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 book is to describe the procedures for preparing biological material in such a way that the ultrastructure of the specimen is preserved with as little alteration as possible when compared with the living organism. A basic essential is to be fully conversant with the appearance of both the living and the fixed specimen under the light microscope before embarking on a study of ultrastructure at high magnification in the electron microscope.

A major problem in judging how well ultrastructure has been preserved is in knowing the basis for comparison; that is, in knowing what the ultrastructure of the living organism should look like. It must be emphasized that this judgement can only be made from the examination of electron micrographs at a high magnification, preferably of 20,000 or higher. Here we can apply a number of criteria:

i. The final appearance of the specimen should not conflict with evidence gained from examination of the living specimen viewed with the light microscope. As early as 1927, Strangeways and Canti followed the changes in living cells during fixation using 'dark-ground' microscopy and showed that osmium tetroxide preserved the cells and their internal structures almost perfectly (Fig. 1.1). More recently the higher resolution of the confocal microscope has extended considerably our knowledge of the structural elements in the unstained living cell.

The polarizing microscope can provide important information about the spatial organization of molecules, such as tubulin and actin, in living cells, while X-ray diffraction is a valuable tool in estimating the dimensions of regular repeating structures, such as muscle fibres (Huxley and Brown 1967), in living material. In addition, with X-ray diffraction it is possible to follow changes in dimensions during fixation and subsequent processing, and thus to monitor the effects of each step of the procedure.

ii. The final appearance of the specimen should not depend critically on the method of fixation, except in ways that can be explained by known chemical and physical principles, and then these principles can act as a guide in the choice of the optimum method of fixation. The fact that particular structures and their inter-relationships are not unique to one method of fixation or preparative method is strong evidence against them being completely artefactual. For example, the observation of rapidly frozen material, which is then dehydrated by freeze-substitution before embedding, provides valuable confirmation of many of the results obtained by standard chemical fixation and dehydration methods.

iii. The final appearance of the specimen should be consistent with those biochemical and physiological data that have structural implications. Thus it is known that mitochondria must have structurally intact outer and inner membranes for oxidative phosphorylation to occur. Any imperfections in the appearance of these membranes in electron micrographs are likely to be the result of damage caused during processing. The degree to which these three criteria can be applied to any new problem is bound to van7 widely, but an attempt should always be made to apply them as far as possible before deciding on the precise details of specimen preparation to be used.

1.3 Artefacts in electron micrographs

It must be emphasized from the outset that every electron micrograph is, in a sense, an artefact. At each stage, from the initial fixation onwards, changes in ultrastructure are inevitable: material is extracted; dimensions are altered (Fig. 1.2); and molecular rearrangement occurs. The best that can be done is to keep these changes to a minimum and this is only possible when the processes involved are fully understood, so that an informed choice of preparative procedures can be made.

The creation of artefacts occurs for several reasons, and the most important of these are:

i. Autolysis before a tissue is fully fixed.

ii. Incorrect osmolarity of the fixative.

iii. Absence of divalent cations from the fixative.

iv. Extraction of lipids during dehydration.

v. Lack of uniformity in the curing or polymerization of the embedding medium.

vi. Instability of the embedding medium under the electron beam.

All these problems are discussed fully in the following chapters. The identification of artefacts in a particular electron micrograph is not always straightforward. Much can often be learnt from previous work on closely related problems, and a knowledge of the types of artefact found by other workers is invaluable (see, for example, the book edited by Crang and Klomparens 1988.) In practice, the best test is a comparison of the same or similar specimens prepared in the same or a different way. Specific indications of possible artefacts include:

i. Disparity in the appearance of adjacent cells of the same type (Fig. 1.3).

ii. Loss of continuity in any membranes (Fig. 1.4a).

iii. Any distortion or disorganization of organelles (Fig. 1.4b).

iv. Presence of empty spaces in the cytoplasm of a cell (Fig. 1.4c).

v. Swollen and empty spaces between the two nuclear membranes (the perinuclear space) (Fig. 1.4d).

vi. Irregularities in the apposition of adjacent cells.

vii. Sharp bends or curves in filamentous structures that are usually straight, such as microtubules.

