SPECIMEN PREPARATION FOR ELECTRON MICROSCOPY By J. A. GARO Chemistry Dept. Aberdeen University. [Read 4th November, 1955] ABSTRACT General techniques for specimen preparation are described, including formation of supporting films and surface replicas, and methods for dispersion of clays. ]INTRODUCTION General techniques for specimen preparation are based on the following considerations. (a) The penetrating power of the electron beam is poor, so the specimens and supports must be very thin. (b) The specimen may be affected by the high vacuum of the instrument, and may be heated to temperatures up to 300~ by the electron beam, so volatile liquids must be removed, and care taken in interpretation of electron micrographs. (c) Cleanliness is essential, as impurities may be misleading. Micropipettes should be freshly drawn from glass tubing, and used only once. The instrument may be used for examination of specimens in the following ways. (1) A fine powder may be dispersed as discrete particles on a thin supporting film, which is in turn supported on a grid of thin metal foil of 3 mm diameter, which has about 300 holes, 80/z square through which the electron beam passes. (2) A thin replica may be formed of the surface of a thick specimen. (3) The surface may be examined directly by oblique reflection of electrons. (4) In modern instruments, an electron rnicrograph and an electron diffraction pattern of the same area may be readily obtained. The area may be as small as 1-2tz diameter, and may therefore consist of an individual crystal. Special preparation of specimens is unnecessary. METHODS OF PR~PARATIOY OF SUePORTIN~ FILMS (1) One or more drops of a 2 or 3% solution of collodion in arnyl acetate are allowed to fall on a clean water surface from a pipctte. After evaporation of the solvent, a large area is covered by a thin even film. (2) A few drops of 0.5% formvar solution in anhydrous chloroform are spread quickly on a clean microscope slide, using the pipette or a 14
SPECIMEN PREPARATION FOR ELECTRON MICROSCOPY 15 clean glass rod. After evaporation of the solvent, the film is cut into squares rather larger than a specimen grid, breathed on, and the slide is slowly immersed in clean water. Surface tension pulls the film off tile slide. These films are often rather uneven, and better films may be made in a simple apparatus due to Revell and Agar (1955). The slide is immersed in formvar solution in a cylindrical tap funnel, and the solution drained through a capillary tube. The thickness of the film is determined by the concentration of the formvar solution. (3) A bulb of borosilicate glass may be blown ot~t in a flame, and the flakes allowed to settle on a water surface. The films are smooth and heat-resisting, but the thickness cannot readily be controlled. (4) Films may be evaporated in vacuum on to a smooth support which is later dissolved. Silica or silicon monoxide (Gerould, 1947), beryllium, aluminium (later oxidised to alumina), and carbon from an arc (Bradley, 1954), have been used. The thickness of plastic films is of the order of 400 3,, but carbon films may be less than 40 X thick. A specimen grid is usually placed on each square of the film, and picked up from under the water surface by means of a brass washer soldered to a length of wire, so that the grid is over the hole in the washer. The water is removed from under the film by means of a capillary, and the grid reversed on to a peg of 81-" diameter. DISPERSION OF CLAYS Clays are usually examined in the form of individual particles, which may be dispersed either by wet or dry methods, of which the most commonly used are described below. (1) The sample is ground or shaken with water. The clay fraction is normally pipetted from a depth of 10 cm after settling for 8 hours, but fractionation in a centrifuge may be preferable. The appropriate dilution is found by trial, and a drop of the suspension transferred to a film-coated grid in a desiccator. Surface tension during drying causes the particles to adhere strongly to the film, so soluble impurities may be dissolved by addition and removal of drops of water from a pipette. If flocculation occurs during drying, it may sometimes be prevented by using a dilute solution of the hydroxide of the appropriate base for dispersion, or by vibration during drying. It is sometimes sufficient to attach the specimen to the moving coil of a loudspeaker energised by 50-cycle A.C. current from a Variac. (2) A dilute suspension of the sample is sprayed on the film-coated
