8 Plastic embedding for light microscopy
Paraffin wax is a suitable embedding medium for most tissues, combining adequacy of tissue support with ease of sectioning on a standard microtome. Section thicknesses of about 5 µm are satisfactory for most diagnostic purposes, although with skill and experience thinner sections may be produced. However, there are three main areas where paraffin wax is an unsuitable embedding medium for light microscopy studies. Firstly, it may not offer sufficient support for some tissues. Secondly, it does not permit very thin sections to be cut (these two factors are inter-related). Thirdly, labile substances such as enzymes are destroyed. In these circumstances, the use of plastic instead of paraffin wax may provide superior histological preparations. This chapter will focus mainly on the use of plastic techniques for light microscopy, as details of those for electron microscopy demand specific and different protocols, which are described in Chapter 22. The main applications of the use of plastic embedding are outlined below.
In the early development of electron microscopy, extremely hard ester waxes were used with limited success. They were unsuitable for ultrastructural studies as they did not offer sufficient support for ultra-thin sections (approximately 30–80 nm), and because they were unable to withstand the high-energy electron beam that passes through the section within the electron microscope (see Chapter 22). The introduction of plastic/resin embedding media during the 1950s provided improved results and stimulated the development of electron microscopy. Nunn (1970) and Glauert (1987) discuss the properties of embedding media suitable for ultrastructural studies.
In extremely hard tissues such as undecalcified bone, especially where the sample is large and/or when dense cortical bone is present, the difference in hardness between the tissue and the medium in which it is embedded may be so great that sectioning is exceptionally difficult, resulting in only poor-quality fragmented sections being obtained. Therefore the use of a harder embedding medium such as a plastic can enable superior sections to be cut, compared to the use of paraffin wax. This may be achieved either in the form of a section using a motorized microtome, or as a slice which is then ground down to the required thickness. The latter (known as a ground section) requires specialized equipment and procedures that are different from those used for conventional microtomy. Ground sections are useful if inorganic material such as a stent (vascular implant) is present, or the tissue is tooth, as these are virtually impossible to section by traditional means. These applications are discussed more fully in Chapter 16.
It has long been appreciated that where an accurate diagnosis and prognosis may depend on the detection of some subtle histological or cytological change, sections thinner than the usual 5 µm greatly facilitate accurate examination. The two best-known examples are for renal biopsy and interpretation of hematopoietic tissue, where the reduction of paraffin section thickness to about 2 µm has led to easier and more accurate diagnosis through the detection of minor histological abnormalities which are obscured in thicker sections. Experience has proved that even thinner sections, combined with high-quality optics, will provide a more accurate assessment by light microscopy of minor histological abnormalities in the renal biopsy. Unfortunately, even with the greatest skill and experience, it has (until recently with the waxes available) been extremely difficult to produce good-quality sections thinner than about 3 µm. As a result the possibilities for high-resolution light microscopy have been limited, and though slightly thinner sections can be produced nowadays with superior waxes and microtomes, the artifacts produced in wax sections limits potential morphological improvements. These artifacts are often less obvious if a plastic is employed as the embedding medium.
Pathologists experienced in the use of electron microscopy have long been aware of the increased amount of cytological detail detectable in 0.5–1 µm plastic-embedded tissue sections produced prior to ultra-thin sectioning for ultrastructural studies. It was the realization of the diagnostic value of high-resolution light microscopy in identifying certain nuclear and cytoplasmic characteristics, which are usually obscured in thicker sections, that led to an interest in plastic embedding for specific uses in diagnostic histopathology. In practice, tissue sections other than those from a renal biopsy are frequently cut at 2–3 µm (referred to as semi-thin sections), to combine satisfactory resolution with sufficient staining intensity and contrast. As applications and histological procedures have developed, there have been a variety of techniques and uses applied to plastic sections for high-resolution light microscopy, some of which will be discussed in this chapter.
