullivant, S., 1973, Freeze-etching and freeze-fracturing, in: Advanced Techniques in Biological Electron Microscopy ( J. Koehler, ed.), pp. 67–112, Springer-Verlag, Berlin.CrossRefGoogle Scholar
  8. Bullivant, S., 1977, Evaluation of membrane structure facts and artefacts produced during freeze- fracturing, J. Microsc. (Oxford) 111: 101–116.CrossRefGoogle Scholar
  9. Bullivant, S., and Ames, A., 1966, A simple freeze-fracture replication method for electron microscopy, J. Cell Biol. 29: 435 - 447.PubMedCrossRefGoogle Scholar
  10. Giddings, T. H., and Staehelin, L. A., 1979, Changes in thylakoid structure associated with the differentiation of heterocysts in the cyanobacterium, Anabaena cylindrica, Biochim. Biophys. Acta 546: 373–382.CrossRefGoogle Scholar
  11. Giesbrecht, P., 1968, Zur Darstellung der DNS von Bakterien and plastischer biologischer Struk- turen mit Hilfe Gefrieratzung, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I Orig. 207: 198–205.Google Scholar
  12. Gilkey, J. R., Staehelin, L. A., 1986, Advances in ultrarapid freezing for the preservation of cellular ultrastructure, J. Electron Microsc. Tech. 3: 177–210.CrossRefGoogle Scholar
  13. Gross, H., 1979, Advances in ultrahigh vacuum freeze fracturing at very low specimen temperature, in: Freeze Fracture: Methods, Artifacts, and Interpretations ( J. Rash and C. S. Hudson, eds.), pp. 127–139, Raven Press, New York.Google Scholar
  14. Haggis, J. H., 1961, Electron microscope replicas from the surface of a fracture through frozen cells, J. Biophys. Biochem. Cytol. 9: 841–852.PubMedCrossRefGoogle Scholar
  15. Hall, B. E., 1950, A low temperature replica method for electron microscopy, J. Appl. Phys. 21: 61–62.CrossRefGoogle Scholar
  16. Herrmann, C., Staehelin, L. A., 1965, Licht- und elektronenmikroskopische Untersuchungen an Pediococcus cerevisiae, Schweiz. Brau. Rundsch. 76: 76–79.Google Scholar
  17. Heuser, J. E., Reese, T. S., Landis, D. M. D., 1976, Preservation of synaptic structure by rapid freezing, Cold Spring Harbor Symp. Quant. Biol. 40: 17–24.CrossRefGoogle Scholar
  18. Hirokawa, N., Heuser, J. E., 1981, Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells, J. Cell Biol. 91: 399–409.PubMedCrossRefGoogle Scholar
  19. Holt, S. C., Leadbetter, E. R., 1969, Comparative ultrastructure of selected aerobic spore- forming bacteria: A freeze-etching study, Bacteriol Rev. 33: 346–378.PubMedGoogle Scholar
  20. Janisch, R., 1972, Pellicle of Paramecium caudatum as revealed by freeze-etching, J. Protozoul. 19: 470–472.Google Scholar
  21. Jost, M., 1965, Die ultrastruktur von Oscillatoria rubescens D.C., Arch. Mikrobiol. 50: 211–245.CrossRefGoogle Scholar
  22. Jost, M., and Matile, P., 1966, Zur Charakterisierung der Gasvacuolen der Blaualge Oscillatoria rubescens, Arch. Mikrobiol. 53: 50-58.Google Scholar
  23. Koehler, J., 1972, The freeze-etching technique, in: Principles and Techniques of Electron Microscopy, Vol. 2 ( M. Hyatt, ed.), pp. 53–98, Van Nostrand-Reinhold, Princeton, New Jersey.Google Scholar
  24. Krah, S., Staehelin, L. A., Nettesheim, G., 1973, A new type of storage container for freeze - etch specimens, J. Microsc. (Oxford) 99: 349–352.CrossRefGoogle Scholar
  25. Lickfeld, K. G., 1968, Der frostgeàtzte Bakterienkern: Ein Beitrag zur Klàrung seiner Tertiàrstruk- tur, Z. Zellforsch. 88: 560–564.PubMedCrossRefGoogle Scholar
  26. Lickfeld, K. G., 1976, Transmission electron microscopy of bacteria, in: Methods in Microbiology, Vol. 9 ( J. R. Norris, ed.), pp. 127–176, Academic Press, New York.CrossRefGoogle Scholar
  27. Under, J. C., Staehelin, L. A., 1979, A novel model for fluid secretion by the trypanosomatid contractile vacuole apparatus, J. Cell Biol. 83: 371–382.CrossRefGoogle Scholar
  28. Margaritis, L., Elgsaeter, A., Branton, D., 1977, Rotary replication for freeze-etching, J. Cell Biol. 72: 47–56.PubMedCrossRefGoogle Scholar
  29. Matile, P., Jost, M., and Moor, H., 1965, Intrazellulàre Lokalisation proteolytischer Enzyme von Neurospora crassa, Z. Zellforsch. 68: 205–216.PubMedCrossRefGoogle Scholar
