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Low-Temperature Embedding

  • Chapter
Ultrastructure Techniques for Microorganisms

Abstract

The examination of cell structure by means of electron microscopy is severely limited by several factors: (1) preparation artifacts as living specimens are subjected to the rigors of chemical fixation, dehydration in organic solvents, and embedment in various plastics and resins (most involving high-temperature curing); (2) distortion generated during sectioning; and (3) beam damage during observation. Suitable preparation methods must be developed before the pursuit of high-resolution data from thin-sectioned material can generate meaningful results. Sjöstrand (1976) has considered the parameters necessary to minimize conformational changes: (1) peptide chain freedom of movement is lessened by inter- and intramolecular cross-linking, (2) low temperature should reduce the extent of conformational changes (i. e., rearrangement of the peptide chains) that are due to the actions of organic liquids, (3) complete dehydration should be avoided to maintain hydration shells, and (4) the specimen environment should be polar.

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References

  • Acetarin, J. D., 1981, Nouvelles recherches sur les résinés d’inclusion pour la microscopie électronique, Thesis, Univ. Louis Pasteur, Strasbourg, France.

    Google Scholar 

  • Acetarin, J. D., Villiger, W., Carlemalm, E., 1986, A new heavy metal containing resin for low temperature embedding and imaging of unstained sections of biological material, J. Electron Micro. Tech. (submitted).

    Google Scholar 

  • Altman, L. G., Schneider, B. G., Papermaster, D. S., 1984, Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy, J. Histochem. Cytochem. 32: 1217–1223.

    Article  PubMed  CAS  Google Scholar 

  • Amako, K., Murata, K., Umeda, A., 1983, Structure of the envelope of Escherichia coli observed by the rapid-freezing and substitution fixation method, Microbiol. Immunol. 27: 95–99.

    PubMed  CAS  Google Scholar 

  • Angelopoulos, E., 1970, Pellicular microtubules in the family Trypanosomatidae, J. Protozool. 17: 39–51.

    PubMed  CAS  Google Scholar 

  • Arborgh, B., Bell, P., Brunk, U., Collins, V. P., 1976, The osmotic effect of glutaraldehyde during fixation: A transmission electron microscopy, scanning electron microscopy and cytochemical study, J. Ultrastruct. Res. 56: 339–350.

    Article  PubMed  CAS  Google Scholar 

  • Armbruster, B. L., Carlemalm, E., Chiovetti, R., Garavito, R. M., Hobot, J. A., Kellenberger, E., Villiger, W., 1982, Specimen preparation for electron microscopy using low temperature embedding resins, J. Microsc. (Oxford) 126: 77–85.

    Article  CAS  Google Scholar 

  • Armbruster, B. L., Garavito, R. M., Kellenberger, E., 1983a, Dehydration and embedding temperatures affect the antigenic specificity of tubulin and immunolabeling by the protein A- colloidal gold technique, J. Histochem. Cytochem. 31: 1380–1384.

    Article  PubMed  CAS  Google Scholar 

  • Armbruster, B. L., Wunderli, H., Turner, B. M., Raska, I., Kellenberger, E., 1983b, Immu- nocytochemical localization of cytoskeletal proteins and histone 2B in isolated membrane- depleted nuclei, metaphase chromatin, and whole Chinese hamster ovary cells, J. Histochem. Cytochem. 31: 1385–1393.

    Article  PubMed  CAS  Google Scholar 

  • Bachhiiber, K., Frôsch, D., 1983, Melamine resins, a new class of water-soluble embedding media for electron microscopy, J. Microsc. (Oxford) 130: 1–9.

    Article  Google Scholar 

  • Bashong, W., Bashong, C., Wurtz, M., Carlemalm, E., Kellenberger, C., and Kellenberger, E., 1984, Preservation of protein structures for electron microscopy by fixation with aldehydes and/or osmium tetroxide. Eur. J. Cell Biol. 35: 21–26.

    Google Scholar 

  • Bayer, M. E., Remsen, B. C., 1970, Structure of Escherichia coli after freeze-etching, J. Bacteriol. 101: 304–313.

