An introduction to fixation and embedding procedures and their safe use in the laboratory

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 studied exhaustively and, because structure and function are intimately related, these studies have helped to explain how many cells fulfil their functions. In fact, the existence of previously unsuspected functions can be revealed. For example, the presence of secretory granules may well indicate that a cell has endocrine potentialities. Transmission electron microscopy is also an invaluable method of observing the consequences of an experimental procedure, either alone, or in combination with cytochemical and immunocytochemical studies. Thus the electron microscope continues to make an invaluable contribution, not only in anatomy, but also in physiology and pathology.

1.1 The scope of this book

For conventional transmission electron microscopes, operating at accelerating voltages up to 100 kV, it is necessary for specimens to be no thicker than 100 nm. Only with specimens this thin is the resolution sufficient to provide sharp images of ultrastructure at high magnification; and even thinner specimens are preferable, provided enough contrast can be obtained. Consequently, ultrathin sections must be prepared of most tvpes of biological material, and it is therefore necessary to take specimens carefully through a long series of operations, ending with the specimen embedded in a resin block which is capable of being cut on an ultramicrotome. It is this necessary series of operations that forms the subject matter of this book, which includes a full discussion of the underlying physics and chemistry of all the procedures, so that the reader can understand the theoretical basis of established techniques and can amend them to suit individual requirements.

Fixation is the most important step in the preparative procedure, since failure at this stage renders the whole project useless. After the present introduction and discussion of safety, Chapter 2 therefore contains a basic discussion of the properties required of fixatives for electron microscopy and advice on their correct preparation. In this first critical stage, the living specimen has to be immobilized and all biochemical processes must be halted in such a way that there are minimal changes in ultrastructure, while at the same time the fixation must be sufficiently strong to prevent, as far as possible, any adverse effects of the subsequent dehydration and embedding procedures. The range of fixatives available for electron microscopy is extensive and each fixative has its advantages and disadvantages. Experience has shown, however, that primary fixation with glutaraldehyde and formaldehyde, followed by osmium tetroxide, and then uranyl acetate, is the preferred sequence for the best preservation of the ultrastructure of the majority of biological specimens.

As well as choosing the best formulation for a fixative, it is even more important, in many ways, to choose the correct method of applying the fixative to living biological material, and the various procedures for achieving this are described in detail in Chapter 3. The overall requirement is to ensure that the primary fixative reaches all parts of the specimen as rapidly as possible and before any significant changes in ultrastructure have had time to occur. Consequently the method of fixation depends critically on the type of specimen and is very different, for example, for tissues in a whole animal than for isolated cells.

Following fixation, the specimen must be dehydrated before it is transferred to an embedding resin. This dehydration stage, which is considered in Chapter 4, is less critical than fixation, so long as the specimen has been adequately fixed to minimize the extraction of components of the specimen, particularly lipids, by the organic solvents that have to be used.

In the final stages the dehydrating agent is replaced in a series of steps by the liquid monomer of a resin embedding medium, which is then cured or polymerized to produce a block suitable for sectioning, as described in detail in Chapter 5. The aim here is to ensure that the resin, which is often quite viscous, infiltrates uniformly throughout the specimen. The resins chosen for ultrastructural studies are epoxy resins, which change very little in volume when the monomer is cured to produce the final hardened block, and are stable during examination in the electron microscope. These excellent properties arise from the nature of the resins and the way in which they interact with hardeners during curing. The molecular details of this process are described fully in Chapter 6 and act as a basis for understanding the reasons for the selection of the components of epoxy resin embedding media. This account is followed by detailed advice on the preparation and use of both the Araldite and Epon resins, and of the low viscosity epoxy resins, such as Spurr s resin.

The properties of acrylic resins are described in Chapter 7 and it is stressed that they are unsuitable for studies of ultrastructure, as a result of problems during polymerization and lack of stability in the electron beam. They are of value, however, in light microscopy and the procedures for preparing and staining semithin sections are described. Acrylic resins are also popular in immunocytochemical studies, because of their ability to polymerize at very low temperatures. Consequently the procedures for embedding in the cold are outlined in Chapter 8, with particular reference to the Lowicryl resins which are described in detail. Other resins of continuing interest for electron microscopy, such as the polyester and melamine resins, are described briefly in Chapter 9.

The book ends with Chapter 10, which contains schedules for the whole procedure from fixation to the final embedded specimen, starting with a standard schedule for the best preservation of ultrastructure and continuing with schedules in which the modifications required for special types of investigation are indicated.

1.2 Criteria for the good preservation of ultrastructure

The main aim of this...