preparation of sections of plastics and polymers for the electron microscope

Farmer & Little: Prepara!ion of Sections of Plastics and Polymers for the Electron Microscope 259

PREPARATION OF SECTIONS OF PLASTICS AND POLYMERS FOR THE ELECTRON MICROSCOPE

Hy JOAN FARMER and KITTY LITTLE*

Experimental details of a rrethod for sectioning rubbers and plastics at room temperature are presented. Usually specimens are embedded in a butyl-isobutyl methacrylate mixture (provided that they do not dissolve in the monomer) which can be polymerised by ultra-violet light to varying hardness. This enables sections through fibres and surfaces lo be obtained. Because of distortion of the surface layers the angle between the knife and specimen is critical. The thickness of these distorted surface layers rather than the total thickness of the specimen provides a limit to the resolution obtained. Choice of the sectioning angle was made primarily by means of sense of touch, so that a microtome design incorporating a direct transmission of pressure from specimen to hand is needed.

Introduction

In the preparation of sections of pol:ymers for the electron microscope, the primary need is to obtain a section thin enough for electrons to penetrate, but at the same time of a sufficient thickness for the greater part of the material to remain undisturbed by the preparative techniques used. In this paper the main stages in the preparation of sections will be described and discussed. The sections illustrating the paper were required for the examination of materials which are used in medicine in contact with the tissues, E O that devices ranging from anaesthetic tubes to plastic heart valves, together with experimental samples of polymers of known composition and properties, have been examined.

Specimen preparation The first requirement is to prepare the specimen in a

shape which is suitable for being held in the chuck of the microtome. After a section has been CL t, it is necessary for it to flatten on a liquid surface ready to be picked up on the supporting grid.

Three methods have been considered Tor the initial specimen preparation.

Direct preparation A few polymers can be directly prepared in a suitable shape

and, after sectioning, will flatten on a liquid surface ready for being picked up on an electron microscope grid. The materials used for embedding biological specimens come into this category.

Difficulties in using this simplest apxoach arise from two principal causes. The first occurs whm there is a need to section a textile fibre or similar specimen in a definite direction, whether transverse, longitudinal or oblique, or to obtain a uniform section through the surface of a specimen where that surface has different hardness, morphology and other properties from the bulk of the material. The second is that as a section is cut it tends to curl up or deat, and while some plastics will subsequently flatten on it water or a dioxane/ water surface, others will not. One of the factors involved here is the contact angle between the liquid and the material.

Embedding in methacrylate To overcome these two difficulties, most of the materials

examined have been embedded in butyl -isobutyl methacrylate. The methacrylate polymers themselves, (methyl, ethyl, butyl, isobutyl, octyl) were the only polymers for which successful sections could be regularly obtained without embedding.

The monomer wets most plastics, so that there is good adhesion, while the duration of the polymerisation process may be varied to produce a methacrylate with a texture varying from that of a rubber to a brittle solid with a glass-like fracture. This method has the additional advantage that specimens of uniform size may be produced easily for holding in the chuck of the microtome.

There may be difficulties with methacrylate embedding. Some polymers swell or dissolve in the monomer. The swelling action sometimes results in internal distortion, and so the method is not suitable for these compounds unless, of course, the need is to examine a plastic normally used in the swollen state. The monomer mixture penetrates other plastics slowly. In some cases the difficulty was overcome by partly polymer- king the methacrylate to a viscous consistency before the specimen was placed on it. Observation has shown that there is a fairly sharp boundary between the unreacted plastics and the altered surface layer (Fig. 1). The surfaces of these materials cannot be examined by this method, but valid results may be obtained from the interior of a block.

Fig. 1 . Polyvinyl chloride In the surface layers it was found that the embedding medium had dissolved away the plasticiser from between the PVC granules.

x 4500

*Presenl address: Wantage Research Laboratory, Wantage, Berks.