    PubMed  CAS  Google Scholar 

  • Bendayan, M., Shore, G., 1982, Immunocytochemical localization of mitochondrial proteins in the rat hepatocyte, J. Histochem. Cytochem. 30: 139–147.

    Article  PubMed  CAS  Google Scholar 

  • Bernhard, W., 1969, A new staining procedure for electron microscopical cytology, J. Ultrastruct. Res. 27: 250–265.

    Article  PubMed  CAS  Google Scholar 

  • Beveridge, T. J., 1981, Ultrastructure, chemistry and function of the bacterial wall, Int. Rev. Cytol. 72: 229–317.

    Article  PubMed  CAS  Google Scholar 

  • Bilinski, M., Plattner, H., Matt, H., 1981, Secretory protein decondensation as a distinct Ca + 2- mediated event during the final steps of exocytosis in Paramecium cells, J. Cell Biol. 88: 179–188.

    Article  PubMed  CAS  Google Scholar 

  • Birbeck, M. S. C., Mercer, E. H., 1956, Applications of an epoxide embedding medium to electron microscopy, J. R. Microsc. Soc. 76: 159–161.

    Article  Google Scholar 

  • Bordier, C., Garavito, R. M., Armbruster, B., 1982, Biochemical and structural analyses of microtubules in the pellicular membrane of Leishmania tropica, J. Protozool. 29: 560–565.

    PubMed  CAS  Google Scholar 

  • Borysko, E., 1956, Recent developments in methacrylate embedding. I. A study of the polymerization damage phenomenon by phase contrast microscopy, J. Biophys. Biochem. Cytol. 2: 3–14 (Suppl.).

    Google Scholar 

  • Borysko, E., and Sapranauskas, P., 1954, A new technique for comparative phase-contrast and electron microscope studies of cells grown in tissue culture, with an evaluation of the technique by means of time-lapse cinematographs, Bull. Johns Hopkins Hosp. 95: 68-79.

    Google Scholar 

  • Carlemalm, E., Kellenberger, E., 1982, The reproducible observation of unstained embedded cellular material in thin sections: Visualization of an integral membrane protein by a new mode of imaging for STEM, EMBO J. 1: 63–67.

    PubMed  CAS  Google Scholar 

  • Carlemalm, E., Garavito, R. M., Villiger, W., 1980, Advances in low temperature embedding for electron microscopy, in: Proceedings of the 7th European Congress on Electron Microscopy, Vol. 2 (P. Brederoo and W. de Priester), pp. 656–657.

    Google Scholar 

  • Carlemalm, E., Garavito, R. M., Villiger, W., 1982a, Resin development for electron microscopy and an analysis of embedding at low temperature, J. Microsc. (Oxford) 126: 123–143.

    Article  CAS  Google Scholar 

  • Carlemalm, E., Acetarin, J. D., Villiger, W., Colliex, C., Kellenberger, E., 1982b, Heavy metal-containing surroundings provide more “negative” contrast by Z-imaging in STEM than with conventional modes, J. Ultrastruct. Res. 80: 339–343.

    Article  PubMed  CAS  Google Scholar 

  • Carlemalm, E., Colliex, C., and Kellenberger, E., 1985a, Contrast formation in electron microscopy of biological material, in: Advances in Electronics and Electronphysics ( P. W. Hawkes, ed.), pp. 269 - 334, Academic Press, New York.

    Google Scholar 

  • Carlemalm, E., Villiger, W., Hobot, J. A., Acetarin, J. D., Kellenberger, E., 1985b, Low temperature embedding with Lowicryl resins: Two new formulations and some applications, J. Microsc. (Oxford) 140: 55–63.

    Article  CAS  Google Scholar 

  • Cope, G. H., 1968, Low-temperature embedding in water-miscible methacrylates after treatment with antifreezes, J. R. Microsc. Soc. 88: 235–257.

