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Ultramicrotomy of carbon-graphite materials for transmission electron microscopy
METALLOGRAPHY 9, 43-49 (1976)
43
Ultramicrotomy of Carbon-Graphite Materials for Transmission Electron Microscopy E. J. WATTS Materials Sciences Laboratory, The Aerospace Corporation, El Segundo, Cali]ornia
Tile biological techniques relating to the encapsulation of materials for ultramicrotomy prior to TEM observation were applied to some specific graphites to which metals had been added to modify the carbon structure. Ultrathin cross sections were prepared and examined in a high-resolution transmission electron microscope in order to characterize the internal morphology of the materials. Particle size and shape and relative particle size distribution were determined for each of the graphite species and identification of the major crystalline constituents was made by means of electron diffraction.
Introduction The microstructure of carbon-graphite systems has been studied extensively by means of optical microscopy, X-ray diffraction, scanning electron microscopy, and ion microprobe analysis. When impurities occur in these materials either naturally or are intentionally added and they are in the submicron size range, then the transmission electron microscope must be employed to locate, measure, and identify them. Sample preparation methods in T E M work are generally tailored to fit the desired endresults for each individual sample, the only requirement being that no artifacts be introduced along the way. In order to characterize submicron impurities a method must be selected such that (a) no further impurities are added, (b) the crystalline arrangement remains undisturbed, and (c) the material is thin enough for transmission electron diffraction. A highly successful method for m a n y years for the preparation of biological specimen materials for examination in the T E M has been ultramicrotomy. This is simply the technique of encapsulating a small sample in a suitable resin for support, cutting sections on the order of 500-2500/~ in thickness, and mounting these slices on standard T E M grids for observation. In order to provide very thin sections of materials such as tissues, bone, and cartilage with sufficient contrast for T E M work, extensive specialized (~) American Elsevier Publishing Company, Inc., 1976
44
E. J. Watts
preparation techniques have been devised. Some of these techniques include fixation, dehydration, staining with electron dense materials, and impregnation with any of a multitude of resin compositions formulated to match the hardness of the material under investigation. Despite the wealth of information available on sectioning techniques, the use of ultramicrotomy to prepare TEM samples for the physical sciences has been limited. This may in part be the result of the current interest in improved scanning electron microscopes, in ion thinning machines, and in various new and improved jet thinning devices. For the study of submicron particles in layers in situ, however, serial sectioning of the material and reconstruction of the sample morphology from the examination of each successive layer may best be accomplished using ultramicrotome sectioning techniques. The method is rather simple since nonbiological samples usually need not be dehydrated or fixed and may be mounted in the resin at precisely the correct position when a particular cutting direction is of interest as in the case of oriented crystallites or certain single-crystal materials. Some interesting work of this type has been done at Eastman Kodak Research Laboratories [1]. Layers of photographic film were sectioned and a study of the distribution of developed silver particles was made. Studies of this kind are quite important for image tone and/or scattering power. Other industrial applications include studies of microdefects in materials, of the orientation and dispersion of iron oxide particles in magnetic tape, and investigations of paint films to solve problems of flotation of pigments and additives which could lead to poor weathering properties. Differential staining methods may be applied to distinguish between phases in materials, particularly if the stain can be easily absorbed. Some of the useful electron dense stains include phosphototungstic acid, uranyl acetate, potassium permanganate, and certain of the lead compounds, most of which are applied in alcoholic solutions. Although most of the inorganic microtome work in the past was applied to relatively soft materials which section fairly easily, it was decided to try this method for sectioning the extremely hard carbon-graphite materials. After much experimentation, ultramicrotome techniques were perfected and direct observations of submicron crystallites in carbon-graphite materials were made in the TEM.
