High-pressure freezing of tissue obtained by fine-needle biopsy

High-pressure freezing (HPF) permits adequate cryoimmobilization (without detectable ice crystals after freeze-substitution) of biological tissue up to a thickness of about 200 μm. Until now the preparation of tissue prior to freezing has been unsatisfactory: sizing of the tissue to the required dimensions takes minutes, during which structural alterations must occur. We demonstrate that the use of a fine-needle biopsy technique minimizes tissue damage and guarantees sample dimensions close to the optimal thickness for HPF. The tissue cores can be cryoimmobilized within 40 s of excision.     

Transmission electron microscopic X-ray quantitative analysis of human dentin at 200 kV accelerating voltage

Qualitative and quantitative x-ray energy dispersive spectroscopy is now used successfully to analyze many features and processes in inorganic samples. When applied to inorganic samples, however, the results are often less satisfactory due to problems of preparation of organic samples, difficulty of measuring x-rays from organic samples, damage of the sample by the electron beam, and other practical problems. In the present study we used a high voltage transmission electron microscope equipped with an energy dispersive x-ray spectrometer to examine accurate quantitative standardless analysis of thin sections of an organic sample, human dentin. Based on our experiments we found the important parameters for quantitative analysis were sample thickness and appropriate choice of model sample. Further, we show that the method of Cliff and Lorimer can be used with biological samples at 200 kV, and we show that quantitative analysis of human dentin can be carried out at 200 kV. Finally, we show that areas of human dentin can be differentiated by their morphological characteristics and x-ray analyses obtained in the transmission electron microscope.     

Quantitative electron microscopic analysis of DNA-protein interactions

Electron microscopy offers a unique potentiality to visualize individual molecules. For the last 30 years it has been used to study the structure and the interactions of various biological macromolecules. The contribution of electron microscopy is important because of its capacity to demonstrate the existence of conformational structures such as kinks, bents, loops, etc., either on naked DNA, or on DNA associated with various proteins or ligands. Increasing interest was given to such observations when it was found that they provide a direct visualization of interacting molecules involved in DNA metabolism and gene regulation. Technical advances in the preparation of the specimens, their observation in the electron microscope, and the image processing by computers have allowed the shifting from qualitative to quantitative analysis, as illustrated by a few examples from our laboratory.     

Freeze-fracture electron microscopic analysis of solutions of biological molecules

Freeze-fracture electron microscopy was used to study the morphology of proteins in solution. The sizes of the particles appearing on the fractured surfaces, replicated with tungsten-tantalum, were measured in a direction perpendicular to the shadowing angle. The distributions of the measured particle sizes could be correlated with the known shape and dimensions for each protein. It is concluded that freeze-fracture electron microscopy is a useful technique to study the morphology of biological molecules in solution, particularly hydrophobic proteins which may be difficult to study by other microscopic techniques.     

Transmission electron microscopic observations of membrane effects of antibiotic cecropin B on Escherichia coli

The pathway of cell membrane lysis by the peptide antibiotic cecropin B (CB), which contains both a hydrophobic and an amphipathic alpha-helix, was analysed by assessing the morphological changes of Escherichia coli following treatment with the peptide. Exposure of green fluorescent protein (GFP)-expressing E. coli to CB does not lead to an efflux of GFP. Moreover, transmission electron microscopic (TEM) examination of cecropin B-treated cells showed that severe swelling precedes cell death and that the outer membrane becomes distended away from the plasma membrane. Using immuno-gold staining and TEM of E. coli expressing the maltose-binding protein in the cytoplasm, it was apparent that the protein remains restricted to the cytoplasmic compartment. These observations suggest that CB causes gross disruption of the outer membrane of Gram-negative bacteria. Circular dichroism measurements of CB in the presence of cell membrane-mimicking liposomes showed that CB forms secondary structure dependent on the ratio of [lipid]/[peptide]. These observations from this study are important for the future design of custom antimicrobial peptides.     