    Article  PubMed  CAS  Google Scholar 

  • Cope, G. H., and Williams, M. A., 1968, Quantitative studies on neutral lipid preservation in electron microscopy, J. R. Microsc. Soc. 88: 259–277.

    Article  PubMed  CAS  Google Scholar 

  • Crewe, A. V., Langmore, J. P., Isaacson, M. S., 1975, Resolution and contrast in the scanning transmission electron microscope, in: Physical Aspects of Electron Microscopy and Microheam Analysis ( M. Siegel and D. R. Beaman, eds.), pp. 47–62, Wiley, New York.

    Google Scholar 

  • de Haller, G., Kellenberger, E., Rouiller, B., 1964, Etude au microscope électronique des plasmas contenant de l’acide desoxyribonucleique. III. Variations ultrastructurales des chromosomes d’Amphidinium, J. Microsc. (Paris) 3: 627–642.

    Google Scholar 

  • Douzou, P., 1977, Cryobiochemistry: An Introduction, Academic Press, New York.

    Google Scholar 

  • Dubochet, J., McDowall, A. W., Menge, B., Schmid, E. N., Lickfeld, K. G., 1983, Electron microscopy of frozen-hydrated bacteria, J. Bacteriol. 155: 381–390.

    PubMed  CAS  Google Scholar 

  • Erlandsen, S. L., Parson, J. A., and Rodning, B. B., 1979, Technical parameters of immunostaining of osmicated tissue in epoxy sections, J. Histochem. Cytochem. 27: 1286–1289.

    Article  PubMed  CAS  Google Scholar 

  • Fryer, P. R., Wells, C., and Ratcliffe, A., 1983, Technical difficulties overcome in the use of Lowicryl K4M electron microscopy embedding resin, Histochemistry 77: 141–143.

    Article  PubMed  CAS  Google Scholar 

  • Garavito, R. M., Carlemalm, E., Villiger, W., 1980, Low temperature embedding of spermatids, in: Proceedings of the 7th European Congress on Electron Microscopy, Vol. 2 (P. Brederoo and W. de Priester), pp. 658–659.

    Google Scholar 

  • Garavito, R. M., Carlemalm, E., Colliex, C., Villiger, W., 1982, Septate junction ultrastructure as visualized in unstained and stained preparations, J. Ultrastruct. Res. 80: 344–353.

    Article  PubMed  CAS  Google Scholar 

  • Giesbrecht, P., 1965, Ùber das Ordnungsprinzip in den Chromosomen von Dinoflagellaten und Bakterien, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. 196: 516–519.

    CAS  Google Scholar 

  • Glauert, A. M., Glauert, R. H., 1958, Araldite as an embedding medium in electron microscopy, J. Biophys. Biochem. Cytol. 4: 191–194.

    Article  PubMed  CAS  Google Scholar 

  • Glauert, A. M., Rogers, G. E., Glauert, R. H., 1956, A new embedding medium for electron microscopy, Nature (London) 178: 803.

    Article  CAS  Google Scholar 

  • Hayat, M. A., 1970, Principles and Techniques of Electron Microscopy, Vol. 1, Van Nostrand- Reinhold, Princeton, New Jersey.

    Google Scholar 

  • Herzog, M., Soyer, M. O., 1983, The native structure of dinoflagellate chromosomes and their stabilization by Ca2+ and Mg2+ cations, Eur. J. Cell Biol. 30: 33–41.

    PubMed  CAS  Google Scholar 

  • Hobot, J. A., Carlemalm, E., Villiger, W., Kellenberger, E., 1984, Periplasmic gel: New concept resulting from the reinvestigation of bacterial cell envelope ultrastructure by new methods, J. Bacteriol. 160: 143–152.

    PubMed  CAS  Google Scholar 

  • Hobot, J. A., Villiger, W., Escaig, J., Maeder, M., Ryter, A., Kellenberger, E., 1985, The shape and fine structure of the nucleoid observed on sections of ultra-rapid frozen and cryosubstituted bacteria, J. Bacteriol. 162: 960–971.