Experimental Specimens approximately 1 mm across were cut and inserted into the pyramid end of standard 5-mm BEEM capsules [2], embedded in Epon
Thin Sections o/Carbon
45
epoxy resin mix and cured for 48 hours in a vacuum oven at 60°C. Many resin formulations were tried but the Epon epoxy combination according to Luft [-31, using the harder ratios of Mix A to Mix B gave the best cutting properties with carbon-graphite materials. Mix A consists of 31 ml Epon 812 resin and 50 ml of DDSA (dodecenyl succinic anhydride), while Mix B contains 25 ml of Epon 812 and 22 ml of NMA (nadic methylanhydride). Fifteen hundredths ml of the accelerator DMP-30 (dimethyl aminomethylphenol) is added for every 10 ml of the above mix prior to curing. This resin set uniformly with almost no shrinkage, produced blocks with a hardness most closely approaching the carbonaceous material to be sectioned, and yielded thin sections which withstood the intensity of the electron beam at 80 kV better than any other medium. The cured capsules were manually trimmed with a small diamond blade saw and mounted in a Reichert OMU-2 ultramicrotome fitted with a DuPont diamond edged knife and boat {-4"]. The most critical parameter in cutting very hard materials is the tilt of the knife edge. If the clearance angle is negative, the sections will be torn or vary in thickness; if it is too large, they will be wrinkled and again vary in thickness. For carbon a tilt of 10° was found to give consistent, continuous sections 800/~ in thickness. The sections coming off the knife float into a trough of water and will form a continuous ribbon when the technique is perfected. These ribbons of sections were retrieved from the boat onto formvar coated standard ~-inch 150-mesh copper grids, dried, and examined in a Philips EM-300 transmission electron microscope equipped with a high-resolution stage. In order to record all the minute carbide particles most of the observations were made at approximately 100,000 diameters. The astigmatism correction at this magnification is rather critical and great care was taken to use single layers and flat samples in order to ensure accuracy in the measurement of particle sizes. It was found that using coated grids on which to collect the sections was far better than placing them on grids with no support. Aside from the obvious prevention of damage to the epoxy and sample from the beam itself, there was considerable improvement in image "flatness" at high magnification. The use of liquid nitrogen to cool the specimen stage reduced contamination and improved accuracy. One thousand particles were measured from photographs taken of each of the different carbon-graphite samples and particle size plots of number versus diameter in microns were made. This was unfortunately done manually, but there are many new systems available where optics and electronics are combined to give rapid, quantitative image analyses either from photographs or by direct electronic interface with the TEM. The peak particle diameter for the metallic carbide deposits in the set of samples
(a)
(b)
(c) FIG. 1. Typical T E M photographs of thin sections of carbon/graphite materials. All 125,000X (shown here at 60% of original size). (a) and (b) Sections of uniform thickness showing dark carbide crystallites of various sizes. (c) and (d) Sections showing tearing because of incorrect angle setting on knife. (e) and (f) Sections illustrating agglomeration in layers from some of the carbides.
v v
48
E. J. Watts
studied ranged from 0.004 to 0.040 micron with some crystallites as small as 0.0005 micron and some as large as 0.130 micron. Figures l ( a ) - l ( f ) illustrate some of the results of this work on carbon-graphite thin sectioned materials. Magnification is 125,000X in all cases. A further advantage of ultramicrotomy of materials containing crystalline deposits is that electron diffraction photographs may be made of selected areas without changing specimens. Microdefects and foreign deposits may be easily located and identified. Quantitative studies are not generally possible because of the nature of this form of analysis where the scattering factor is so different from that associated with X-ray analysis. The Ewald sphere in electron diffraction is represented by a plane and is huge because of the short wavelength of electrons, while in X-ray diffraction the wavelength is of the order of atoms and hence the radius of the Ewald sphere is comparable with lattice spacings. The main bulk of the impurities may, nevertheless, be identified qualitatively with electron diffraction transmission techniques and, in addition, identification may be made of deposits which might be too small to yield measurable X-ray
FIG. 2. Selected area transmission electron diffraction pattern of MC type carbide deposit in carbon-graphite thin section. 2.8 X.
Thin Sections of Carbon
49
patterns. A typical electron diffraction pattern of one of the MC type carbides studied is shown in Fig. 2.
Summary A method for measuring particle size of submicron impurities in carbongraphite materials by direct observation in the transmission electron microscope is described. Samples are made sufficiently thin by encapsulating them in epoxy resin, curing the capsules, and then sectioning them to 800/~ in thickness in an ultramicrotome using special handling methods. The technique of observing particles in situ has the obvious advantages of permitting determination of the nature of the different kinds of materials in the sample, the distribution, state of agglomeration, and the particle sizes directly.
The author wishes to express sincere appreciation to Dr. Lyle C. Dearden and his staff in the Department of Anatomy, College of Medicine, University of California at Irvine for their technical assistance in this work.
References 1. c.F. Oster, "The application of ultra-thin sectioning techniques to non-biological samples," Proc. of the EMSA, New Orleans, La. (1968). 2. Available from Ernest F. Fullam, Inc., Schenectady, N.Y. 12301. 3. J.H. Luft, "Improvements in epoxyresin embeddingmethods," J. Biophys. Biochem. Cytol. 9, 409 (1961). 4. Available from E. I. DuPont, Wilmington, Del.