1.4 Safety precautions in the electron microscope laboratory

In recent years laboratory workers, and especially laboratory managers, have had to become much more aware of the safety aspects of their work and of their working environment. It is assumed that everyone reading this book understands the basic need for safety awareness in the laboratory.

By and large electron microscope (EM) laboratories world-wide have a good safety record, exposure to chemicals being the gravest cause for concern, and that is largely preventable. Considerations of safety, here and throughout this book, are therefore aimed largely at the risks of handling chemicals, and the hazards peculiar to fixation, dehydration and embedding are emphasized. Hazards associated with the individual procedures are discussed in more detail at the beginnings of the relevant chapters and where the procedures are described.

1.4.1 General safety precautions

Laboratories of all descriptions are potentially dangerous places. They may be dangerous for those who work in them, they are especially dangerous for the casual uneducated visitor and they can present a danger to the environment. In recent years most countries and government agencies have become much more safety and health conscious. Often there is now a wealth of relevant legislation, which the laboratory worker has to be familiar with, and even small organizations now have an official Safety Officer, who should be well known and should be consulted regularly. The particular safety precautions required in an EM laboratory are discussed in detail in a companion volume in this series (Alderson 1975).

It is essential that everyone who enters an EM laboratory should be aware of their obligation to maintain a safe working environment, and this applies to the casual visitor as well as to the routine worker in the laboratory. Maintenance of a safe working environment is important on three different levels. Firstly there is the individual research worker's own personal safety. Secondly there is the safety of the laboratory itself, of other workers who use it regularly and of those who merely enter it occasionally. Thirdly there is the safety of the general public and of the outside environment. At all three levels there are clear moral obligations placed on the individual. In most countries there are specific legal requirements, which vary so widely that no attempt is made to deal with them here, but every individual must find out precisely what his or her legal and moral responsibilities are.

A wide range of literature is available on laboratory safety and should be consulted. Legal requirements are covered by government publications. Advice on the use and disposal of chemicals is available in the form of leaflets and booklets from most of the major manufacturers and suppliers of laboratory chemicals. Specific advice should be sought from the Safety Officer or local authority as appropriate.

1.4.2 Hazards from electrical equipment and fire

The likelihood of getting an electric shock from a modern electron microscope can be discounted. There is always a risk from other electrical equipment in the laboratory, however, particularly if there has been a spillage of aqueous solutions. Everyone should therefore be familiar with the correct emergency treatment for electrocution. Equipment that poses a special danger should always be wired via a protecting trip switch if, as is mandatory in some countries, the whole laboratory is not so protected.

Fire is an important hazard to be aware of in any laboratory and an EM laboratory is no exception. The main risk is from faulty electrical equipment and from volatile solvents, such as those used for dehydration. Portable electrical equipment must be inspected annually and any faults rectified. Flammable solvents must be handled and stored with the precautions described below. Everyone should be briefed on what to do in the event of fire.

1.4.3 Safe procedures for handling animals

All tissues of animal or human origin are a potential source of infection, and must be handled and disposed of accordingly. Unfixed material from human sources is particularly hazardous, and so too is material from other primates. The local Safety Officer must be consulted before work on specimens from any primate is started. All material from human or animal sources should be handled over a surface that can be disinfected, preferably a large enamel tray. Material remaining after samples have been taken must be disposed of safely. Small volumes of liquid (such as excess tissue culture medium) and small pieces of tissue should be added to a ten-fold excess of domestic bleach for 2 hours. Larger pieces of tissue should be placed in a plastic bag with sawdust (to absorb any liquid) and then sealed into two further bags ready for incineration. The outermost bag must carry a clear hazard warning. All non-disposable labware and all contaminated surfaces must be adequately disinfected. Your safety officer or local hospital should be consulted about the approved method of disinfection to be used. Treatment with a solution of either domestic bleach or a strong oxidizing agent is normally acceptable, but it is extremely important that domestic bleach should never come into contact with a strong oxidizing agent since this will release toxic chlorine gas. After treatment all surfaces should be swabbed down with 70% ethanol (alcohol). Once any material has been fixed, it can be treated as nonhazardous, unless the presence of a highly resistant pathogen is suspected.