Wear, 50 (1978) 95 - 103 95

A COMPARATIVE STUDY OF REPLICATION TECHNIQUES FOR USE IN SCANNING ELECTRON MICROSCOPY OF ORTHOPAEDIC IMPLANTS

PHILIP ROBBINS and JAMES PUGH

Biomechanics Laboratory, Department of Orthopaedics, Hospital for Joint Diseases and Medical Center, 1919 Madison Avenue, New York, N.Y. 10035 (U.S.A.)

(Received August 12, 1977; in final form December 9, 1977)

Summary

Three replication techniques for use in scanning electron m icroscopy of orthopaedic implants are described and compared. The three consist of one type of silicone replica (Xantopren@ Blue) and two types of plastic film replicas (collodion in amyl acetate and Formvar in ethylene dichloride). The clearest image with the highest resolution and contrast is obtained with a Formvar replica. Collodion produces almost as good an image and the replicas are easier to produce. However, neither Formvar nor collodion can be used on implant surfaces which are grossly curved or rough textured since the replicas are not rigid enough to retain the true shape of the sur- face. Xantopren Blue is considerably easier to use than either of the plastic films, gives a true reproduction of specimen shape and allows replication of rough surfaces. Its only deficiencies relative to the plastic films are resolution and contrast at high magnification. If the sample to be replicated is relatively flat and smooth, and high magnification is desired, plastic film replication materials are recommended.

1. Introduction

Often the actual specimen to be studied is not suitable for direct observation in the scanning electron m icroscope (SEM). The specimen may be too delicate and thus will deform when subjected to either a vacuum or the electron beam [ 11. Alternatively, when dealing with orthopaedic im- plants, the area to be studied cannot be placed in the specimen chamber because either (1) the specimen is subject to continuing wear and cannot be removed for observation or (2) the specimen may be too large to be con- tained in the specimen chamber and cannot be sectioned for medico-legal reasons. In these cases it is most convenient to replicate the surface of the sample and study its negative replica in the SEM.

It is important to pick a suitable replicating material. Various plastic films and silicone rubbers are the most widely used because they are easy to

0043-1648/78/0050-0095$2.25 0 Elsevier Sequoia S.A.. Lausanne/Printed in the Netherlands

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apply and to strip from the surface. This study deals with surfaces or ortho- paedic implants which are relatively smooth with either a matt or a shiny finish. The implants are fabricated from 316L stainless steel, cast chrome cobalt alloy or high density polyethylene.

This paper illustrates the use of one type of silicone replica (Xantopren Blue) and two types of plastic replicas (collodion and Formvar) of implant surfaces.

2. Replication techniques

2.1. Collodion replication (1) Collodion replication is best performed with a 2 - 4% solution of

collodion in amyl acetate as discussed later. (2) All surfaces to be replicated must be cleaned thoroughly using

either alcohol or acetone with fine-grain tissue such as Kimwipes@ followed by a Freon duster in order to eliminate any loose particles on the surface.

(3) The 4% collodion solution is applied with either an eyedropper or a syringe. Two or three drops are usually sufficient. The collodion should be spread as thinly as possible on the surface by tilting the sample at an angle in order to allow the excess to drain.

(4) The collodion dries in 10 - 20 min depending upon the thickness of the application and the room temperature.

(5) The collodion replica is best removed from the surface of the sample using a cellulose tape (a 2 in long piece of.+ in Scotch@ tape is recommended). The tape is firmly applied to the dried collodion and the replica is removed by peeling off the Scotch tape. It is advisable to begin lifting the tape at the end where the collodion accumulated while draining. The tape should be pulled with minimal bending and tension while main- taining an angle of about 30” to the surface.

Fig. 1. A 1% collodion replica of a glass slide damaged by Duco cement (magnification 50X).

Fig. 2. A 4% collodion replica of a glass slide damaged by Duco cement. Naked Scotch tape is shown at the upper right with the rep!ica below and to the left of the diagonal line (magnification 50X).

(6) Specimens are usually attached to the SEM specimen stud with adhesive. It is important to realize that, if an adhesive material is used which dries and bonds by solvent evaporation, care must be taken that this will not “out-gas” excessively and result in contamination of the vacuum in the specimen chamber of the SEM. Another consideration is that many of the solvents in glues are destructive to both the Scotch tape (on which the collo- dion replica is attached) and the collodion. The result of a collodion replica being bonded to a stud by means of Duco@ cement is shown in Figs. 1 and 2. Double-sided masking tape appears to be a safe bonding adhesive for collo- dion replicas. The Scotch tape with the attached replica must first be trimmed to the size of an SEM stud with sharp scissors. The double-sided masking tape is also trimmed to size and then attached to the stud. The replica is simply attached to the other side of the double-sided masking tape by gently pressing down in the corners with a small blunt-ended tool.