Data acquisition system for maximum-likelihood analysis of electron microscopic autoradiographs

EMAMAP is a program for the data acquisition phase of maximum-likelihood analysis of electron microscope autoradiographs. This program is written in C and has been implemented on a Masscomp MC-500 which supports a graphics processor and a digitizing tablet. The image analysis is automated at a low level: the program operator outlines the edges of the structures of interest using the digitizing tablet, while contiguous regions formed by closed contours are automatically filled by the software. The resulting image is compressed for efficient storage by a quadtree encoding technique for which data compression ratios of greater than 25:1 have been achieved. In practical terms this implies that the data from a typical experiment of 50 autoradiographs could be stored on a single floppy disk. The system is currently in use for acquiring actual biological experimental data.     

Transmission and scanning electron microscopic examination of intracellular organelles in freeze-substituted Kloeckera and Saccharomyces cerevisiae Yeast Cells

The application of the conventional double-fixation method (glutaraldehyde and osmium tetroxide) to whole yeast cells is difficult because the thick cell wall of the yeast prevents the penetration of osmium tetroxide. However, this problem was solved by using the freeze-substitution fixation method. Therefore, it was possible to examine the intracellular structures of the yeast cells without digestion of the cell wall. In the present method, specimens for transmission electron microscopy and for scanning electron microscopy were prepared simultaneously. By scanning electron microscopic observation, three-dimensional information about internal structures was obtained. In the cytological analysis of the yeasts, intracellular structures were well preserved by using the freeze-substitution fixation method. On the outer leaflet of the nuclear envelope, many ribosomes were attached. The rough endoplasmic reticulum and Golgi apparatus were clearly seen in the yeast cytoplasm. The Golgi stack appeared to consist of smooth membranes, and small vesicles were present beside it. The details of other structures such as the nuclear division apparatus, actinlike filaments, and viruslike particles were also revealed. The present technique can be applied to most species of yeast cells. With this new information, the previous model of a yeast cell was modified.     

Localization of opioid binding sites in rat brain by electron microscopic radioautography

Two met-enkephalin analogs (FK 33–824 and FW 34–569, Sandoz) were utilized for in vitro labeling of opioid binding sites in the rat central nervous system. Binding kinetics determined in 20-μm-thick frozen tissue sections of the striatum revealed that both pentapeptides bind to a single population of sites at 20°C with an apparent dissociation constant (K_D ) of approximately 1–2 nM and a maximum capacity (B max) of 65–170 fmoles/mg protein. Radioautographic data suggest that this population is the same for iodinated and tritiated forms of the FK compound and the iodinated FW analog. Fixation of labeled sections with high concentrations of glutaraldehyde allowed proportional retention of more than 50% of specifically bound ¹²⁵I-FK molecules in all brain regions after histological processing for high-resolution radioautography. In contrast, glutaraldehyde fixation did not prevent the loss of bound ¹²⁵I-FW molecules. These differences are attributed to the presence in FK, but not in FW molecules, of a free primary amino group considered essential for cross-link formation between aldehydes and proteins, and imply that a majority of FK-receptor complexes may be stabilized by glutaraldehyde. Consistent with this observation is the fact that the radioautographic distribution of specifically bound ¹²⁵I-FK was unchanged after fixation and dehydration. In electron microscopic radioautographs prepared from prefixed, vibratome-cut striatal sections that were incubated with ¹²⁵I-FK and fixed with glutaraldehyde, silver grains were found to be mostly associated with neuronal plasma membrane interfaces. The present methodological approach thus appears to be compatible with electron microscopic localization of opioid binding sites in the central nervous system and might be applicable to the localization of other types of binding sites using radioligand molecules that contain a free primary amino group.     

