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PubMed1995

Ultramicrotomy of semiconductors and related materials.

Ultramicrotomy has found novel application in materials research using transmission electron microscopy, for both analytical and high resolution work. Specimens 20-50 nm in thickness with lateral dimensions of up to 100 microns may routinely be prepared, with the additional advantage of precise area location for cross-sectional samples from real devices (e.g., VLSI structures). Much of this work is possible through the availability of diamond knives with various included angles and lengths at reasonable cost. This paper reports on various applications of ultramicrotomy, in particular, lattice imaging of surfaces and interface regions from epilayers of II-VI compound semiconductors and related materials. Ultramicrotomed cross-sections have enabled modern electron beam imaging, diffraction and analytical techniques to be brought to bear near surfaces and across interfaces of multilayer structures, yielding high spatial resolution information on crystallography, defect structure and composition.

Microscopy, Electron↗
PubMed1995

Electrochemical and microstructural study of oxide films formed electrochemically at microcrystalline Al-Fe-V-Si alloys.

A recent advance in metallurgical technology has been the application of rapid solidification techniques to Al alloy production. FVS0812 is the designation given to a microcrystalline Al-based alloy consisting of 8 wt% Fe, 1 wt% V and 2 wt% Si. It is a two-phase alloy, consisting of ca. 27 vol percent of approximately spherical Fe-V-Si-rich dispersoids in an essentially pure Al matrix. The high strength, low density properties of this advanced material, and other related alloys, have not yet been realized, however, due, in part, to the inability of the alloy to form a thick, adherent, abrasion-resistant outer surface oxide film, a feature readily achieved at conventional Al alloys by normal anodizing methods. The present research has involved an electro-chemical study of oxide film growth at the 812 alloy, with the specific goals being to seek an understanding of the origin of the oxide film growth problem and ultimately to propose alternative approaches to the formation of a thick, stable oxide film at this material. The techniques used in this research have included electrochemical methodologies such as cyclic voltammetry and electrochemical impedance spectroscopy. Crucial information has been obtained through transmission electron microscopy (TEM) of ultramicrotomed specimens. Experiments were carried out initially in neutral borate solutions to characterize the compact barrier oxide film formed in this environment and expected to be present beneath the porous oxide film formed in the normal sulfuric acid anodizing medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Alloys↗
PubMed1995

Development and application of a dry ultramicrotomy technique for the preparation of galvanneal sheet coatings.

The formability of galvanneal steel sheets used in the automotive industry is influenced by the presence and distribution of brittle and difficult to distinguish Zn-Fe intermetallics in the coating. Characterization of these intermetallics requires a high spatial resolution technique such as analytical transmission electron microscopy (ATEM). Sample preparation by ion milling is impossible due to iron redeposition, and traditional ultramicrotomy using water affects the coating chemistry. A technique based on dry ultramicrotomy has therefore been developed. To optimize the technique, different parameters (knife angle, cutting medium, thickness setting on the ultramicrotome, cutting speed) have been investigated for the preparation of galvanneal coatings and pure A1 sections. Results show that dry cutting does not affect the coating chemistry but shortens the life of the knife. Knife quality (cleanliness, sharpness and absence of defects) is a major factor to obtain good dry sections. The best results for the more ductile pure A1 are obtained with a 35 degrees knife whilst for the harder galvanneal coating it is recommended to use a 55 degrees knife. These results suggest that the sectioning mechanism for the harder material involves more a cleavage-fracture mechanism whilst a greater amount of shear is involved when sectioning relatively ductile A1. The optimum parameters for sectioning galvanneal coatings are established and results obtained by parallel electron energy loss spectrum imaging and energy dispersive X-ray spectrometry in the TEM are given. This study shows that with a good control of all the sectioning parameters it is possible to obtain good sections repeatedly and rapidly.

Electron Probe Microanalysis↗
PubMed1995

Ultramicrotomy of industrial alumina.

Industrial materials, such as alumina, often pose difficulties in their preparation for TEM examination. Composite and particulate materials are particularly difficult to prepare using conventional thinning techniques, i.e., ion beam and chemical jet thinning. Ultramicrotomy (UM) can be used to produce TEM specimens with a uniform thickness and an unaltered composition. Some crystalline materials, i.e., alumina hydrate, were difficult to section due to conflict between the cutting direction and cleavage planes. Sectioning was successful when these two directions were mutually parallel or perpendicular. At other orientations shattering occurred. Microcrystalline particulate materials, i.e., calcined alumina, were sectioned successfully in particles < 70 microns in diameter. The phases found in industrial alumina particles were gamma, delta, theta, and alpha alumina. Gamma alumina consisted of fine-grained, equiaxed crystallites. The selected area electron diffraction (SAED) patterns indicated poor crystallinity with a distinct hexagonal texture. Delta and theta alumina appeared as an undifferentiated intermediary microstructure of elongated grains. The SAED patterns indicated poor crystallinity, but without a distinct texture. Alpha alumina was found to be a coarse-grained crystalline phase with high diffraction contrast. SAED patterns consisted of fine, randomly oriented spots. Considerable variation was observed in the distribution of phases. In some specimens, discrete particles of gamma and alpha predominated. In others, particles were a mixture of phases representative of the bulk composition. To characterise these samples, TEM of numerous whole particles was required. Ultramicrotomy was the only preparation technique capable of producing such samples.

