The development of cancer is characterized by a long latency between the initial exposure to a carcinogenic substance and the final appearance of a macroscopic tumor. Histochemistry has provided the means to identify specific cells involved in this process and many of the changes that take place during the transformation of normal cells to malignant ones. Putative premalignant lesions have been studied extensively in liver carcinogenesis. Histological sections of grossly normal appearing liver a few weeks after treating animals with carcinogen contain discrete areas or “islands” of aberrant enzyme activity that can be detected with enzyme histochemistry and are commonly called “enzyme-altered foci.” These foci display altered activity of one or more enzymes; one of the most common is increased γ-glutamyl transpeptidase (GGT) activity. Since the activities of most enzymes are destroyed by the heat and solvents used for paraffin-embedding, most of this work was done with frozen sections that are often 10 μm in thickness

Abstract: To optimize specimen-processing protocols for investigations of fertilization in mammals, with high-resolution, low-voltage scanning electron microscopy (LVSEM), bovine oocytes matured in vitro were prepared by either aldehyde fixation, conductive staining, or high-pressure cryoimmobilization with freeze-substitution. Samples prepared by these different preparative techniques were coated with either platinum or gold and imaged with an LVSEM operating at 1.5 keV. Additionally, aldehyde-fixed oocytes, sputter-coated with gold, were imaged with a conventional scanning electron microscope (cSEM). The results show that bovine oocytes prepared by aldehyde fixation or cryoimmobilized by high-pressure freezing produced superior images of the zona pellucida (ZP) compared with those from the conductive stained samples. A comparison of LVSEM and cSEM images suggests that the gold-coating employed in cSEM preparation obscures the ZP detail seen with the LVSEM, but not with the cSEM. Application of these procedures provide not only a new view of the ZP but may be useful in elucidating the molecular mechanism of gamete interactions during the fertilization process.

Abstract: A method is described to prepare a high-resolution electron micrograph for quantitative comparison with a simulated high-resolution image. The experimental data are converted from the darkening of film used to acquire the image to units of electrons per incident electron, the same units used in the simulation. Also, distortions in the image arising from distortions in the image-forming lenses of the electron microscope are removed to improve the quality of the data. Finally, an alignment procedure is described which gives precise, pixel-by-pixel alignment of the experimental image with the simulated image. Examples of the procedure are shown to illustrate how actual data are prepared for quantitative analysis.

Introduction

Abstract: In this study, we investigate the principle of the annular aperture and polarization gating methods for microscopic imaging through a turbid medium using images simulated by a modified Monte Carlo program. Significant improvement in image quality is shown when thin annular apertures are used in the illumination and detection paths of a microscopic imaging system. The polarization gating method is proven to be an efficient gating method for selecting ballistic photons that carry the information of an object embedded in a turbid medium. We also demonstrate that the combination of the annular aperture and polarization gating methods is highly efficient in suppressing the scattered light in microscopic imaging through a turbid medium. It is also revealed that a pinhole used in a confocal microscope plays an independent angle-gating role.

High Resolution Electron Microscopy In Situ Observation of Dynamic Behavior of Grain Boundaries and Interfaces at Very High Temperatures

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Abstract: All lenses, whether for light or for electrons, have aberrations that limit their performance. In light optics the invention of the achromat over 200 years ago solved the problem of correcting spherical and chromatic aberrations; however, correcting aberrations of electron lenses remains a challenge. Spherical and chromatic aberrations constitute a fundamental barrier to improving resolution and reducing probe size in various types of electron-optical instruments. Recently, efforts have intensified to reduce probe size in microanalysis and lithography, and to improve resolution in the new emission microscopes being built for synchrotron light sources. The experiments described in this report show how an electron mirror can achieve simultaneous correction of spherical and chromatic aberrations.

In Situ Transmission Electron Microscopy Observations of Alloying of Nanoscale Pb Inclusions by Implantation with Cd Ions

Abstract: It has long been debated whether the 30-nm fiber of chromatin is packed in an orderly array. The fiber may be condensed by supercoiling, producing structures of varying diameter. Alternatively, technical problems may have prevented the detection of higher-order structures. We developed a strategy to distinguish between these two possibilities. One potential obstacle to studying the order of packing was the effect of fixatives, dehydrating agents, heat, and embedding polymers on the native structure of chromatin prepared for viewing by electron microscopy. The known tendency of proteins to be degraded by osmium tetroxide and subsequently to be extracted in the conventional protocols for embedding might be particularly damaging. To avoid such denaturants and ensure the retention of proteins in chromatin, the embedding resin HACH was employed. Drosophila mimica polytene chromosomes were thin sectioned, stained with uranyl acetate, and viewed in the transmission electron microscope. Images were digitized and subjected to computerized image processing. Raw data files, containing boundary coordinates of all closed figures in the image, were edited to retain only those regions of interest (ROIs) that exhibited dimensions similar to those of 30-nm fibers in projection views. Euclidean distances between the centroids of such structures were calculated to obtain linear intercepts between recognizable 30-nm fibers. According to stereology theory, the dimensions of a lamellar structure can be determined from the volume distribution function of such intercepts. Therefore, intercept values were pooled for final data files from five processed images of chromatin. The resulting frequency histogram, showing the number of observations at different intercept values, had a sigmoidal inflection that was diagnostic of a major, new spacing at 95 nm. The 95-nm minimum was sandwiched between maxima in the 85 to 90 nm interval and throughout the range 105 to 120 nm. The results suggest that established stereological theory will be a useful tool for investigating the intractable problem of higher-order chromatin structure.