Plastics are classified according to their chemical composition into epoxy, polyester, or acrylic. The change in the physical state of an embedding medium from liquid to solid is called polymerization and is brought about by joining molecules together to produce a complex macromolecule made up of repeating units. The macromolecule, termed polymer (derived from the Greek word poly meaning ‘many’ and mer meaning ‘part’), is synthesized from simple molecules called monomers (‘single part’). Several ingredients are required to produce a plastic suitable as an embedding medium for biological material and subsequent histological examination. Some of these ingredients can present potential health and safety problems, and it is important that all chemicals used in the formulation of plastics are handled in accordance with local and legal safety requirements. The chemical reactions between the various ingredients in plastic embedding kits, including the process of polymerization, are complex and more akin to data found in the polymer industry. However, as it is useful to understand the fundamental role of particular components, an outline is discussed below.
Various epoxy plastics have found their widest application as embedding media for ultrastructural studies, because the polymerized plastic is sufficiently hard to permit sections as thin as 30–40 nm to be cut, and it is stable in an electron beam. Embedding schedules for the different epoxy resins used in electron microscopy are given in Chapter 22, and only a brief outline of their properties and uses is given here. Epoxy plastics derive their name from the active group through which they polymerize (Fig. 8.1).
Epoxide or oxirane groups can be attached to an almost infinite number of chemical structures in single or multifunctional conformations. Three types of epoxy plastic are used in microscopy: those based on either bisphenol A (Araldite), glycerol (Epon), or cyclohexene dioxide (Spurr). The names in parentheses are those in common usage by microscopists and do not convey any structural information.
Epoxy embedding plastics are a carefully balanced mixture of epoxy plastic, catalyst, and accelerator, each component having a direct influence on the physical and mechanical properties of the cured plastic. The catalyst system used is of the anhydride/amine type and causes curing of the resin through the formation of ester cross-links. The anhydride catalyst can be either a long-chain aliphatic anhydride, e.g. dodecenyl succinic anhydride (DDSA), or an aromatic fused ring anhydride, e.g. methyl nadic anhydride (MNA). The long-chain anhydrides act as internal plasticizers which make blocks more flexible and generally tough, whereas MNA is a rigid molecule that results in stiffening and hardening of cured blocks. Both catalysts increase the hydrophobicity of the plastic, but this property can be reduced by oxidizing the alkyl chains and aromatic rings with peroxides.
The amines used as accelerators are either mono- or poly-functional. As amines form adducts with epoxide groups, the use of poly-functional amines, e.g. dimethylamino methyl phenol (DMP 30), can result in the formation of three-dimensional structures, which are slow to diffuse and hence slow to infiltrate tissue. Thus the mono-functional amines, ethanolamine and benzyl dimethyl amine (BDMA), are more conducive to faster infiltration times. The only other common additive in epoxy plastic mixtures is dibutyl phthalate (DBP), which is present as an external plasticizer to soften the blocks, especially in ‘Araldite’ formulations.
The rate at which each epoxy plastic infiltrates the tissue depends on the density of the tissue and the size of the diffusing molecules. Infiltration by Araldite is slow, partly because of the formation of amine adducts, but also because the epoxy plastic itself is a large molecule. The glycerol-based epoxy plastics (Epon) have a lower viscosity but are often sold as mixtures of isomers, and care should be exercised in choosing the most suitable fraction of isomers. Cyclohexene dioxide-based plastics (Spurr) can be obtained pure and infiltrate the fastest, having a low viscosity (7 centipoise at 25°C). Infiltration at higher temperatures is faster, although agglomerates can form and these negate any increase in the diffusion coefficient.
The physical properties of epoxy plastics are considerably affected by their rate of polymerization. In industry, the same formulations as used in microscopy are cured at temperatures in excess of 150°C for up to several days, whereas tissue blocks at 60°C are considerably undercured. Rapid curing for 1 hour at 120°C produces large numbers of cross-links, but hard brittle blocks; 18 hours at 60°C results in tougher blocks that are more suitable for sectioning and subsequent microscopy. Care is needed to provide a section with the right level of cross-linking to allow staining later. Sodium methoxide can be used to reduce the cross-link density of the cured plastic by trans-esterifying the ester cross-links. This allows the plastic to expand more in solvents and improves access to the tissue for stains and antibodies.
Stability in an electron beam arises from two main factors. Both aromatic and unsaturated groups can stabilize the radicals formed by electron impact, either within the aromatic ring or along an unsaturated aliphatic chain, thus preventing chain fission and depolymerization. Further stability is induced by cross-links that minimize the effects of any depolymerization that occurs by preventing creep.
