Skip to main content
SpringerLink
Log in

Introduction to freeze-fracture method in retinal research

  • Published:
Albrecht von Graefes Archiv für klinische und experimentelle Ophthalmologie Aims and scope Submit manuscript

Summary

The freeze-fracture method employs platinum-carbon replicas prepared from the exposed faces of fractured, deep-frozen material. Preparation technique, advantages and disadvantages of this method are described and briefly discussed. The applicability of the method is demonstrated on replicas of freeze-fractured human retinas: Pigment epithelial cells with their peculiar organelles are illustrated. At the level of Verhoeff's membrane, intercellular junctions composed of zonulae occludentes and gap junctions are identified. The so-called outer limiting membrane is confirmed to be composed of zonulae adhaerentes. Bidimensionally arranged particles, characteristic for astroglial cells in the central nervous system, are shown on the exposed faces of the plasma membrane of Müller cells.

Zusammenfassung

Die Gefrierbrechungsmethode arbeitet mit Platin-Kohle-Abdrücken der exponierten Flächen von gebrochenem, tiefgefrorenem Material. Präparationsmethode, Vorteile und Nachteile dieser Methode werden beschrieben und kurz diskutiert. Anschließend wird die Anwendbarkeit der Methode an Abdrücken von gefriergebrochenen menschlichen Netzhäuten demonstriert: Pigmentepithelzellen mit ihren charakteristischen Organellen werden demonstriert. Im Niveau der Verhoeffschen Membran finden sich Intercellularverbindungen, die sich als sog. Zonulae occludentes und Gap junctions identifizieren lassen. Es wird bestätigt, daß die sog. Membrana limitans externa aus Zonulae adhaerentes aufgebaut ist. Auf den exponierten Flächen der Plasmamembran von Müllerschen Zellen werden bidimensional angeordnete Partikel gezeigt, die im Zentralnervensystem für Astrogliazellen charakteristisch sind.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bell, L. G. E.: The application of freezing and drying techniques in cytology. Int. Rev. Cytol. 1, 35–63 (1952)

    Google Scholar 

  • Benedetti, E. L., Dunia, I., Diawara, A.: The organization of the plasma membrane in mammalian cells. Europ. J. Cancer 9, 263–272 (1973)

    Google Scholar 

  • Benedetti, E. L., Emmelot, P.: Hexagonal array of subunits in tight junctions separated from isolated rat liver plasma membranes. J. Cell Biol. 38, 15–24 (1968)

    Google Scholar 

  • Benedetti, E. L., Favard, P.: Freeze-etching, techniques and applications. Paris: Soc. Franç. Microscopie Electronique 1973

    Google Scholar 

  • Bennett, M. V. L.: Function of electrotonic junctions in embryonic and adult tissues. Fed. Proc. 32, 65–75 (1973)

    Google Scholar 

  • Branton, D.: Fracture faces of frozen membranes. Proc. nat. Acad. Sci. (Wash.) 55, 1048–1056 (1966)

    Google Scholar 

  • Branton, D.: Freeze-etching studies of membrane structure. Phil. Trans. B 261, 133–138 (1971)

    Google Scholar 

  • Brightman, M. W., Reese, T. S.: Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol. 40, 648–677 (1969)

    Google Scholar 

  • Brindley, G. S.: The passive electrical properties of the frog's retina, choroid and sclera for radial fields and currents. J. Physiol. (Lond.) 134, 339–352 (1956)

    Google Scholar 

  • Bullivant, S.: Freeze-etching and freeze-fracturing. In: Advanced techniques in biological electron microscopy, J. K. Koehler, ed., p. 67–112. Berlin-Heidelberg-New York: Springer 1973

    Google Scholar 

  • Chalcroft, J. P., Bullivant, S.: An interpretation of liver cell membrane and junction structure based on observation of freeze-fracture replicas of both sides of the fracture. J. Cell Biol. 47, 49–60 (1970)

    Google Scholar 

  • Claude, P., Goodenough, D. A.: Fracture faces of zonulae occludentes from “tight” and “leaky” epithelia. J. Cell Biol. 58, 390–400 (1973)

    Google Scholar 

  • Cohen, A.I.: A possible cytological basis for the “R” membrane in the vertebrate eye. Nature (Lond.) 205, 1222–1223 (1965)

    Google Scholar 

  • Dermietzel, R.: Visualization by freeze-fracturing of regular structures in glial cell membranes. Naturwissenschaften 60, 208 (1973)

    Google Scholar 

  • Farquhar, M. G., Palade, G. E.: Junctional complexes in various epithelia. J. Cell Biol. 17, 375–412 (1963)

    Google Scholar 

  • Friend, D. S., Gilula, N. B.: Variations in tight and gap junctions in mammalian tissues. J. Cell Biol. 53, 758–776 (1972)

    Google Scholar 

  • Frömter, E.: The route of passive ion movement through the epithelium of Necturus gallbladder. J. Membrane Biol. 8, 259–301 (1972)

    Google Scholar 

  • Frömter, E., Diamond, J.: Route of passive ion permeation in epithelia. Nature (Lond.) 235, 9–13 (1972)