Br. Polym. J., 1969, Vol. 1, Novemlber

260 Farmer & Little: Preparation of Sections of Plastics and Polymers for the Electron Microscope

Embedding in other media Where there has been reaction, swelling or solution in the

methacrylate monomer, other embedding media must be sought. In the present work this need has not arisen, and consequently the topic has not been pursued. Rusnock & Hansen' have reported good results with an epoxy resin.

Embedding

A mixture of n-butyl and isobutyl methacrylate was used to embed the specimens. Methyl and ethyl methacrylate had to be kept out of the mixture, since in their presence surfaces of many plastics were not wetted so effectively, and the greater decrease of volume during polymerisation tended to manifest itself as a shrinking away from the specimen. To prepare the methacrylate monomer, a mixture of 20% isobutyl and 80% n-butyl methacrylate was prepared, and the inhibitor was removed by washing the mixture several times with approximately 10% sodium carbonate solution. The mixture was dried over sodium sulphate, benzoin was added as an acceler- ator for the polymerisation process, and then the mixture was spread over anhydrous sodium sulphate. After being left in the light for several days until a slight increase in viscosity was noticed, the mixture of prepared monomer was stored in the dark. The prepared monomer has, on occasion, been kept successfully for as long as six months.

A strip of the plastic to be examined (about 1 mm cross- section) was placed in a gelatine capsule (size 00) which was then filled up with methacrylate monomer and immediately placed in front of an ultra-violet lamp for polymerisation. This could be done conveniently by attaching it to Sellotape held in a frame. Strips were normally taken because many plastics float, so that if a small piece of material was placed at the bottom of the capsule, there was nothing to hold it in place. A further advantage is that the strips were held more firmly in the embedding medium than were smaller pieces. Polymerisat ion was commenced immediately to minimise any swelling action of the polymer, but if such swelling had been noticed the method would have been considered to be inappropriate for that particular material. The rate of polymerisation could be varied by altering the distance

between specimen and lamp. A SOOW high-pressure ultra- violet lamp was used in the present work. At a distance of approximately 6-8 in, the methacrylate was hard and could even become brittle, while the slower rate of polymerisation at greater distances resulted in a rubber-like texture. When the specimen was held at distances of about 25-30 in, the polymer obtained was flexible. For the majority of specimens slight resilience is to be preferred. Use of the S500 ultra-violet lamp at a distance of about 15 in and an exposure of 10 h was satisfactory. An additional few hours made no apparent difference to the texture or suitability for sectioning.

After polymerisation, the gelatine capsule was removed by being soaked in water, and the end of the block was suitably shaped. This was best effected by grinding it on a stone with a right-angled edge driven by a variable speed, low horsepower motor (1/30 h.p.), taking care to leave a rim of the methacrylate. The flat side of the stone was used to prepare a flat surface on the base of the block for labelling with the serial number. When sufficient care was taken during this trimming process, almost any plastic, apart from those that are soluble in methacrylate monomer, could be sectioned.

Sectioning

Sections were cut at room temperature using a Servall Porter-Blum microtome (MTI), which gives direct transmission of pressure from the specimen to the hand of the operator. Cutting speed and pressure could be adjusted as the knife penetrated the block. This is necessary for a hetero- geneous specimen with components that differ in hardness and elasticity. Fig. 2, for example, shows a soft silicone rubber with a diatomaceous earth as a filler. As the hard silica diatom shells are cut the surrounding softer medium should be torn as little as possible, while inclusions in the surface (Fig. 3) need to be retained. For this stabilisation of the surface the surrounding embedding medium is almost essential. The rate of cutting varied with hardness, the harder polymers usually requiring a swifter movement.

The knife used was a diamond knife prepared by Ge-Fe-Ri. For plastics a suitable knife angle was 53-55", and sections of up to 1)-2 mm across could be cut with a 3 mm diamond edge.

Fig. 2. Silicone sponge containing a diatomaceous earth with fragments of diatoms and other materials dispersed through the silicone

Coherent sections could be obtained without damage to the diamond knife. x 4500

Fig. 3. Two diatoms in the surface of the sponge These enabled the filler to be identified as being from a Californian

deposit. x 7500

Br. Polym. J., 1969, Vol. 1, November

Farmer & Little: Prepara!iorc of Sections of Plastics and Polymers for the Electron Microscope 261

The knives used in the present work haw rarely been chipped, and were used regularly for a year or more before resharpening was needed.

