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Biological Specimen Preparation for Transmission Electron Microscopy

Biological Specimen Preparation for Transmission Electron Microscopy

Audrey M. Glauert
Peter R. Lewis
Copyright Date: 1998
Pages: 348
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  • Book Info
    Biological Specimen Preparation for Transmission Electron Microscopy
    Book Description:

    This book contains all the necessary information and advice for anyone wishing to obtain electron micrographs showing the most accurate ultrastructural detailin thin sectionsof any type of biological specimen.

    The guidelines for the choice of preparative methods are based on an extensive survey of current laboratory practice. For the first time, in a textbook of this kind, the molecular events occurring during fixation and embedding are analysed in detail. The reasons for choosing particular specimen preparation methods are explained and guidance is given on how to modify established techniques to suit individual requirements.

    All the practical methods advocated are clearly described, with accompanying tables and the results obtainable are illustrated with many electron micrographs.

    Portland Press Series:Practical Methods in Electron Microscopy, Volume 17, Audrey M. Glauert, Editor

    Originally published in 1998.

    ThePrinceton Legacy Libraryuses the latest print-on-demand technology to again make available previously out-of-print books from the distinguished backlist of Princeton University Press. These paperback editions preserve the original texts of these important books while presenting them in durable paperback editions. The goal of the Princeton Legacy Library is to vastly increase access to the rich scholarly heritage found in the thousands of books published by Princeton University Press since its founding in 1905.

    eISBN: 978-1-4008-6502-4
    Subjects: Biological Sciences

Table of Contents

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  1. Front Matter
    (pp. i-vi)
  2. Editor’s preface to the series
    (pp. vii-viii)
  3. Authors’ preface and acknowledgements
    (pp. ix-xii)
    Audrey M. Glauert and Peter R. Lewis
  4. Table of Contents
    (pp. xiii-xxii)
  5. 1 An introduction to fixation and embedding procedures and their safe use in the laboratory
    (pp. 1-20)

    The techniques of fixation, embedding and sectioning for the examination of biological specimens by transmission electron microscopy enable us to appreciate the detailed ultrastructure of all types of cells and tissues. The facts that electron micrographs of ultrathin sections are used to illustrate practically every textbook and monograph in cell biology, anatomy and pathology, and that they are now a common feature of scientific programmes on television, are sufficient indication of the importance of this technique as one of the basic methods of modern biological and medical science.

    The normal ultrastructure of a wide range of cell types has been...

  6. 2 Fixatives
    (pp. 21-76)

    The first step in the preparation of most biological specimens for electron microscopy is the application of a chemical fixative. Cryoimmobilization fixation of non-chemically treated tissue is now a viable alternative (Robards and Sleytr 1985), and initial irradiation with microwaves to produce physical fixation has recently been advocated, but some form of chemical fixation is still by far the most popular procedure. The aim of fixation is “to stabilize cellular organization to such an extent that ultrastructural relations are preserved despite the subsequent rather drastic treatments of dehydration, embedding and exposure to the electron beam” (Riemersma 1968). Inevitably, however, the...

  7. 3 Fixation methods
    (pp. 77-128)

    There are many ways of fixing specimens for electron microscopy and they are as varied as the types of specimen to be fixed. It is possible, therefore, to lay down only very general guide-lines for the best method or methods to be followed. In most investigations it is advisable to first try a method that has been used by previous workers with satisfactory results on similar specimens and then to make such empirical modifications as seem appropriate in the light of the results. This chapter covers a wide range of methods, tissues and specimens. It should normally be possible to...

  8. 4 Dehydration methods
    (pp. 129-146)

    Since most embedding media are not miscible with water, it is necessary to ‘dehydrate’ fixed specimens by taking them through a sequence of solutions leading up to one which is fully miscible with the embedding medium. Ethanol and acetone are the two dehydrating agents most commonly used in ultrastructural studies, and are usually followed by an intermediate solvent, such as propylene oxide, before embedding in an epoxy resin. In the standard method well-fixed specimens are dehydrated at room temperature. Alternatively, excellent preservation of ultrastructure is obtained after dehydration of cryofixed specimens by freeze-substitution and embedding in an epoxy resin.