2.2. Formvar replication Formvar replication is similar to the collodion replication procedure

outlined in Section 2.1 since both are plastic film replicas. A 1 - 4% solution of Formvar in ethylene dichloride is slightly more difficult to use than the solution of collodion in amyl acetate. The Formvar dries very quickly (approximately 1 min), and if it is not applied in an extremely thin even coat cloudy areas tend to form in the replica upon drying. This is illustrated in Fig. 3. Therefore, as low a concentration as possible of Formvar in ethylene dichloride is used and only one drop is applied followed by imme- diate spreading and draining by tilting and rotating the sample.

With both collodion and Formvar replication there is a tendency when stripping the replica from the surface of a specimen with Scotch tape not to pull off the entire replica. The bare Scotch tape can be recognized in the microscope. Figure 4 shows micrographs of Scotch tape to aid in the recogni- tion of unacceptable specimens.

Fig. 3. A 3% Formvar replica of a glass slide, showing cloudiness and imperfections resulting from poor technique (magnification 5000x).

Fig. 4. Scotch tape (adhesive side) under the SEM (magnification 500x).

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2.3. Xantopren Blue* replication (1) The surface to be replicated is first thoroughly cleaned. (2) The Xantopren Blue is prepared by thoroughly mixing the.proper

quantity of Xantopren silicone base with the “soft hardener”. (3) The mixture of hardener and base is applied to the surface in as

thin a coat as possible. (4) The Xantopren is allowed to dry for approximately 5 - 10 min

depending upon the amount of “soft hardener” used and the room tempera- ture.

(5) The Xantopren replica is removed from the sample surface by pulling off gently with a pair of forceps.

(6) The replica is trimmed to size with scissors. (7) The specimen is secured to the stud using Xantopren as a glue.

3. Notes on specimen preparation

3.1. Choice of collodion or Formvar concentration A general rule is that, regardless of the type of surface, the range of

concentration for both collodion in amyl acetate and Formvar in ethylene dichloride is between 1% and 5% plastic in solution.

It is more difficult to strip a plastic replica from a rougher surface. Therefore, rough surfaces require films of greater strength (i.e. greater con- centration of plastic) than smooth surfaces. A general rule is that the greater the concentration of plastic (which means a thicker plastic film), the easier it is to strip the film from the surface of the specimen. However, if the sur- face is porous or so rough that the plastic enters deep cracks and crevices, stripping by the methods previously outlined becomes practically impossible.

Thinner films give better contrast in the SEM [ 21. Thus although thicker films promote ease of stripping, thinner films give a sharper image in the microscope. A compromise must be reached. Shadow casting brings out finer structure through increased resolution and thus aids in the interpreta- tion of heights and depressions [ 21.

Collodion has a tendency to charge up in small patches while being viewed in the SEM at high magnification. The result is destruction of the surface in the area of these patches. The replica literally “fries” as is shown in Fig. 5(a). A replica made of a 1% solution fries considerably more easily than one of a 4% solution. Although vacuum evaporation of a metallic layer considerably reduces charging, it is still difficult to examine 1% collo- dion replicas at magnifications about 2000X while operating at a normal high-voltage-mode accelerating potential of 17 kV.

Formvar tends to cloud when too great a concentration is used (see Fig. 3). A lower concentration, such as 1% or 2%, markedly reduces the

*Xantopren Blue is a silicone substance manufactured by Unitek@ for use as a precision dental impression material.

(a) (b) Fig. 5. 4% collodion replicas of a piece of machined high density polyethylene: (a) replica showing the result of charging up on the surface -the specimen literally fries (magnifi- cation 1400x); (b) area of replica adjacent to area where silver conductive paint was used, illustrating the destruction of the surface by the solvent base of the paint (250x).

propensity to cloud but increases the difficulty of stripping the replica from the surface of the sample.

It may be concluded that the best workable range of concentrations for collodion in amyl acetate is 2 - 4% because of the ease with which a 1% solution charges up (fries). However, Formvar in ethylene dichloride is not as sensitive to charging up and therefore a range of concentrations of 0.5 - 3% is recommended. It is generally a good procedure when using Formvar to start with higher concentrations and then to reduce the concentration to find empirically the lowest concentration which can be stripped with ease from the particular sample [ 21.

3.2. Final steps of replica preparation Shadow casting with heavy metals such as gold, platinum, chromium or

gold-palladium* provides a means of greatly improving the contrast and thus the sharpness of the image in the SEM [ 31. It also renders the image relatively independent of film thickness, thus aiding in the interpretation of heights and depressions. Once evaporated, the final step in replica prepara- tion is to use conductive paint in order to obtain a good electrical conduc- tion line to the stud. It is strongly recommended that only a small corner be painted for the plastic film replicas because the solvent base of silver conductive paint easily dissolves the collodion or Formvar. This greatly restricts the type of destruction that is illustrated in Fig. 5(b).

*The authors have found success using gold-palladium for vacuum evaporation of both silicone and plastic film replicas.