A statistical analysis of the variation in measured crystal orientations obtained through electron backscatter diffraction

Electron backscatter diffraction (EBSD) techniques are used to determine the crystallography of individual metal grains. This paper examines the variability in the orientation of measurements obtained by EBSD. Although precision and statistics of orientation have been explored in the literature, little attention has been paid to formal statistical inference for quantifying variation in orientation measurements. Our intention is to study precision by developing statistical analyses for quantifying multiple sources of orientation variation, given repeat scans of a metal sample. Three sources of variability are simultaneously explored: variation in repeat measurements at a fixed location, variation among locations within a grain, and grain-to-grain variation. Bayes statistical methods will be applied to a hierarchical model with the uniform-axis-random-spin (UARS) components of Bingham et al. [1] to quantify these sources of variation. Repeat scans of a Inconel 600 specimen will be used to provide an illustrating example of how the statistical methods can be used to arrive at precision estimates.     

Immunogold electron microscopic localization of antigenic sites in the outer dense fiber region of rat sperm tail obtained by using antisera to two Sertoli cell secretory proteins

Polyclonal antisera raised against polypeptide components of two rat Sertoli cell secretory proteins, designated protein S70 and S45–S35 heterodimeric protein on the basis of cell origin and estimated molecular weight, were used to identify antigenic sites in (1) rat testis, () cultured Sertoli cells, (3) developing spermatids (collected from spermatogenic stage-specific seminiferous tubular segments), and (4) epididymal sperm. Indirect immunofluorescence, immunoper-oxidase, and immunogold electron microscopy (single and double labeling) were used. Immunocytochemical techniques have detected antigenic sites in (1) the cytoplasm of Sertoli cells in the intact seminiferous tubule and in culture in the form of a punctuate, granular-like pattern, and (2) the acrosome (but not the Golgi region) and tail of developing spermatids and sperm. In developing spermatids, the principal piece of the tail displays a characteristic apical-to-distal immunoreactive banding pattern that correlates both temporally and spatially with the reported multistep assembly of outer dense fibers along the axoneme. The immunoreactivity of the acrosome, connecting piece, and outer dense fibers of the sperm tail was confirmed by immunogold electron microscopy. A precise identification of the component(s) of the outer dense fiber region responsible for the antigenic homology with Sertoli cell secretory proteins is under investigation.     

Transmission electron microscopy analysis of phase separation in GaInAsSb films grown on GaSb substrate

The GaSb‐based quaternary alloys are a good choice for thermophotovoltaic applications. The thermophotovoltaic cell converts infrared radiation to electricity, using the same principles as photovoltaic devices. The aim of the present work was the microstructural study of such an alloy, namely Ga_0.84In_0.16As_0.12Sb_0.88. A thin film of the material was grown by metal organic vapour phase epitaxy on a (100)α→[111]B (α = 2°, 4°, 6°) GaSb substrate. The GaInAsSb alloy has an appropriate band gap, but suffers from a phase separation consisting of GaAs‐rich and InSb‐rich regions that is disadvantageous for cell efficiency. In this work, we employed a morphological approach to phase separation, with the use of conventional transmission electron microscopy and atomic force microscopy. The phase separation occurs in two different orientations: parallel to the growth direction (vertical) and inclined (lateral). After application of fast Fourier transformation filtering, the vertical periodicity was found to be λ = 5 nm for the pair (black and white) of layers independently of the cut‐off angle, whereas the lateral periodicity was related to it.     

Transmission electron microscopy investigations of AZ91 alloy deformed by equal-channel angular pressing

The microstructure of transverse and longitudinal sections of a commercial AZ91 alloy processed by equal‐channel angular pressing was examined by transmission electron microscopy. A high dislocation density and large number of deformation twins were observed in the investigated material. The {102}_matrix ‖ {012}_twin twinning system was determined by selection area diffraction patterns obtained from the twin and matrix. Transmission electron microscopy analyses also revealed that the twins interacted with each other and pile‐ups of dislocations occurred near the twin boundary.     