  9. 5 Embedding methods
    (pp. 147-174)

    The aim of embedding is to replace the dehydrating agent or intermediate solvent with a liquid resin monomer which can then be cured or polymerized to produce a solid block with good sectioning properties.

    Two types of embedding media are in widespread use in electron microscopy. The earliest to be developed were the acrylic resins,n-butyl and methyl methacrylate, but these were superseded in the late 1950s by the epoxy resins for ultrastructural studies. More recently, advantage has been taken of the fact that some acrylic resins can be polymerized at low temperatures and consequently they have now become popular...

  10. 6 Embedding in epoxy resins
    (pp. 175-224)

    The epoxy resins have considerable advantages as embedding media for electron microscopy, in comparison with the acrylic resins, as discussed in Sect. 5.1. They change very little in volume during curing, harden uniformly, have excellent sectioning properties and are very stable in the electron microscope. Consequently they are the resins of choice when the accurate preservation of ultrastructure is paramount.

    These embedding media consist basically of the epoxy resin itself and an anhydride hardener, which reacts with the epoxide and hydroxyl groups of the resin molecules to give three-dimensional, so-called cross-linked, structures (Glauert and Glauert 1958). This process is not...

  11. 7 Embedding in acrylic resins
    (pp. 225-250)

    Acrylic resins are transparent, colourless polymers formed from substituted derivatives of acrylic acid (Fig. 7.1). Acrylic monomers have a very low viscosity and are polymerized by radical chain reactions in which the double bond in the acryl group is the centre of the reaction. Monomers having one acryl group, such as methyl methacrylate, form linear polymers, while polyfunctional monomers, such as ethylene glycol dimethacrylate, form cross-linked, three-dimensional, insoluble structures (Fig. 7.1). Both types of polymer are thermoplastic and are brittle below their glass transition temperature (Tg), which is defined as the temperature at which glassy amorphous polymers become flexible or...

  12. 8 The Lowicryl resins and embedding at low temperatures
    (pp. 251-278)

    The procedures described in this chapter for embedding at low temperatures cannot provide good preservation of ultrastructure (Fig. 8.1), which is the main topic of this book, as a result of problems during the polymerization of acrylic resins and their instability under the electron beam. The Lowicryl resins are, however, of value in immunocytochemical studies and consequently details of Lowicryl embedding media and an outline of their use are included here. Further practical information is given by Griffiths (1993).

    A reduction of temperature during processing minimizes the extraction and modification of cell components, particularly when specimens are only lightly fixed...

  13. 9 Other embedding media
    (pp. 279-292)

    The epoxy and acrylic resins have proved to be the most popular embedding media for electron microscopy. Various other polymers have been investigated, but of these only the polyester and melamine resins give satisfactory preservation of ultrastructure. Most water-miscible embedding media have not lived up to their initial promise, and only those which have value for special purposes are mentioned here. It is sometimes an advantage to be able to remove the embedding medium before the sections are examined in the electron microscope and a short account of these ‘reversible’ media is included.

    Polyester resins were introduced as embedding media...

  14. 10 Processing schedules
    (pp. 293-312)

    In this chapter the standard schedule for the whole process, from fixation to embedding, is summarized. This is followed by a selection of schedules that indicate the type of modification that has to be made to the standard schedule for special purposes. Details of the preparation and use of the various fixatives, embedding media and stains are given in earlier chapters.

    The sequence of fixation with an aldehyde, post-fixation with osmium tetroxide and then with uranyl acetate, followed by dehydration and embedding in an epoxy resin is recommended as the standard method of preparing tissues for ultrastructural studies. Specimens for...

  15. Appendix: List of suppliers
    (pp. 313-318)
  16. Subject index
    (pp. 319-326)