16 J.A. GARD grid, so that each drop covers a discrete area. Unintentional fractionation due to surface tension is thereby avoided. (3) The powder may be dispersed in the medium normally used in practice, then diluted in a volatile solvent. After drying, the residual medium is washed away with more solvent. Examples of this technique are paint pigments in linseed stand oil, and rubber fillers in crepe rubber. (4) There are numerous dry dusting techniques. In the simplest methods, the powder is sprinkled on the grid, and dispersed by vibration, or electrostatic repulsion using a Tesla coil. A better method is that of Cuckow, in which a stream of air is drawn through the powder, in the bend of a J-tube, by means of a water-pump, so that a cloud of dust is elutriated into the long arm. After allowing to settle, a film-coated grid is placed in the cloud. The fraction collected depends on the settling times and the height of the grid. SURFACE REPLICAS The orientation of clay particles on the bulk has been studied by making replicas of the surface. There are two principal types of replica. (a) Single-stage, or negative replicas, in which the surface is coated with a dilute solution of a plastic, such as formvar, and drained. The apparatus of Revell and Agar (1955), already described, is suitable. When the solvent has evaporated, the film is stripped, either dry or on to a water surface, and supported on a grid. (b) Two-stage, or positive replicas, in which a comparatively massive negative cast is made of the surface, in a thermoplastic material, and a thin film evaporated on to the surface of the cast after separation from the specimen. Silica or silicon monoxide on polystyrene, and carbon on formvar or Bedacryl resin are the most usual combinations. Comer and Turley (1955) found that bulk clays were too porous for ordinary methods, as particles were removed from the surface. To prepare true replicas, the surface was pre-shadowed with platinum, which was backed with a layer of evaporated carbon. The clay was then dissolved in hydrofluoric acid. REFLEXION ELECTRON MICROSCOPY Electrons reflected at glancing angles from the surface of the specimen are imaged by the lenses. An oblique view of the surface is obtained, and the resolution is comparatively poor. Insulating surfaces must be coated with a thin film of evaporated silver to dis-
SPECIMEN PREPARATION FOR ELECTRON MICROSCOPY 17 perse the charge which would otherwise accumulate and deflect the electron beam. The technique does not appear to have been applied to clay minerals. ANCILLARY TECHNIQUES In order to measure the height of particles, and to increase contrast, especially of surface replicas, a loop of heavy metal may be evaporated, in a high vacuum, from a tungsten filament or basket, on to specimens supported at an oblique angle, so that shadows clear of metal are thrown on the supporting film. An alloy of 60% goldpalladium, and a ratio between the length of the shadow and the height of the particle of four or six, are suitable for most specimens. The deposit must be thin in order to avoid apparent distortion of the particles, as the sides facing the filament receive a much thicker coating than the supporting film. Heights of particles may be calculated from the length of shadow. A negative print has the appearance of a specimen illuminated obliquely by a beam of light, giving rise to dark shadows. The objective lens of the electron microscope has a great depth of field, and this makes stereoscopy possible. Two micrographs of the same area of specimen are recorded, with the specimen at different angles of tilt. The prints are mounted as a pair after correcting their orientation for rotation of the image by the lenses, and viewed in a stereoscope. Contrast in micrographs of clays with poor scattering power may sometimes be improved by base exchange of the cations of the specimen for those of a heavier metal, such as caesium, barium and lanthanum. REFERENCES Revell, R. S. M. and Agar, A. W. 1955. Brit. J. appl. Phys., 6, 23. Gerould, C. H. 1947. J. appl. Phys., 18, 333. Bradley, D. G. 1954. Brit. J. appl. Phys., 5, 65. Comer, J. J. and Turley, J. W. 1955. J. appl. Phys., 26, 346. DISCUSSION Mr J. Cartwright asked whether amongst the various metals used in shadowing, chromium had been tried. Mr Gard replying, stated that he had not tried this particular metal, but had found that gold-palladium alloy gave good definition. Mr Cartwright then said his question had been prompted by the fact that there was considerable variation from operator to operator in the efficiency of chromium shadowing. Mr Cartwright then
18 J.A. GARD showed a micrograph of a specimen which had had violent agitation in methanol but which still showed clusters of crystals. He asked whether Gard thought his vibrator would aid the dispersion or whether these aggregates were, in reality, single crystals. Mr R. H. S. Robertson then stated that due to parallelism during settling of clay particles, the twisting forces needed to dislodge particles were extremely high and consequently normal methods of agitation frequently failed to break up aggregates. In this respect, it had been noticed that there was considerable variation even in the same mineralogical species, due to degree and perfection of crystallinity. Dr G.F. Walker asked Mr Gard his opinion on the future of carbon replica techniques, for it seemed that good resolutions could be obtained and Gard in reply stated that Bradley had had considerable success in this field. Dr D. M. C. MacEwan stated that considerable success has been obtained at Pennsylvania State University with a combined technique of metal and carbon shadowing. The resolutions obtained were excellent.