Microwave-accelerated cytochemical stains for the image analysis and the electron microscopic examination of light microscopy diagnostic slides

Recent studies in our laboratories have shown how microwave (MW) irradiation can accelerate a number of tissue-processing techniques, especially staining, to aid in the preparation of single specimens on glass microscope slides or coverslips for examination by light microscopy (and electron microscopy, if required) for diagnostic purposes. Techniques have been developed, which give permanently stained preparations, that can be studied initially by light microscopy, their areas of interest mapped, and computer-automated image analysis performed to obtain quantitative information. This is readily performed after MW-accelerated staining with silver methenamine by the Giammara-Hanker PATS or PATS-TS reaction. This variation of the PAS reaction gives excellent markers for specific infectious agents such as lipopolysaccharides for gram-negative bacteria or mannans for fungi. It is also an excellent stain for glycogen and basement membranes and an excellent marker for type III collagen or reticulin in the endoneurium or perineurium of peripheral nerve or in the capillary walls. Our improved MW-accelerated Feulgen reaction with silver methenamine for nuclear DNA is useful to show the nuclei of bacteria and fungi as well as of cells they are infecting. Improved coating and penetration of tissue surfaces by thiocarbohydrazide bridging of ruthenium red, applied under MW-acceleration, render biologic specimens sufficiently conductive for SEM so that sputter coating with gold is unnecessary. The specimens treated with these highly visible electron-opaque stains can be screened with the light microscope after mounting in polyethylene glycol(PEG) and the structures or areas selected for EM study are mapped with a Micro-Locator? slide. After removal of the water soluble PEG the specimens are remounted in the usual EM media for scanning electron microscopy (SEM) or transmission electron microscopy (TEM) study of the mapped areas. By comparing duplicate smears from areas of infection, such as two coverslips of buffy coat smears of blood from a patient with septicemia, the microorganisms responsible can occasionally be classified for antimicrobial therapy long before culture results are available; gram-negative bacteria are positive with the Giammara-Hanker PATS-TS stain, and gram-positive bacteria are positive with the SIGMA HT40 Gram stain. The gram-positive as well as gram-negative bacteria are both initially stained by the crystal violet component of the Gram stain. The crystal violet stain is readily removed from the gram-negative (but not the gram-positive) bacteria when the specimens are rinsed with alcohol/acetone. If this rinse step is omitted, the crystal violet remains attached to both gram-negative and gram-positive bacteria. It can then be rendered insoluble, electron-opaque, and conductive by treatment with silver methenamine solution under MW-irradiation. This metallized crystal violet is a more effective silver stain than the PATS-TS stain for a number of gram-negative spirochetes such as Treponema pallidurn, the microbe that causes syphilis     

Silver-enhanced colloidal-gold labelling of rabbit kidney collecting-duct cell surfaces imaged by scanning electron microscopy

The luminal cell surfaces of rabbit kidney cortical collecting-duct cells were labelled with peanut lectin (PNA) and investigated by scanning electron microscopy. Labelling was performed either on 20-μm-thick cryostat sections from prefixed and cryoprotected rabbit kidney tissue or on cultured collecting-duct epithelium using biotinylated PNA and a 6-nm colloidal-gold-coupled antibody against biotin. Colloidal-gold labels were detected at low magnification (2000–4000 x) using silver enhancement. Coating with chromium allowed simultaneous imaging of both cell-surface morphology and labelling topography in the backscattered electron imaging mode. Our results show that PNA binding is specific for a subtype of intercalated cells equipped with microvilli on the luminal surface. The presented method promises to be useful for the identification of specific cell types in heterogeneous tissues.     