The angle between specimen and knife varied from one specimen to another and was sometimes as high as 30-35" (Fig. 4) although 18-20' was the most usual. The angle chosen was that which was felt to cut the mosi easily. The value of this angle is critical. One reason is that if there is a deviation to above the optimum, knife shiver may occur (Fig. 5). Sometimes one component may be affected but another may not, or the harder surface layer may be revealed; in the case illustrated in Fig. 5 the folds perpendicular to the cutting direction show a higher frequency in the harder surface layer. The more exacting reason is that distortton of the cut surface,

Fig. 4. Specimen embedded in methacrylate held in chuck of Pouter- Bkm MTi microtome

usually caused by too low an angle, tends to destroy the structure, so that the thickness of the distorted layer must be kept to a minimum. These two effects need to be balanced, and use of the sense of touch has proved to be the most reliable criterion. Each polymer felt different. The most difficult to cut were the very soft rubber (Fig. 6) and the very hard nylon (Fig. 7), with both of which the knife tended to slide away from the specimen.

Sectioning at an unsuitable angle increases the thickness of the distorted surface layer. Andrews2 has partly overcome this difficulty by cooling the specimen with liquid nitrogen, but found that a different temperature range was required for each type of specimen. It would seem to be simpler to adjust the angle of the knife than to have the additional complication

Fig. 5. Surface of nylon cut with SON

The dialnond knife ( G ~ - F ~ - R ~ ) is heid on a brass block, This photograph demonstrates a hlgh cutting angle.

There is an altered surface zone which is harder and more ordered

x 4500 than the interior. The results of knife shiver may be seen

Fig. 6 . Soft natural rbbber x 7500

Br. Polym. J., 1969, Vol. 1, November 2

Fig. 7 . A hard, highly crystalline nylon The portion of a crystalline area seen in the section has bulged out

as strain was released. x 4500


of working at such low temperatures. In general, the softer the section the greater is the angle required.

After sectioning the section must be transferred from the knife to the specimen grid. Each section was lifted separately from the knife with a soft brush and placed on the surface of a mixture of dioxane and distilled water in a small dish. A pig’s eyelash is also useful for handling sections. The optimum concentration of dioxane varied, and was determined by trial and error. Even with the rim of methacrylate around the section it may take several hours for a section to unroll or unpleat completely. Rusnock & Hansenl have gone to considerable trouble to devise conditions which would allow sections to float directly from the cutting edge to a liquid surface in an attached boat, but this direct method considerably limits the range of specimens that can be successfully sectioned. In the present work, for specimens with high internal stresses, and particularly some of the nylons, sections were even left for 24 hours in a dry container before being floated on the dioxane and water mixture. In the majority of cases, sections which showed purple or blue interference colours when floating on the liquid surface were found to be of a suitable thickness, although with some polymers thicker sections were required. They were then picked up in the usual way on to electron microscope grids.

Section thickness

In choosing an optimum section thickness, which again may vary from one specimen to another, several factors need to be borne in mind, the most important of these being the surface distortion effect. Rusnock & Hansen’ and Andrews et d2 partly compensated for the loss of resolution due to this distortion by staining the sections, but the distortion still remained, and for plastics, ultra-thin sections will always be liable to create more problems than they solve.

The appearance of surfaces, as seen in section, has varied according to the method of preparation of the surface and according to the structure and composition of the plastic. This latter aspect will be discussed more fully in a separate paper. Fig. 8 shows the surface of a cut nylon film. The film is still partly folded, and the plastic has slightly expanded in the electron beam. The distoited surface layer was less

affected, and shows clearly. The surface layer in the air-dried gelatin of a photographic emulsion (Fig. 9) is apparent for a different reason - there are no silver grains in it. The methacrylate embedding medium is seen at the top of the photograph. A different appearance again is seen in the section through the surface of a ‘polished’ polypropylene heart valve shown in Fig. 10.