    PubMed  CAS  Google Scholar 

  • Hoppe, W., 1974, Towards three-dimensional “electron microscopy” at atomic resolution, Naturwissenschaften 61: 239–249.

    Article  PubMed  CAS  Google Scholar 

  • Kellenberger, E., 1962, The study of natural and artificial DNA-pIasms by thin sections, in: The Interpretation of Ultrastructure, Vol. 1, pp. 233–249, Symp. Int. Soc. Cell Biol., Berne.

    Google Scholar 

  • Kellenberger, E., Schwab, W., Ryter, A., 1956, L’inclusion d’un copolymere du groupe des polyesters comme materiel d’inclusion en ultramicrotomie, Experientia 12: 421–422.

    Article  PubMed  CAS  Google Scholar 

  • Kellenberger, E., Carlemalm, E., Villiger, W., Roth, J., Garavito, R. M., 1980, LowDenatura- tion Embedding for Electron Microscopy of Thin Sections, Chemische Werke Lowi, G. m. b. H., Waldkraiburg.

    Google Scholar 

  • Kellenberger, E., Carlemalm, E., Stauffer, E., Kellenberger, C., Wunderli, H., 1981, In vitro studies of the fixation of DNA, nucleoprotamine, nucleohistone and proteins, Eur. J. Cell Biol. 25: 1–4.

    PubMed  CAS  Google Scholar 

  • Kersken, H., Tiggemann, R., Westphal, C., Plattner, H., 1984, The secretory contents of Paramecium tetraurelia trichocysts: Ultrastructural cytochemical characterization, J. Histochem. Cytochem. 32: 179–192.

    Article  PubMed  CAS  Google Scholar 

  • Lange, R. H., Blôdern, J., Magdowski, G., Trampish, H. J., 1979, Crystalline preparations of rhombohedral porcine insulin as studied by electron diffraction, J. Ultrastruct. Res. 68: 81–91.

    Article  PubMed  CAS  Google Scholar 

  • Leduc, E. H., Bernhard, W., 1967, Recent modifications of the glycol methacrylate embedding procedure, J. Ultrastruct. Res. 19: 196–199.

    Article  PubMed  CAS  Google Scholar 

  • Leduc, E. H., Holt, S. J., 1965, Hydroxypropyl methacrylate, a new water-miscible embedding medium for electron microscopy, J. Cell Biol. 26: 137–155.

    Article  PubMed  CAS  Google Scholar 

  • Livolant, F., 1980, Mise en evidence d’actine dans les trichocystes de Prorocentrum micans (Dinoflagelle), Biol. Cell 39: 10a.

    Google Scholar 

  • Luft, J. H., 1973, Embedding media—Old and new, in: Advanced Techniques in Biological Electron Microscopy ( J. K. Koehler, ed.), pp. 1–34, Springer, Berlin.

    Chapter  Google Scholar 

  • Michel-Salamin, L., Gautier, A., Soyer-Gobillard, M. O., de Billy, F., Dubochet, J., McDowall, A. W., Kellenberger, E., Carlemalm, E., 1984, Appearance of “arch-shaped” chromosomes in dinoflagellates as observed in thin sections and cryofractures, following various preparation procedures, in: Proceedings 8th European Congress Electron Microscopy, Vol. 3 ( A. Csanady, P. Rohlich, and D. Szabo), pp. 1803–1804.

    Google Scholar 

  • Millonig, G., 1961, A modified procedure for lead staining of thin sections, J. Biophys. Biochem. Cytol. 11: 736–739.

    Article  PubMed  CAS  Google Scholar 

  • Moncany, M. L. J., 1982, Determination des conditions intracellulaires chez E. coli: Consequences biologiques de leur modification, Thesis, University of Basel.

    Google Scholar 

  • Morel, F. M. M., Baker, R. F., H. Wayland, 1971, Quantitation of human red blood cell fixation by glutaraldehyde, J. Cell Biol. 48: 91–100.

    Article  PubMed  CAS  Google Scholar 

  • Newman, S. B., Borysko, E., Swerdlow, M., 1949, New sectioning techniques for light and electron microscopy, Science 110: 66–68.