Epoxy plastics have some disadvantages: they are hydrophobic and subsequent oxidation by peroxide to correct this may produce tissue damage. Both epoxide groups and anhydrides can react under mild conditions with proteins, which may reduce the antigenicity of embedded tissue, and may in addition cause sensitization of workers who absorb them by skin contact or inhalation. The components of many epoxy plastics are toxic, and one, vinylcyclohexane dioxide (VCD), is known to be carcinogenic. Hence gloves should always be worn when handling these plastics, and adequate facilities must be provided for the removal of the chemical vapors and the disposal of toxic waste (Causton 1981).
Ultra-thin section cutting and staining for electron microscopy are discussed in detail in Chapter 22. It is not possible to obtain satisfactory sections of thickness 0.5–1 µm on a standard microtome using a steel knife, so semi-thin sections are produced using a glass or diamond knife, and cut on a motorized microtome. Using specialized strips of glass and equipment, two types of glass knives can be prepared: either the more common triangular shape Latta-Hartmann knife or the Ralph knife, which has a longer cutting edge.
To an observer experienced in its interpretation, there is little doubt that for high-resolution light microscopy, toluidine blue is the most useful and informative stain applied to tissue sections embedded in an epoxy plastic. If the stain is heated and used at high alkaline pH, it easily penetrates the plastic and stains various tissue components a blue color of differing shades and intensities, with no appreciable staining of the embedding medium. The staining intensity of tissue components by toluidine blue largely reflects its electron density, and the ultrastructural appearances on subsequent electron microscopy can be partly predicted by the appearances at light microscopy level. For those who prefer polychromatic stains, various formulations, e.g. Paragon, can be used which resemble H&E staining. Many staining techniques can be applied after the surface resin has been ‘etched’ using alcoholic sodium hydroxide (Janes 1979), but the results are not always reliable. Another type of pre-treatment consists of oxidizing osmium-fixed tissue without etching (Bourne & St John 1978) so that aqueous solutions can be stained more consistently.
A few reports have described the application of immunohistochemistry to epoxy sections for light microscopy studies following treatment with sodium ethoxide/methoxide (Giddings et al. 1982; McCluggage et al. 1995; Krenacs et al. 2005), but this practice is seldom used. In general, for the majority of occasions when high-resolution light microscopy is required, it is preferable to use acrylic plastic sections because of their potential easier handling and quality of staining achieved, although some techniques such as immunohistochemistry (as discussed later) have presented tough challenges.
These plastics were originally introduced for electron microscopy in the mid-1950s, but were soon superseded by the superior epoxides for ultrastructural purposes. Nowadays they are rarely used for microscopy, although Mawhinney and Ellis (1983) have reported on the use of embedding undecalcified bone for light microscopy studies.
The acrylic plastics used for microscopy are esters of acrylic acid (CH2··CH·COOH) or more commonly methacrylic acid (CH··C(CH3)·COOH), and are often referred to as acrylates and methacrylates, respectively. They are used extensively for light microscopy, but some have been formulated so that electron microscopy can be performed, either in addition or exclusively. Numerous mixes can be devised to produce plastics that provide a wide range of properties, and consequently there is a diverse range of potential uses. Butyl, methyl, and glycol methacrylate (the latter is chemically the monomer 2-hydroxyethyl methacrylate or HEMA) were all introduced for electron microscopy, but are now rarely used (unless as a component of a mix) because the plastic disrupts in the electron beam. However, greater success has been achieved in the production and staining of semi-thin sections for high-resolution light microscopy.
Conventional acrylics are cured by complex free-radical chain reactions. The intermediate chemicals formed have an incomplete number of electrons. The monomer is exposed to a source of radicals, usually produced from the breakdown of a catalyst such as benzoyl peroxide. This decomposes to produce phenyl (benzoyl-peroxy) radicals that transfer to the double bond of the acrylic monomer, and which then itself breaks to become a radical in turn. This now acts as an active site, attracting and joining another monomer by repeating the process of opening the carbon-carbon double bond and forming a covalent link. In this way a polymer is formed by joining monomeric units together to produce a long aliphatic chain. Finally, to complete the polymer, a phenyl radical instead of another monomer molecule attaches to the active site to block and terminate further reactions. Both oxygen and acetone prevent attachment of radicals and therefore should be avoided during the curing process.