Fig. 6. A 4% collodion replica of a glass slide at low magnification. The replica is at the lower right, while a piece of Scotch tape not covered by the replica is shown at the upper left (32x ).

Fig. 7. A 4% collodion replica of a glass slide at relatively high magnification showing a blemished surface with a piece of dust on top (magnification 1250x).

Fig. 8. A Xantopren Blue replica of a glass slide (magnification 500x).

Fig. 9. A 2% Formvar replica of a glass slide showing surface defects (magnification 3600x).

4. Replica comparison

4.1. Comparison using glass replication In order to make an initial comparison of these three types of surface

replicas a plain glass slide was replicated with each method. Comparison at a superficial level revealed that both Xantopren Blue

and collodion were relatively simple to use, while there were complications with Formvar because of the production of cloudy replicas. However, with some less concentrated solutions the cloudiness was eliminated and a satis- factory replica was finally achieved.

In the SEM, differences between replication methods were made evident. The first sample examined was a piece of the glass slide itself. Viewing for several m inutes revealed absolutely nothing and not even a

Fig. 10. A Xantopren Blue replica of HDPE at low magnification (200x).

Fig. 11. A 4% collodion replica of HDPE at low magnification (360x).

picture was taken (i.e. no image of the surface of the glass slide could be ob- tained in the SEM). However, examination of the replicas revealed some useful information. The collodion replica looked extremely smooth and flat at low magnification as was expected (see Fig. 6), but upon closer examina- tion at considerably higher magnification the replica appeared to be spotted with small dark patches scattered throughout (Fig. 7). It appeared almost as though the replica had blemishes since the spots were perceived as level with the surface when viewed at all angles. In contrast, the Xantopren Blue replica looked almost completely defect free (Fig. 8). With Formvar the surface looked smooth in most areas at low magnification, but at high magnification defects were prevalent throughout most of the specimen. These defects appeared as small protuberances and depressions (Fig. 9). Clearly, this Formvar replica of a glass slide is rather poor in quality.

4.2. Comparison using high density polyethylene replication A more conclusive comparison involving the replication of a substance

with a more detailed surface was undertaken. The substance chosen was a section of machined high density polyethylene (HDPE) which was part of a knee prosthesis. The results proved somewhat different from those obtained in the comparison using a glass slide replica. All three replicas were made with relative ease and without complications.

At low magnification all the replicas appeared to have virtually no defects. Surface features from the three different replicas looked comparable (Figs. 10, 11, 12). At high magnification (Figs. 13,14,15) the specimens showed much detail, especially the plastic film replicas. The Formvar replicas undoubtedly produced the best image in the SEM. The picture was extremely sharp and clear with a great deal of contrast (Fig. 15). There appeared to be few surface defects in comparison with the collodion replica (Fig. 14). The Xantopren also produced a good image, although not nearly as sharp as the Formvar specimen. On the whole, the detail in the Xantopren replica was not as good as in either of the plastic films.

Fig. 14. A 4% collodion replica of HDPE at high magnification (2500x).

Fig. 15. A 3% Formvar replica of HDPE at high magnification (3000~).

4.3. Shape characteristics It is also important to consider the shape of the sample to be replicated

when deciding upon a replication material. The replication reported in this paper was only of flat surfaces. However, with orthopaedic implants there are usually many grossly curved sections3 (e.g. the head of the femoral component of a hip prosthesis). If the curves are very severe, forming such shapes as small gulleys or spheres, then collodion or Formvar replication is not feasible. However, if the curves are more gently sloping, plastic films can be used. Plastic film replicas do not retain the shape of the surface from which they were stripped. Rather, they usually conform to the curved shape of the Scotch tape or sometimes just lie flat. This generates artefacts simply because the original shape to which the plastic is molded is then altered for viewing in the m icroscope. Thus, surface features can be somewhat distorted by this unnatural bending. However, Xantopren Blue is able to conform to gross curves or small crevices and then to retain their original shape when the replica is removed from the surface of the specimen.

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5. Conclusions

Three replication materials were compared. The clearest image with the highest resolution and contrast is obtained with a Formvar replica. Collodion produces almost as good an image, but is less difficult to use. Xantopren Blue is considerably easier to use than either of the plastic films, gives true reproduction of specimen shape and allows replication of rough surfaces. However, if the sample to be replicated is relatively flat and smooth and high magnification is desired, then either of the two plastic film replication materials is recommended.

References

1 J. W. S. Hearle, J. T. Sparrow and P. M. Cross, The Use of the Scanning Electron Microscope, Pergamon Press, Oxford, 1972, p. 85.

2 C. E. Hall, Introduction to Electron Microscopy (2nd edn), McGraw-Hill, New York, 1966, pp. 310 - 315.

3 G. Thomas, Transmission Electron Microscopy of Metals, Wiley, New York, 1966, pp. 133 - 134.