Aluminum Oxide↗
PubMed1995

Ultramicrotomy of diamond films for TEM cross-section analysis.

Ultramicrotomy has been used to prepare TEM cross-sections of typical hard dielectric, semiconductor, and metal coatings, providing a critical capability in the study of structure-property relationships of thin films. Ultramicrotomy of thin film coatings requires meticulous attention to technique and handling. The sample to be microtomed must be very small, well bonded to the epoxy embedding medium, and precisely oriented. In this article we report the ability to microtome TEM cross-sections of diamond and cubic boron-nitride (cBN) coatings.

Diamond↗
PubMed1995

Microscopy of the gap junction: a historical perspective.

Gap junctions were discovered more than three decades ago, and since this time, enormous strides have been made in understanding their structure and function. This article summarises the part played by microscopy, within the context of multidisciplinary research, in the historical development of our knowledge of the gap junction.

Animals↗
PubMed2004

From cells to tissues: fluorescence confocal microscopy in the study of histological samples.

Our knowledge of the genetic mechanisms controlling cell proliferation and differentiation usually originates from in vitro cultured cell line models. However, the definition of the molecular switches involved in control of homeostasis and the understanding of the changes occurring in neoplastic transformation require looking at single cells as the components of a complex tissue network. Histological examination of tissue samples can gain a substantial amount of information from high-resolution fluorescence analysis. In particular, confocal microscopy can help in the definition of functional pathways using multiparameter analysis. In this report, we present acquisition and analysis procedures to obtain high-resolution data from tissue sections. Confocal microscopy coupled to computational restoration, statistical evaluation of spatial correlations, and morphological analysis over large tissue areas were applied to colorectal samples providing a molecular fingerprint of the biological differences inferred from classical histological examination.

Animals↗
PubMed2004

Bioimaging TOF-SIMS of tissues by gold ion bombardment of a silver-coated thin section.

The imaging time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) method was utilized to address the problem of cholesterol localization in rat tissues. Rat kidneys were fixed, cryoprotected by sucrose, frozen, sectioned by cryoultramicrotomy, and dried at room temperature. The samples were either covered with a thin silver layer or analyzed uncovered in an imaging TOF-SIMS instrument equipped with an Au1-3(+)-source. The yield of desorbed secondary ions for some species was up to 600-fold higher after silver coating of the samples. Reference samples of cholesterol were silver-coated and analyzed by TOF-SIMS to define significant peaks, specific for cholesterol. Such peaks were found at m/z = 386 (C27H46O+), m/z = 493 (C27H46O107Ag+), m/z = 495 (C27H46O109Ag+), m/z = 879 (C54H92O2 107Ag+), and m/z = 881 (C54H92O2 109Ag+). The silver-cationized cholesterol (493 < or = m/z < or = 495) signal was localized by imaging TOF-SIMS in the kidney sections and showed a high cholesterol content in the kidney glomeruli. A more diffuse distribution of cholesterol was also found over areas representing the cytoplasm or plasma membrane of the epithelial cells in the proximal tubules of rat kidney.

Animals↗
PubMed2006

Two distinct events, section compression and loss of particles ("lost caps"), contribute to z-axis distortion and bias in optical disector counting.

Deformation of tissue sections in the z-axis can bias optical disector counting. When samples of particle densities are not representative for the entire tissue section, significant bias of estimated numbers can result. To assess the occurrence, prevalence, extent, sequence of events, and causes of z-axis distortion, the distribution of neuronal nucleoli in thick paraffin and vibratome sections was determined in chicken, rodent, and human brain tissues. When positions of neuronal nucleoli were measured in the z-axis, nucleoli were more frequent at the surfaces (bottom and top) of tissue sections than in the core. This nonlinear z-axis distribution was not lab-, equipment-, or investigator-specific, and was independent of age, fixation quality, coverslipping medium, or paraffin melting temperature, but in paraffin sections, was highly correlated with the tilt of the knife (cutting) angle. Manipulation of subsequent tissue processing steps revealed that two events contribute to z-axis distortion. Initially, a higher density of particles results at surfaces after sectioning, apparently due to section compression. Subsequently, particles can be lost to varying degrees from surfaces during floating or staining and dehydration, resulting in "lost caps." These results may explain different degrees of z-axis distortion between different types of sections and different labs, and reinforce the importance of checking z-axis distributions as a "quality control" prior to selection of guard zones in optical disector counting. Indirect approaches to assess section quality, such as resectioning in a perpendicular plane, yield additional artifacts, and should be replaced by a direct quantitative measurement of z-axis distribution of particles.

