Epoxy plastics

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).

Figure 8.1 The active epoxide group in all epoxy plastics.

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).

Polyester plastics

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.

Acrylic plastics

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.

Tinctorial 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.