    Google Scholar 

  • Geisweid, G., Wermbter, G.: Die Feinstruktur des Nexus zwischen glatten Muskelzellen der Taenia coli im Gefrierätzbild. Cytobiologie 9, 121–130 (1974)

    Google Scholar 

  • Gilula, N. B., Reeves, O. R., Steinbach, A.: Metabolic coupling, ionic coupling and cell contacts. Nature (Lond.) 235, 262–265 (1972)

    Google Scholar 

  • Hudspeth, A. J., Yee, A. G.: The intercellular junctional complexes of retinal pigment epithelia. Invest. Ophthal. 12, 354–365 (1973)

    Google Scholar 

  • Koehler, J. K.: The technique and application of freeze-etching in ultrastructure research. Advanc. biol. med. Phys. 12, 1–84 (1968)

    Google Scholar 

  • Kreutziger, G. O.: Freeze-etching of intercellular junctions of mouse liver. Proc. 26th Ann. Meet. Electron Microscopical Soc. Amer., p. 234–235, C. J. Arceneaux, ed. Baton Rouge: Claitor's Publ. Div. 1968

    Google Scholar 

  • Landis, D. M. D., Reese, T. S.: Arrays of particles in freeze-fractured astrocytic membranes. J. Cell Biol. 60, 316–320 (1974)

    Google Scholar 

  • McNutt, N. S., Weinstein, R. S.: The ultrastructure of the nexus. A correlated thin-section and freeze-cleave study. J. Cell Biol. 47, 666–688 (1970)

    Google Scholar 

  • McNutt, N. S., Weinstein, R. S.: Membrane ultrastructure at mammalian intercellular junctions. Progr. Biophys. Membr. Biol. 26, 45–101 (1973)

    Google Scholar 

  • Moor, H.: Die Gefrier-Fixation lebender Zellen und ihre Anwendung in der Elektronenmikroskopie. Z. Zellforsch. 62, 546–580 (1964)

    Google Scholar 

  • Moor, H.: Use of freeze-etching in the study of biological ultrastructure. Int. Rev. exp. Path. 5, 179–216 (1966)

    Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

  • Moor, H., Mühlethaler, K., Waldner, H., Frey-Wyssling, A.: A new freezing ultramicrotome. J. biophys. biochem. Cytol. 10, 1–13 (1961)

    Google Scholar 

  • Peyman, G. A., Spitznas, M., Straatsma, B. R.: Chorioretinal diffusion of peroxidase before and after photocoagulation. Invest. Ophthal. 10, 489–495 (1971)

    Google Scholar 

  • Reale, E., Luciano, L., Franke, K., Pannese, E., Wermbter, G., Iurato, S.: Fracture faces of the junctional complex between the cells limiting the cochlear duct. I. The cell junctions in the stria vascularis and spiral ligament. (In preparation)

  • Revel, J.P., Karnovsky, M. J.: Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J. Cell Biol. 33, C7-C12 (1967)

    Google Scholar 

  • Spitznas, M.: Zur Feinstruktur der sog. Membrana limitans externa der menschlichen Retina. Albrecht v. Graefes Arch. klin. exp. Ophthal. 180, 44–56 (1970)

    Google Scholar 

  • Spitznas, M.: The fine structure of the chorioretinal border tissues of the adult human eye. Adv. Ophthal., vol. 28, p. 78–174. Basel: Karger 1974

    Google Scholar 

  • Spitznas, M., Joussen, F.: New observations on the nuclear envelope of human retinal pigment epithelial cells. Albrecht v. Graefes Arch. klin. exp. Ophthal. 188, 71–78 (1973)

    Google Scholar 

  • Staehelin, L. A.: Further observations on the fine structure of freeze-cleaved tight junction. J. Cell Sci. 13, 763–786 (1973)

    Google Scholar 

  • Staehelin, L. A., Mukherjee, T. M., Williams, A.W.: Freeze-etch appearance of the tight junctions in the epithelium of small and large intestine of mice. Protoplasma 67, 165–184 (1969)

    Google Scholar 

  • Steere, R. L.: Electron microscopy of structural detail in frozen biological specimens. J. biophys. biochem. Cytol. 3, 45–60 (1957)

    Google Scholar 

  • Wade, J. B., Karnovsky, M. J.: The structure of the zonula occludens. A single fibril model based on freeze-fracture. J. Cell Biol. 60, 168–180 (1974)

    Google Scholar 

  • Weinstein, R. S., Someda, K.: The freeze-cleave approach to the ultrastructure of frozen tissues. Cryobiol. 4, 116–129 (1967)

    Google Scholar 

  • Zingsheim, H. P.: Membrane structure and electron microscopy. The significance of physical problems and techniques (freeze etching). Biochim. biophys. Acta (Amst.) 265, 339–366 (1972)

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut für Anatomie, Abteilung Elektronenmikroskopie, Medizinische Hochschule, Hannover

    E. Reale & L. Luciano

  2. Universitäts-Augenklinik, Essen

    M. Spitznas

Authors
  1. E. Reale
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. L. Luciano
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Spitznas
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Reale, E., Luciano, L. & Spitznas, M. Introduction to freeze-fracture method in retinal research. Albrecht von Graefes Arch. Klin. Ophthalmol. 192, 73–87 (1974). https://doi.org/10.1007/BF00410695

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00410695

Keywords

Search

Navigation