Animals↗
PubMed1985

Altered orientation of glycosaminoglycans and cellular changes in the tibial cartilage in the first two weeks of experimental canine osteoarthritis.

Changes in the cellularity and in the nature of the matrix were studied in the cartilages of the tibial plateau in experimentally induced arthritis in the dog, 7 and 14 days after section of the anterior cruciate ligament. Samples from the different regions of these cartilages were chilled and sectioned in a cryostat, with a variable microtome chuck to allow precise orientation of the specimens. The samples were examined by normal light microscopy, by microscopic interferometry, and by quantitative polarized light microscopy. The orientation of the glycosaminoglycans was assessed by the new "induced birefringence" method. The results indicated that only the region of the medial tibial cartilage that was unprotected by the meniscus was affected, showing increased water content, loss of superficial cells, and a decrease in orientation of the glycosaminoglycans. Whereas the birefringence of the collagen was unaffected, the superficial area that lacked oriented glycosaminoglycans was markedly increased; this may be a useful indicator of early osteoarthritic changes.

Alcian Blue↗
PubMed1988

A reflection scanning acoustic microscope for bone and bone-biomaterials interface studies.

A relatively simple scanning acoustic microscope (SAM) that operates in the reflection mode has been constructed. The system uses a 20 MHz spherically focused transducer, acting both as transmitter and as detector, to obtain acoustic impedance information on a thin surface layer at a maximum resolution of approximately 100 micron. The specimen is mounted on an X-Y driving system (precision, 5 micron) under computer control in order to scan a grid of 256 x 256 points across areas ranging from 6.5 to 1300 mm2. An algorithm is used to reference the data against standards; specially developed software provides for pseudo-color mapping, three-dimensional images, zooming to 16 x magnification, contouring, and single line profiles of the data. The system has been used to determine inhomogeneities in surface acoustic properties of mineralized tissues and implant materials, in many cases as a complement to using ultrasonic wave propagation techniques to measure the bulk anisotropic properties.

Adult↗
PubMed2000

Efficiency of systematic sampling in histomorphometric bone research illustrated by hydroxyapatite-coated implants: optimizing the stereological vertical-section design.

The sampling efficiency of the unbiased stereological vertical-section method was analyzed in five hydroxyapatite-coated implants. They were inserted into humans and harvested after 1 year. To find an optimal sampling design for histomorphometric analyses, sampling efficiency was estimated by variance analyses at different sampling levels (humans, sections, fields of view, and number of counting items) and intensities. Only minor changes in variance were observed when the initial scheme was reduced to include just one of the two possible implant sides, every third field of view, and half the density of the probe; this reduced the total workload at the microscope to less than 10% for all sections. In addition, the number of sections for analysis could be reduced to every fourth section per implant (three to four sections for evaluation) without significantly increasing variance. The study demonstrated that biological variation contributed to the majority of the total observed variance. Optimizing the sampling design could significantly reduce the workload at the hard-tissue microtome and the microscope without reducing the quality of the data that were unbiased and that had low sampling variance as compared with the true biological variation.

Adult↗
PubMed1986

Methods for in vitro percutaneous absorption studies. VI: Preparation of the barrier layer.

The skin membrane for in vitro percutaneous absorption studies was prepared so that it was similar in thickness to the in vivo barrier layer. A dermatome section from the skin surface produced a layer of skin that included the epidermis and papillary dermis (location of capillary loops) but without most of the dermal tissue. Improved absorption measurements were then obtained with hydrophobic compounds with the use of a polyethylene glycol 20 oleyl ether (PEG-20 oleyl ether) receptor fluid. With the haired rat, preparing a skin section 300-micron thick (and pretesting for damage to the barrier with H3-water) resulted in a membrane that gave values in good agreement with in vivo results for 3-phenyl-2-propenyl 2-aminobenzoate (cinnamyl anthranilate) (1) and benzo(a)pyrene absorption. When sparsely haired fuzzy rat skin was used, a section of skin 200-micron thick could be prepared without the need for pretesting for damage. Good agreement was obtained between in vivo and in vitro values for 1-(3-ethyl-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)etha none (acetyl ethyl tetramethyl tetralin) (2) and DDT with 0.5% PEG-20 oleyl ether in water as the receptor fluid. The skin of the fuzzy rat seemed more similar in permeability to human skin than did the skin of the hairless mouse when the absorption of six compounds was compared.

Animals↗