    Article  PubMed  CAS  Google Scholar 

  • Petsko, G. A., 1975, Protein crystallography at sub-zero temperatures: Cryo-protective mother liquors for protein crystals, J. Mol. Biol. 96: 381–392.

    Article  PubMed  CAS  Google Scholar 

  • Plattner, H., Westphal, C., Tiggemann, R., 1982, Cytoskeleton-secretory vesicle interactions during the docking of secretory vesicles at the cell membrane in Paramecium tetraurelia cells, J. Cell Biol. 92: 368–377.

    Article  PubMed  CAS  Google Scholar 

  • Rauh, J. J., Nelson, D. L., 1981, Calmodulin is a major component of extruded trichocysts from Paramecium tetraurelia, J. Cell Biol. 91: 860–865.

    Article  PubMed  CAS  Google Scholar 

  • Reichelt, R., Engel, A., 1984, Monte Carlo calculations of elastic and inelastic electron scattering in biological and plastic materials, Ultramicroscopy 13: 279–294.

    Article  CAS  Google Scholar 

  • Reichelt, R., Carlemalm, E., Villiger, W., Engel, A., 1985, Concentration determination of embedded biological matter by scanning transmission electron microscopy, Ultramicroscopy 16: 69–80.

    Article  CAS  Google Scholar 

  • Reynolds, E. S., 1963, The use of lead citrate at high pH as an electron-opaque stain in electron microscopy, Cell Biol. 17: 208–213.

    Article  CAS  Google Scholar 

  • Rosenberg, M., Bartl, P., Lesko, J., 1960, Water-soluble methacrylate as an embedding medium for the preparation of ultrathin sections, J. Ultrastruct. Res. 4: 298–303.

    Article  PubMed  CAS  Google Scholar 

  • Roth, J., 1982, The protein A-gold (pAg) technique—A qualitative and quantitative approach for antigen localization on thin sections, in: Techniques in Immunocytochemistry, Vol. 1 ( G. R. Bullock and P. Petrusz, eds.), pp. 107 - 133, Academic Press, New York.

    Google Scholar 

  • Roth, J., 1983a, Application of lectin-gold complexes for electron microscopic localization of glycoconjugates on thin sections, J. Histochem. Cytochem. 31: 987–999.

    Article  PubMed  CAS  Google Scholar 

  • Roth, J., 1983b, The colloidal gold marker system for light and electron microscopy: Theory and application, in: Techniques in Immunocytochemistry, Vol. 2 ( G. R. Bullock and P. Petrusz, eds.), pp. 217–284, Academic Press, New York.

    Google Scholar 

  • Roth, J., 1984, Light and electron microscopic localization of antigenic sites in tissue sections by the protein A-gold technique, Acta Histochem. 29: 9–22.

    CAS  Google Scholar 

  • Roth, J., Bendayan, M., Carlemalm, E., and Villiger, W., 1980, Immunocytochemistry in thin sections with the protein A-gold (pAg) technique, Experientia 36: 757.

    Google Scholar 

  • Roth, J., Bendayan, M., Carlemalm, E., Villiger, W., Garavito, R. M., 1981, Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue, J. Histochem. Cytochem. 29: 663–671.

    Article  PubMed  CAS  Google Scholar 

  • Ryter, A., and Kellenberger, E., 1958, L’inclusion au polyester pour l’ultramicrotomie, J. Ultrastruct. Res. 2: 200–214.

    Article  PubMed  CAS  Google Scholar 

  • Schreil, W. H., 1964, Studies on the fixation of artificial and bacterial DNA-plasms for the electron microscopy of thin sections, J. Cell Biol. 22: 1–20.

    Article  PubMed  CAS  Google Scholar 

  • Schwab, M. E., Thônen, H., 1978, Selective binding, uptake, and retrograde transport of tetanus toxin by nerve terminals in the rat iris, J. Cell Biol. 77: 1–13.