  30. Merryman, H. T., 1950, Replication of frozen liquids by vacuum evaporation, J. Appl. Phys. 21: 68.Google Scholar
  31. Merryman, H. T., and Kafig, E., 1955, The study of frozen specimens, ice crystals, and ice crystal growth by electron microscopy, Res. Rep. Nav. Med. Res. Inst., Natl. Nav. Med. Ctr. 13: 529–544.Google Scholar
  32. Miller, K. R., Prescott, C. S., Jacobs, T. L., and Lassignol, N. L., 1983, Artifacts associated with quick-freezing and freeze-drying, J. Ultrastruct. Res. 82: 123-133.Google Scholar
  33. Moor, H., 1969, Freeze-etching, Int. Rev. Cytol. 25: 391–412.PubMedCrossRefGoogle Scholar
  34. Moor, H., 1971, Recent progress in the freeze-etching technique, Philos. Trans. R. Soc. London Ser. B 261: 121–131.CrossRefGoogle Scholar
  35. Moor, H., and Miihlethaler, K., 1963, Fine structure in frozen-etched yeast cells, J. Cell Biol. 17: 609–628.PubMedCrossRefGoogle Scholar
  36. Moor, H., Miihlethaler, K., Waldner, H., and Frey-Wyssling, H., 1961, A new freezing ultra- microtome, J. Biophys. Biochem. Cytol. 10: 1–13.PubMedCrossRefGoogle Scholar
  37. Moor, H., Kistler, J., Muller, M., 1976, Freezing in a propane jet, Experientia 32: 805.Google Scholar
  38. Moor, H., Bellin, G., Sandri, C., Akert, K., 1980, The influence of high pressure freezing on mammalian nerve tissue, Cell Tissue Res. 209: 201–216.PubMedCrossRefGoogle Scholar
  39. Muller, M., Meister, N., and Moor, H., 1980, Freezing in a propane jet and its application in freeze- fracturing, Mikroskopie 36: 129–140.PubMedGoogle Scholar
  40. Muller, W., and Pscheid, P., 1981, An improved freeze-fracturing procedure preventing contamina¬tion artifacts at fracturing temperatures below 163 (-110°C) in an unmodified Balzers unit, Mikroskopie 39: 143–148.Google Scholar
  41. Nanninga, N., 1968, Structural features of mesosomes (chondriods [sic]) of Bacillus subtilis after freeze-etching, J. Cell Biol. 39: 251–263.PubMedCrossRefGoogle Scholar
  42. Neushul, M., 1970, A freeze-etching study of the red alga Porphyridium, Am. J. Bot. 57: 1231–1239.CrossRefGoogle Scholar
  43. Niedermeyer, W., 1982, Freeze-fracturing at low temperatures. I. A device for fracturing biological specimens at 77-10 under high vacuum, J. Microsc. (Oxford) 125: 307–318.CrossRefGoogle Scholar
  44. Platt-Aloia, K. A., Thomson, W. W., 1982, Freeze-fracture of intact plant tissue, Stain Technol. 57: 327–334.PubMedGoogle Scholar
  45. Plattner, H., Bachmann, L., 1982, Cryofixation: A tool in biological ultrastructural research, Int. Rev. Cytol. 79: 237–304.PubMedCrossRefGoogle Scholar
  46. Plattner, H., Schmitt-Fumian, W. W., Bachmann, L., 1973, Cryofixation of single cells by spray-freezing, in: Freeze-Etching Techniques and Applications ( E. L. Benedetti and P. Favard, eds.), pp. 81–100, Société Française de Microscopie Electronique, Paris.Google Scholar
  47. Rash, J. E., Hudson, C. S. (eds.), 1979, Freeze Fracture: Methods, Artifacts, and Interpretations, Raven Press, New York.Google Scholar
  48. Remsen, B. C., 1966, The fine structure of frozen-etched Bacillus cereus spores, Arch. Mikrobiol. 54: 266–275.PubMedCrossRefGoogle Scholar
  49. Remsen, B. C., and Lundgren, D. G., 1966, Electron microscopy of the cell envelope of Fer- robacillus ferrooxidans prepared by freeze-etching and chemical fixation techniques, J. Bacteriol. 92: 1765–1771.PubMedGoogle Scholar
  50. Riehle, U., and Hoechli, M., 1973, The theory and technique of high pressure freezing, in: Freeze- Etching Techniques and Applications ( E. L. Benedetti and P. Favard, eds.), pp. 31–61, Société Française de Microscopie Electronique, Paris.Google Scholar
  51. Rodgers, F. G., and Davey, M. R., 1982, Freeze-etch nomenclature for procaryotic bacteria, Micron 13: 419–424.Google Scholar
  52. Satir, B., Schooley, C., Satir, P., 1973, Membrane-fusion in a model system: Mucocyst secretion in Tetrahymena, J. Cell Biol. 56: 153–176.Google Scholar
  53. Schmid, E. N., Sleytr, U. B., Lickfeld, K. G., 1980, Nomenclature of frozen-etched bacterial envelopes, J. Ultrastruct. Res. 71: 22–24.PubMedCrossRefGoogle Scholar