More relevant to the problem of sectioning is the effect of a scissor cut shown in Fig. 1 1 . The width of the hardened surface layer varied from one end of the cut to the other, and so demonstrated the importance of a correct knife angle. In the case of thin sections, a transmission photograph can give a true picture of the internal stiucture of the polymer only if there is a sufficient thickness of unmodified material between the two altered surfaces, and it is fortunate that in nearly every specimen examined there was a sharp boundary between the distorted and unmodified regions.

The sectioning of rubbers, whether natural latex (Fig. 6), synthetic (Fig. 12), or silicone rubber (Fig. 13) is from this point of view simpler, since their resilience prevents surface distortion. In passing, the photograph in Fig. 12 suggests one use to which this technique may be applied. The specimen was an experimental silicone, and the filler particles, inadequately compounded, are not a part of the structure and so would represent a source of weakness rather than strength.

The nylon shown in Fig. 8 had been sectioned at an unsuitable angle and so no unmodified material remained in the section. The section illustrated in Fig. 14 does show structure, but it is rather blurred. Here the two surface layers dominate. The tear has crossed the spherulite with scant regard for its presence, showing that the physical properties of the surface layers were the more important. Moreover, there is a continuous layer of material present, although one of the characteristics of spherulites is the presence of a very low density of material, or actual voids, between the dendritic processes in sections where the internal stresses have been partly or wholly released. This is shown in the nylon spherulite in Fig. 15. With thinner surface layers there is also a considerable increase in the effective resolution. In all cases, the limit- ing factor for resolution is electron scatter within the specimen.

The section thickness must depend partly on the minimum

Fig. 8 . Surface of cast nyIonfilm Folds are seen in the film, which had not completely flattened on the dioxane-water surface. The surface distortion due to sectioning

Fig. 9. Section across emulsion of a photographic bromide paper after developing but not fixing

x 7500 has in this case completely obliterated the nylon structure. x 4500


Farmer & Little: Prepara,'iorr of Sections of Plastics and Polymersfor the Electron Microscope 263

Fig. 10. Section through 'polished' surface of polypropylene heart valve

X 4500 Fig. 11. Hardened surface layer of scissor-cut through anaesthetic

tube x 3000

Fig. 12. Synthetic rubber, partly crystalline Fig. 13. Silicone rubber prepared experimentally, and with filler x 4500 particles inadequately incorporated

x 6000

thickness of surface distortion for the particular specimen. It also depends on the structure which is being sought. The material containing diatoms shown in Figs 2 & 3 requires a sufficient thickness of section to contain the width of a diatom shell in the section. Similarly the material in Fig. 16, containing 10% filler, requires a sufficient thiakness of section to give an adequate idea of the dist-ibution of the filler. With too thin a section, this filler, molybdenum disulphide, would crumble. The carbon black used as filler in the Delrin (polyformaldehyde) in Fig. 17 will, on the olher hand, section easily. With this material a thinner section is an advantage, because of the very fine linear or dend1,itic crystals which are formed when the material is under tension. This structure is seen in one of the grains in Fig. 17.

Examination of sections

Many of the points which require consideration during the examination of sections have already been mentioned, as illustiations of the sectioning technique.

Expansion of many specimens in the electron beam is one important phenomenon and so are other consequences of the interaction of the electron beam and the section. There is electron scatter both in the disturbed layers and also in the undisturbed material. In Fig. 18 an oriented specimen of polytetrafluoroethylene has been sectioned in the crystal direction. The banded structure is easily distinguished and contrast is fairly low. Fig 19 is a section through another granule in the same specimen, this time cut obliquely. The



Fig. 15. Spherulite in a section cut from the same block as theprevious section, but with a more suitable angle between knife and specimen, so that a greater proportion of the section is occupied by unmodified

material x 2400

Fig. 14. Split in nylon section, passing through a spherulite The two hardened surface layers are responsible for the properties exhibited by the section, and consequently the spherulite has failed to deflect the direction of the tear. Spherulites tend to be saucer- shaped, and this section is a transverse one through the spherulite.