Radicals can be produced spontaneously by light or heat, and consequently acrylic plastics and their monomers should be stored in dark bottles in a cool place. Acrylics contain a few parts per million of hydroquinone to prevent premature polymerization, and for most applications this can remain when preparing the embedding mixes. Benzoyl peroxide is the most common source of radicals, since it breaks down at 50–60°C, but the addition of a tertiary aromatic amine, e.g. N,N-dimethylaniline or dimethyl p-toluidine, can cause the peroxide to break down into radicals at 0°C, so that the plastic can be cured at low or room temperature. Dry benzoyl peroxide is explosive and is therefore supplied damped with water, or as a paste mixed with dibutyl phthalate, or as plasticized particles. In some mixes, the water is required to be removed and care must be taken to dry aliquots away from direct sunlight or heat. Azobisisobutyronitrile and Perkadox 16 are other catalysts that have been used, but by far the most popular is benzoyl peroxide. Light-sensitive photocatalysts such as benzil and benzoin (various types) are used for polymerization of acrylics at sub-zero temperature using short wavelength light.
In addition to the monomer and catalyst, several other ingredients are often necessary in acrylic plastics. An amine will stimulate polymerization to proceed at a faster rate, and consequently these chemicals are termed either activators or more commonly accelerators. Other activators include sulfinic acid and some barbiturates. To improve the sectioning qualities of acrylic blocks, softeners or plasticizers are often added to the mix. Examples are 2-butoxyethanol, 2-isopropoxyethanol, polyethylene glycol 200/400 and dibutyl phthalate. Some acrylic mixes require a small amount of a cross-linker to stabilize the matrix of the plastic against physical damage caused by either the electron beam (Lowicryl plastics) or staining solutions (Technovit 8100). An example of a cross-linking agent is the difunctional ethylene glycol dimethacrylate, which provides flexible hydrophilic cross-links. Unlike epoxides, the viscosity of acrylics is low and hence short infiltration times are possible, although the size and nature of tissue, together with the processing and embedding temperature, will affect the times required.
Poly (2-hydroxyethyl methacrylate) ‘glycol methacrylate’ (GMA) has proved to be a popular embedding medium for light microscopy since it is extremely hydrophilic, allowing many tinctorial staining methods to be applied, yet tough enough when dehydrated to section well on most microtomes. Various mixes have been reported, with a result that some may be either prepared from the ingredients or purchased as a commercial kit, but many are based on the recipe published by Ruddell (1967). Although the mixes all contain the monomer HEMA, the proportion and variety of this and other ingredients may be different, leading to dissimilar characteristics between various kits. The monomer can be contaminated with methacrylic acid, which may result in some background staining, but this can be reduced by purchasing low-acid HEMA or a high-quality proprietary kit such as JB4, JB4 Plus (Polysciences Inc., USA), Technovit 7100, or Technovit 8100 (Kulzer, Germany). Butyl methacrylate is now rarely used for any histological purpose unless as an ingredient in an acrylic mix, e.g. Unicryl (British BioCell International, UK), since it has proved unreliable and produces considerable tissue artifact during polymerization.
There are also available aromatic polyhydroxy dimethacrylate resins (Histocryl, LR White & LR Gold from London Resin Company, UK). Histocryl is intended for light microscopy purposes but LR White and LR Gold can be used both for light and electron microscopy since they combine hydrophilicity with electron beam stability. LR White may be polymerized by the addition of dimethyl p-toluidine, whereas LR Gold is cured by the addition of benzil and exposure to a quartz halogen lamp specifically for sub-zero temperature embedding. Other acrylic plastics cured at low temperature include Lowicryl HM20, HM23 (hydrophobic) and K4M, K4M Plus, K11M (hydrophilic), and Unicryl (formerly called Bioacryl). The Lowicryls may be cured by the addition of a benzoin photocatalyst exposed to ultraviolet light. Though in some cases these plastics can be employed for light microscopy studies, they are really intended and are more suitable for electron microscopy. Lowicryl K4M Plus is a light curable epoxy-acrylate product combining rapid polymerization of an acrylic with the high strength of an epoxy. Various plastic embedding kits have been marketed under different names (especially the Technovit range), which has caused confusion (Hand 1995a), and there is a constant introduction of new proprietary kits, each with claims suggesting their suitability for specific studies, which often makes it difficult for the scientist to know which to choose. Currently many of these kits are available from TABB, UK and/or Polysciences, USA.