  54. Sjôstrand, F. S., 1943, Electron-microscopic examination of tissues, Nature (London) 151: 725–726.CrossRefGoogle Scholar
  55. Sleyr, U. B., 1978, Gefrierbruch-Abdruckmethoden: technische Entwicklungen and Interpretation, Mikroskopie 34: 2–5.Google Scholar
  56. Sleytr, U. B., Robards, A. W., 1977, Plastic deformation during freeze-cleavage: A review, J. Microsc. (Oxford) 110: 1–25.CrossRefGoogle Scholar
  57. Sleytr, U. B., Robards, A. W., 1982, Understanding the artefact problem in freeze-fracture replication: A review, J. Microsc. (Oxford) 126: 101–122.CrossRefGoogle Scholar
  58. Southworth, D., Fisher, K., Branton, D., 1975, Principles of freeze fracturing and etching, in: Techniques of Biochemical and Biophysical Morphology, Vol. 2, ( D. Glick and R. Rosenbaum, eds.), pp. 247–282, Wiley, New York.Google Scholar
  59. Speth, V., Wunderlich, F., 1973, Membranes of Tetrahymena. II. Direct visualization of reversible transitions in biomembrane structure induced by temperature, Biochim. Biophys. Acta 291: 621–628.PubMedCrossRefGoogle Scholar
  60. Sprague, S. G., Staehelin, L. A., Fuller, R. C., 1981, Semiaerobic induction of bacteriochlorophyll synthesis in the green bacterium Chloroflexus aurantiacus, J. Bacteriol. 147: 1032–1039.PubMedGoogle Scholar
  61. Staehelin, L. A., 1966, Die Ultrastruktur der Zellwand und des Chloroplasten von Chlorella, Z. Zellforsch. Mikrosk. Anat. 74: 325–350.PubMedCrossRefGoogle Scholar
  62. Staehelin, L. A., 1976, Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro, J. Cell Biol. 71: 136–158.PubMedCrossRefGoogle Scholar
  63. Staehelin, L. A., 1980, Freeze-fracture and freeze-etch techniques, in: Handbook of Phycological Methods. Developmental and Cytological Methods ( E. Gantt, ed.), pp. 355–365, Cambridge University Press, London.Google Scholar
  64. Staehelin, L. A., and Pickett-Heaps, J. D., 1975, The ultrastructure of Scenedesmus (Chlorophyceae). I. Species with the “reticulate” or “warty” type of ornamental layer, J. Phycol. 11: 163–185.Google Scholar
  65. Steere, R. L., 1957, Electron microscopy of structural detail in frozen biological specimens, J. Biophys. Biochem. Cytol. 3: 45–60.PubMedCrossRefGoogle Scholar
  66. Steere, R. L., Erbe, E. F., 1983, Supporting freeze-etch specimens with “lexan” while dissolving biological remains in acids, Proc. 41st Annu. Meet. Electron Microsc. Soc. Am. p. 618.Google Scholar
  67. Steere, R. L., and Schaffer, F. L., 1958, The structure of crystals of purified Mahoney poliovirus, Biochim. Biophys. Acta 28: 241–246.PubMedCrossRefGoogle Scholar
  68. Stolinski, C., and Breathnach, A. S., 1975, Freeze-fracture Replication of Biological Tissues: Techniques, Interpretation and Applications, Academic Press, New York.Google Scholar
  69. Van Harreveld, A., Crowell, J., 1964, Electron microscopy after rapid freezing on a metal surface and substitution fixation, Anat. Rec. 149: 381–385.CrossRefGoogle Scholar
  70. Van Harreveld, A., Crowell, J., Malhotra, S. K., 1965, A study of extracellular space in central nervous tissue by freeze-substitution, J. Cell Biol. 25: 17–137.Google Scholar
  71. Varga, A. R., and Staehelin, L. A., 1983, Spatial differentiation in photosynthetic and non-photo- synthetic membrances of Rhodopseudomonas palustris, J. Bacteriol. 154: 1414–1430.PubMedGoogle Scholar
  72. Willison, J. H. M., Rowe, A. J., 1980, Replica, shadowing and freeze-etching techniques, in: Practical Methods in Electron Microscopy (A. M. Glauert, ed.) Vol. 8, North-Holland, Amsterdam.Google Scholar
  73. Wolf, . V., Stockem, W., and Wohlfarth-Bottermann, E., 1981, Cytoplasmic actomyosin fibrils after preservation with high pressure freezing, Cell Tissue Res. 217: 479–495.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1986

Authors and Affiliations

  • Russell L. Chapman
    • 1
  • L. Andrew Staehelin
    • 2
  1. 1.Department of BotanyLouisiana State UniversityBaton RougeUSA
  2. 2.Department of Molecular, Cellular, and Developmental BiologyUniversity of ColoradoBoulderUSA

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