Received March, I975
43
Ultramicrotomy of Carbon-Graphite Materials for Transmission Electron Microscopy E. J. WATTS Materials Sciences Laboratory, The Aerospace Corporation, El Segundo, Cali]ornia
Tile biological techniques relating to the encapsulation of materials for ultramicrotomy prior to TEM observation were applied to some specific graphites to which metals had been added to modify the carbon structure. Ultrathin cross sections were prepared and examined in a high-resolution transmission electron microscope in order to characterize the internal morphology of the materials. Particle size and shape and relative particle size distribution were determined for each of the graphite species and identification of the major crystalline constituents was made by means of electron diffraction.
Introduction The microstructure of carbon-graphite systems has been studied extensively by means of optical microscopy, X-ray diffraction, scanning electron microscopy, and ion microprobe analysis. When impurities occur in these materials either naturally or are intentionally added and they are in the submicron size range, then the transmission electron microscope must be employed to locate, measure, and identify them. Sample preparation methods in T E M work are generally tailored to fit the desired endresults for each individual sample, the only requirement being that no artifacts be introduced along the way. In order to characterize submicron impurities a method must be selected such that (a) no further impurities are added, (b) the crystalline arrangement remains undisturbed, and (c) the material is thin enough for transmission electron diffraction. A highly successful method for m a n y years for the preparation of biological specimen materials for examination in the T E M has been ultramicrotomy. This is simply the technique of encapsulating a small sample in a suitable resin for support, cutting sections on the order of 500-2500/~ in thickness, and mounting these slices on standard T E M grids for observation. In order to provide very thin sections of materials such as tissues, bone, and cartilage with sufficient contrast for T E M work, extensive specialized (~) American Elsevier Publishing Company, Inc., 1976
44
E. J. Watts
preparation techniques have been devised. Some of these techniques include fixation, dehydration, staining with electron dense materials, and impregnation with any of a multitude of resin compositions formulated to match the hardness of the material under investigation. Despite the wealth of information available on sectioning techniques, the use of ultramicrotomy to prepare TEM samples for the physical sciences has been limited. This may in part be the result of the current interest in improved scanning electron microscopes, in ion thinning machines, and in various new and improved jet thinning devices. For the study of submicron particles in layers in situ, however, serial sectioning of the material and reconstruction of the sample morphology from the examination of each successive layer may best be accomplished using ultramicrotome sectioning techniques. The method is rather simple since nonbiological samples usually need not be dehydrated or fixed and may be mounted in the resin at precisely the correct position when a particular cutting direction is of interest as in the case of oriented crystallites or certain single-crystal materials. Some interesting work of this type has been done at Eastman Kodak Research Laboratories [1]. Layers of photographic film were sectioned and a study of the distribution of developed silver particles was made. Studies of this kind are quite important for image tone and/or scattering power. Other industrial applications include studies of microdefects in materials, of the orientation and dispersion of iron oxide particles in magnetic tape, and investigations of paint films to solve problems of flotation of pigments and additives which could lead to poor weathering properties. Differential staining methods may be applied to distinguish between phases in materials, particularly if the stain can be easily absorbed. Some of the useful electron dense stains include phosphototungstic acid, uranyl acetate, potassium permanganate, and certain of the lead compounds, most of which are applied in alcoholic solutions. Although most of the inorganic microtome work in the past was applied to relatively soft materials which section fairly easily, it was decided to try this method for sectioning the extremely hard carbon-graphite materials. After much experimentation, ultramicrotome techniques were perfected and direct observations of submicron crystallites in carbon-graphite materials were made in the TEM.