x 5000

Fin. 17. Delrin containing carbon black asfiller - The Delrin shown in this photograph appeared to have a two-phase structure. In some grains small thread-like crystals may be seen which are almost in the ulane of the section (which was parallel

Fig. 16. Fluon containing 10 % MoS, as filler Each main is filled comparatively uniformly with the MoS, but there 6 a different proportion in each grain.. The particles of-filler have been cut successfully longitudinally, but have broken when

cut transversely. x 4500

to the surface of a tensile-test piece.) Other grains show larger and more diffuse crystalline areas. The particles of carbon black are

also sectioned evenly. x 7500

wide, almost horizontal, bands which can be distinguished show where ordered sheets of the material are traversed. Most striking, however, is the increase in contrast due almost entirely to Bragg-type deflection of the electrons, and not to large density differences within the material. This subject of the interaction of the electron beam with plastic specimens will be discussed in greater detail in a separate paper.

In nearly every case the amount of information available is substantially increased when stereoscopic photographs of the

area of section are viewed, and in some instances this is essential for a correct interpretation. Fig. 7, for example, showed a section through a nylon crystal in a material cooled from the melt to form a three-dimensional mosaic of such crystalline areas. At room temperature there is a very considerable internal strain which is relieved on sectioning by portions of many of the crystalline areas bulging out to form conical humps. On the other hand, the portion of the spherulite in Fig. 15 is still in the plane of the section. The clarity of Fig. 14


Farmer & Little: Preparation of Sections of Plastics and Polymersfor the Electron Microscope

Fig. 18. Highly crystalline Fluon specimen, sectioned in the direction of orientation

The crystalline areas show a regular cross-banded structure. Stereoscopic observation shows that the portion of the section on the bottom and right of the photograph which appears slightly

darker is bent upwards. x 7500

is also considerably enhanced when viewed three-dimension- ally.

Electron scatter nearly always prevents an adequate electron diffraction pattern being obtained from crystalline regions such as those shown in Figs 7, 15, 18 and 19. There has been no real justification for the assumption that a lack of emergent diffracted beams of electrons indicates a lack of crystallinity, and this assumption is now known to be incorrect. This question of electron scatter and electrca diffraction will be considered further in a paper on the interaction of the electron beam and the specimen.

Conclusions

Using the sectioning technique described in this paper it has been found that sections of most polymeric and plastic materials can be obtained provided tk . t the material is not soluble in the butyl-isobutyl methacrylate mixture used as an embedding medium. Sectioning was done at room temperature, the essential requirements being a micrslome which allowed direct transmission of presure from specimen to hand so that the sense of touch could be used to control the conditions and

Fig. 19. Fluon section oblique to the direction of orientation x 7500

265

a sufficiently wide range of available cutting angles. The sectioning angle was found to be fairly critical and a sharp boundary between distorted surface regions and the interior of the section allowed useful observations to be made of the undisturbed structures. The thickness of the distorted surface layers rather than the total thickness of the specimens provided a limit to the resolution obtained.

Acknowledgments

The authors wish to thank the many people who have provided or made specimens for this work, and especially members of the Research Staffs of British Nylon Spinners Ltd. (now I.C.I. Fibres Ltd.) and I.C.I. Plastics Ltd. who made various samples of nylon and PFTE, and Dr. George Wood of the Royal Aircraft Establishment and Mr. A. J. Sherrin of the Dunlop Rubber Company who provided other specially prepared samples. They would also like to thank Mr. T. Rowe who took the photograph shown in Fig. 4.

Nuffield Orthopaedic Centre, Oxford

Received 20 May, 1969; amended manuscript 2 July, 1969

References 1 Rusnock. J. A.. & Hansen. D.. J. Polvm. Sci.. 1965. A3. 647

Andrews; E. H', Bennett, M. *., & Markham; A., i Pobm. Sci., 1967, A2, 5, 1235

Br. Polym. J., 1969, Vol. 1, Novemb,er

preparation of sections of plastics and polymers for the electron microscope

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