For many years methyl methacrylate (MMA) has been widely used because of its hardness as the ideal embedding medium for undecalcified bone, other hard tissues, and tissues with stents or implants, and again there are proprietary kits such as Technovit 9100, (Kulzer) OsteoBed (Polysciences) and Acrylosin (Dorn & Hart Microedge Inc.) for these purposes. However, by using the MMA monomer in specially devised mixes and in specific ways, it has been shown that tinctorial and immunohistochemical staining on semi-thin sections for high-resolution light microscopy is possible.
The development of acrylic plastic embedding media has usually been stimulated by a requirement for a specific application. Most applications are for light microscopy, but as understanding of the formulation of acrylics has increased, so too have various plastics been introduced which may also be useful for some electron microscopy studies. Some of these plastics, such as the Lowicryls, have been developed mainly for electron microscopy alone (Carlemalm et al. 1982; Acetarin et al. 1986), whereas LR White and Unicryl (Scala et al. 1992) can be used for either purpose. However, for various technical reasons, not all dual-purpose plastics are practical for routine high-resolution light microscopy studies.
Hydrophilic plastics such as GMA and LR White allow tissue to be stained without removal of the embedding medium, and have therefore become popular for routine use. Many ‘simple’ staining techniques may be applied, but some may require modification or present special difficulties to those used on paraffin sections. All acrylic hydrophilic media are insoluble, and consequently all staining occurs with the plastic in situ. This can cause problems in two ways, either because the medium itself becomes stained, which may affect the final appearance, or because the matrix acts as a physical barrier to particular molecules. The most obvious example where the latter occurs is the difficulty that large molecules have in penetrating the plastic matrix during immunohistochemical staining. The alternative use of hydrophobic MMA without the addition of cross-linking agents as an embedding medium permits the plastic to be dissolved, and for certain techniques this is an extremely useful property. However, hydrophobic plastics such as Lowicryl HM20 and HM23 that contain cross-linking agents are insoluble.
Acrylic plastics may be polymerized in different ways by using a chemical accelerator, heat, or light. The optimum method depends on several factors, including the study required and the practicality of the method chosen. Polymerization can also be induced at low temperature, and with some Lowicryl plastics, processing and embedding can be accomplished at temperatures down to −70°C. K4M is the most popular, and is said at −35°C to enhance ultrastructural preservation and immunohistochemical staining.
Acrylics have become popular for high-resolution light microscopy mainly because of the ease with which sections can be stained. Excellent staining may be achieved on tissue embedded in any of the various GMA mixes/kits and other acrylics such as LR White, even though the plastic cannot be removed. Numerous (but not all) routine histological staining methods may be applied, including H&E, PAS, van Gieson, alcian blue, Perls’, elastic methods, Giemsa, and silver techniques for reticulin. Modifications from the standard methods for paraffin sections may be necessary and, because London Resins (Histocryl, LR White, and LR Gold) are all softened by alcohol with the possibility of section loss from the slide, alcoholic staining solutions such as those used in elastic methods should be avoided. Consequently, even hematoxylin staining on London Resins should be progressive to avoid differentiation with acid alcohol, whereas regressive staining on GMA is (with care) possible. The plastic embedding medium (especially GMA) may also become stained, but in some techniques this can be reduced by various washing procedures.
An alternative approach is to use MMA where the plastic can easily be removed prior to staining, using similar procedures and solutions but with slightly extended times to those used routinely for dewaxing paraffin sections. It is beyond the scope of this chapter to describe in detail numerous staining methods on different acrylics, but generally best results are achieved using either a method previously published or one that is recommended by other histologists. It should be noted that tinctorial staining of tissue embedded in MMA is possible (where no cross-linker has been added) without removing the plastic, and this is often useful for sections of undecalcified bone MMA prepared as described in Chapter 16. However, this type of procedure is unsuitable for semi-thin sections described in this chapter for high-resolution light microscopy.