Experimental Specimens approximately 1 mm across were cut and inserted into the pyramid end of standard 5-mm BEEM capsules [2], embedded in Epon
Thin Sections o/Carbon
45
epoxy resin mix and cured for 48 hours in a vacuum oven at 60°C. Many resin formulations were tried but the Epon epoxy combination according to Luft [-31, using the harder ratios of Mix A to Mix B gave the best cutting properties with carbon-graphite materials. Mix A consists of 31 ml Epon 812 resin and 50 ml of DDSA (dodecenyl succinic anhydride), while Mix B contains 25 ml of Epon 812 and 22 ml of NMA (nadic methylanhydride). Fifteen hundredths ml of the accelerator DMP-30 (dimethyl aminomethylphenol) is added for every 10 ml of the above mix prior to curing. This resin set uniformly with almost no shrinkage, produced blocks with a hardness most closely approaching the carbonaceous material to be sectioned, and yielded thin sections which withstood the intensity of the electron beam at 80 kV better than any other medium. The cured capsules were manually trimmed with a small diamond blade saw and mounted in a Reichert OMU-2 ultramicrotome fitted with a DuPont diamond edged knife and boat {-4"]. The most critical parameter in cutting very hard materials is the tilt of the knife edge. If the clearance angle is negative, the sections will be torn or vary in thickness; if it is too large, they will be wrinkled and again vary in thickness. For carbon a tilt of 10° was found to give consistent, continuous sections 800/~ in thickness. The sections coming off the knife float into a trough of water and will form a continuous ribbon when the technique is perfected. These ribbons of sections were retrieved from the boat onto formvar coated standard ~-inch 150-mesh copper grids, dried, and examined in a Philips EM-300 transmission electron microscope equipped with a high-resolution stage. In order to record all the minute carbide particles most of the observations were made at approximately 100,000 diameters. The astigmatism correction at this magnification is rather critical and great care was taken to use single layers and flat samples in order to ensure accuracy in the measurement of particle sizes. It was found that using coated grids on which to collect the sections was far better than placing them on grids with no support. Aside from the obvious prevention of damage to the epoxy and sample from the beam itself, there was considerable improvement in image "flatness" at high magnification. The use of liquid nitrogen to cool the specimen stage reduced contamination and improved accuracy. One thousand particles were measured from photographs taken of each of the different carbon-graphite samples and particle size plots of number versus diameter in microns were made. This was unfortunately done manually, but there are many new systems available where optics and electronics are combined to give rapid, quantitative image analyses either from photographs or by direct electronic interface with the TEM. The peak particle diameter for the metallic carbide deposits in the set of samples
(a)
(b)
(c) FIG. 1. Typical T E M photographs of thin sections of carbon/graphite materials. All 125,000X (shown here at 60% of original size). (a) and (b) Sections of uniform thickness showing dark carbide crystallites of various sizes. (c) and (d) Sections showing tearing because of incorrect angle setting on knife. (e) and (f) Sections illustrating agglomeration in layers from some of the carbides.
v v
48
E. J. Watts
studied ranged from 0.004 to 0.040 micron with some crystallites as small as 0.0005 micron and some as large as 0.130 micron. Figures l ( a ) - l ( f ) illustrate some of the results of this work on carbon-graphite thin sectioned materials. Magnification is 125,000X in all cases. A further advantage of ultramicrotomy of materials containing crystalline deposits is that electron diffraction photographs may be made of selected areas without changing specimens. Microdefects and foreign deposits may be easily located and identified. Quantitative studies are not generally possible because of the nature of this form of analysis where the scattering factor is so different from that associated with X-ray analysis. The Ewald sphere in electron diffraction is represented by a plane and is huge because of the short wavelength of electrons, while in X-ray diffraction the wavelength is of the order of atoms and hence the radius of the Ewald sphere is comparable with lattice spacings. The main bulk of the impurities may, nevertheless, be identified qualitatively with electron diffraction transmission techniques and, in addition, identification may be made of deposits which might be too small to yield measurable X-ray
FIG. 2. Selected area transmission electron diffraction pattern of MC type carbide deposit in carbon-graphite thin section. 2.8 X.
Thin Sections of Carbon
49
patterns. A typical electron diffraction pattern of one of the MC type carbides studied is shown in Fig. 2.
Summary A method for measuring particle size of submicron impurities in carbongraphite materials by direct observation in the transmission electron microscope is described. Samples are made sufficiently thin by encapsulating them in epoxy resin, curing the capsules, and then sectioning them to 800/~ in thickness in an ultramicrotome using special handling methods. The technique of observing particles in situ has the obvious advantages of permitting determination of the nature of the different kinds of materials in the sample, the distribution, state of agglomeration, and the particle sizes directly.
The author wishes to express sincere appreciation to Dr. Lyle C. Dearden and his staff in the Department of Anatomy, College of Medicine, University of California at Irvine for their technical assistance in this work.
References 1. c.F. Oster, "The application of ultra-thin sectioning techniques to non-biological samples," Proc. of the EMSA, New Orleans, La. (1968). 2. Available from Ernest F. Fullam, Inc., Schenectady, N.Y. 12301. 3. J.H. Luft, "Improvements in epoxyresin embeddingmethods," J. Biophys. Biochem. Cytol. 9, 409 (1961). 4. Available from E. I. DuPont, Wilmington, Del.
Received March, I975