    Article  PubMed  CAS  Google Scholar 

  • Shinagawa, Y., Shinagawa, Y., 1978, Melamine resin as water-containing embedding medium for electron microscopy, J. Electron Microsc. 27: 13–17.

    CAS  Google Scholar 

  • Sjôstrand, F. S., 1976, The problems of preserving molecular structure of cellular components in connection with electron microscopic analysis, J. Ultrastruct. Res. 55: 271–280.

    Article  PubMed  Google Scholar 

  • Sjôstrand, F. S., Barajas, L., 1968, Effect of modifications in conformation of protein molecules on structure of mitochondrial membranes, J. Ultrastruct. Res. 25: 121–155.

    Article  PubMed  Google Scholar 

  • Sjôstrand, F. S., Kretzer, F., 1975, A new freeze-drying technique applied to the analysis of the molecular structure of mitochondrial and chloroplast membranes, J. Ultrastruct. Res. 53: 1–28.

    Article  PubMed  Google Scholar 

  • Spurr, A. R., 1969, A low-viscosity epoxy resin embedding medium for electron microscopy, J. Ultrastruct. Res. 26: 31–43.

    Article  PubMed  CAS  Google Scholar 

  • Stàubli, W., 1963, A new embedding technique for electron microscopy, combining a water-soluble epoxy resin ( Durcupan) with water-insoluble Araldite, J. Cell Biol. 16: 197-201.

    Google Scholar 

  • Thomas, R. N., Cox, E. R., 1973, Observations on the symbiosis of Peridinium balticum and its intracellular alga. I. Ultrastructure, J. Phycol. 9: 304–323.

    Google Scholar 

  • Watson, M. L., 1963, Explosionfree methacrylate embedding, J. Appl. Phys. 34: 2507.

    Article  Google Scholar 

  • Weibull, C., Carlemalm, E., Villiger, W., Kellenberger, E., Fakan, J., Gautier, A., Larsson, C., 1980, Low-temperature embedding procedures applied to chloroplasts, J. Ultrastruct. Res. 73: 233–244.

    Article  PubMed  CAS  Google Scholar 

  • Weibull, C., Christiansson, A., Carlemalm, E., 1983, Extraction of membrane lipids during fixation, dehydration and embedding of Acholeplasma laidlawii-cells for electron microscopy, J. Microsc. (Oxford) 129: 201–207.

    Article  CAS  Google Scholar 

  • Weibull, C., Villiger, W., Carlemalm, E., 1984, Extraction of lipids during freeze-substitution of Acholeplasma laidlawii-cells for electron microscopy, J. Microsc. (Oxford) 134: 213–216.

    Article  CAS  Google Scholar 

  • Wunderli, H., Westphal, M., Armbruster, B., Labhart, P., 1983, Comparative studies on the structural organization of membrane-depleted nuclei and metaphase chromosomes, Chromosoma 88: 241–248.

    Article  PubMed  CAS  Google Scholar 

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Author notes

  1. Barbara L. Armbruster

    Present address: Monsanto Company, St. Louis, Missouri, 63167, USA

Authors and Affiliations

  1. Division of Infectious Diseases, Washington University Medical School, St. Louis, Missouri, 63110, USA

    Barbara L. Armbruster

  2. Abteilung Mikrobiologie Biozentrum, Universität Basel, CH-4056, Basel, Switzerland

    Edward Kellenberger

Authors
  1. Barbara L. Armbruster
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  2. Edward Kellenberger
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Editors and Affiliations

  1. Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, USA

    Henry C. Aldrich

  2. Department of Veterinary Science Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana, USA

    William J. Todd

  3. Department of Veterinary Microbiology and Parasitology School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA

    William J. Todd

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Armbruster, B.L., Kellenberger, E. (1986). Low-Temperature Embedding. In: Aldrich, H.C., Todd, W.J. (eds) Ultrastructure Techniques for Microorganisms. Springer, Boston, MA. https://doi.org/10.1007/978-1-4684-5119-1_10

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  • DOI: https://doi.org/10.1007/978-1-4684-5119-1_10

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