In situ analysis of gas composition by electron energy-loss spectroscopy for environmental transmission electron microscopy

We have developed methods for using in situ electron energy-loss spectroscopy (EELS) to perform quantitative analysis of gas in an environmental transmission electron microscope. Inner-shell EELS was able to successfully determine the composition of gas mixtures with an accuracy of about 15% or better provided that some precautions are taken during the acquisition to account for the extended gas path lengths associated with the reaction cell. The unique valence-loss spectrum associated with many gases allowed simple methodologies to be developed to determine gas composition from the low-loss region of the spectrum from a gas mixture. The advantage of the valence loss approach is that it allows hydrogen to be detected and quantified. EELS allows real-time analysis of the volume of gas inside the reaction cell and can be performed rapidly with typical acquisition times of a few seconds or less. This in situ gas analysis can also be useful for revealing mass transport issues associated with the differential gas diffusion through the system.     

Automated analysis of submicron particles by computer-controlled scanning electron microscopy

Automated analysis of submicron particles by computer-controlled scanning electron microscopy is generally possible. The minimum diameter of the detectable particles is dependent on the mean atomic number of the particles and the operating parameters of the scanning microscope. The main limitation with regard to particle size is set by the quality of the particle detection system, which generally is the backscatter electron detector. The accuracy of the results of the x-ray analyses is very often strongly affected by specimen damage, omnipresent especially for environmental particles even at low electron energies and probe currents. With the exception for light elements, the detection limit is approximately 1 wt%. Device-related limitations to automated analysis may be specimen drift and an unreliable autofocus function.     

'Five-parameter' analysis of grain boundary networks by electron backscatter diffraction

This paper describes state‐of‐the‐art analysis of grain boundary populations by EBSD, with particular emphasis on advanced, nonstandard analysis. Data processing based both on misorientation alone and customised additions which include the boundary planes are reviewed. Although commercial EBSD packages offer comprehensive data processing options for interfaces, it is clear that there is a wealth of more in‐depth data that can be gleaned from further analysis. In particular, determination of all five degrees of freedom of the boundary population provides an exciting opportunity to study grain boundaries by EBSD in a depth that was hitherto impossible. In this presentation we show ‘five‐parameter’ data from 50 000 boundary segments in grain boundary engineered brass. This is the first time that the distribution of boundary planes has been revealed in a grain boundary engineered material.     

Transmission path analysis of noise and vibration in a rotary compressor by statistical energy analysis

The hermetic rotary compressor is one of the most important components of an air conditioning system since it has a great effect on both the performance and the noise and vibration of the system. Noise and vibration occurs due to gas pulsation during the compression process and to unbalanced dynamic force. In order to reduce noise and vibration, it is necessary to identify their sources and transmission path and effectively control them. Many approaches have been tried in order to identify the noise transmission path of a compressor. However, identification has proven to be difficult since the characteristics of compressor noise are complicated due to the interaction of the compressor parts and gas pulsation. In this study, the statistical energy analysis has been used to trace the energy flow in the compressor and to identify the transmission paths from the noise source to the exterior sound field.     

The simulation and error analysis of raindrop size distribution obtained by micro rain radar

Observation of raindrop size distribution( DSD) with a vertically pointing micro rain radar( MRR) has important significance in the precipitation measurement field. The Mie scattering of large particle,vertical wind and air turbulence have great influences on the retrieval of DSD measured by MRR. This paper simulates the process of how three factors affect the inversion of DSD and the calculation of other precipitation parameters,then makes the errors analysis. Because the wavelength of MRR is 12.5mm,M ie theory is more suitable for the precipitation particle than Rayleigh approximation,w hich may cause the underestimation of number density of small droplets and the overestimation of that in middle field. The vertical wind results in inaccurate estimation of particle terminal velocity,so the diameter is measured with some errors by empirical relationship,w hich affects the calculating accuracy of radar reflectivity and rain rate. Air turbulence can broaden the pow er spectral density,of which the impact on the inversion of DSD are concentrated in small droplets field. Then the measured data from MRR is analyzed and the results prove the impacts of those factors. Finally,according to the analysis and application limits,the prospect of the future research trend of particle size distribution is conducted.