Three cell types not previously described were revealed by the application of several microscopic procedures in combination with cytochemistry and irnmunocytochemistry in the livers of rats treated according to the Solt et al carcinogenesis protocol. This protocol consists of the administration of an initiating carcinogen (diethynitrosamine), a mitoinhibitory carcinogen, (acethylaminofluorene) and a growth stimulus (partial hepatectomy) (1). Two of the cell types were found in intrahepatic bile ductules and one within the connective tissue stroma surrounding the ductules. These findings have implications for understanding the cell lineage pathways that operate in an experimental rodent hepatocarcinogenesis system in which hepatocyte regeneration is inhibited and in which preneoplastic nodules and hepatomas develop. Our previous studies have determined some of the enzymatic, antigenic and structural properties of these cell types and their interrelations to each other, to parenchymal hepatocytes and to preneoplastic nodules (2).

Proliferation of bile ductule cells occurs early (24-48 hrs) after the partial hepatectomy step of the protocol with extensive branching of the ductules into the hepatic parenchyma.

Abstract: Multiple linear least squares (MLLSQ) and sequential simplex spectral processing procedures were used in an electron probe energy-dispersive (EDS) analysis of a 2223 Bi-Sr-Ca-Cu-O (BSCCO) high Tc superconductor specimen. This phase is normally difficult to quantify by EDS at voltages below 25 kV because of the severe overlap of the BiM lines with the M lines from a small amount of Pb that is added to the phase for stability. Quantitative results from the MLLSQ spectral processing and ZAF matrix correction procedures agreed well with wavelength-dispersive analysis (WDS) of the same specimen.

Abstract: The microstructure of YBCO/SrRuO3/YBCO junctions in the edge geometry have been studied by high resolution electron microscopy. Various defects in the YBCO and SrRuO3 films are observed and analyzed. The most striking defects in the c-axis-oriented YBCO film are a-axis particles, which cause bending and interruption of lattice planes as well as high steps and cracks in the subsequent layers. The SrRuO3 layer in the ramp-edge junction region consists of small domains with slightly different orientations. This leads to the formation of steps on its surface and large variation in SrRuO3 thickness. Mechanisms for forming this defective layer are suggested based on the growth mode and the crystal structure of SrRuO3. We find evidence for strain fields at the SrRuO3/YBCO interface. The oxygen deficiency in YBCO caused by this interface strain may to a large part be responsible for the excess resistivity of this junction. In addition to the defective SrRuO3 layer, the occurrence of various defects in the junction area, including Y2O3 and a-axis YBCO particles, seem to be the cause for the wide scatter in the junction electrical characteristics. The formation of steps and large thickness variation in the barrier layer may be the reason for the rather large critical thickness of the SrRuO3 superconductor-normal-superconductor (SNS) junction. In addition to the junction interfaces, the microstructure of SrRuO3/LaAlO3 and SrRuO3/SrTiO3 interfaces have also been studied.

Abstract: We have developed a method for the examination of atomic force microscopy (scanning force microscopy) tips using a high-resolution transmission electron microscope (TEM). The tips can be imaged in a nondestructive way, enabling one to observe the shape of an atomic force microscope probe in the vicinity of the apex with high resolution. We have obtained images of atomic force microscopy probes with a resolution on the order of 1 nm. The tips can be imaged repeatedly, so one can examine tips before and after use. We have found that the tip can become blunted with use, the rate of wear depending upon the sample and tip materials and the scanning conditions. We have also found that the tips easily accrue contamination. We have studied both commercially produced tips, as well as tips grown by electron beam deposition. Direct imaging in the TEM should prove useful for image deconvolution methods because one does not have to make any assumptions concerning the general shape of the tip profile.

Although ovarian carcinomas are the most lethal gynecologic tumors, their origins remain unclear. Do they develop from malignant transformation of benign neoplasms through a multistep process of tumor progression, or do they arise de novo? The histologically benign and/or low malignant potential (LMP) components in heterogeneous ovarian carcinomas have been considered as evidence supporting the theory of tumor progression. These components are interpretted as the remnants of pre-existing neoplasms that underwent malignant transformation. In two other possible interpretations, however, such components may be clones which developed independently (de novo hypothesis) or they may represent malignant epithelium which underwent focal maturation (maturation hypothesis).

To evaluate genetic relationships of the histological components in heterogeneous ovarian carcinomas, 10 such neoplasms and 5 normal ovary controls were examined using fluorescence in situhybridization (FISH) on intact paraffin sections (6 μm).The retention of tissue architecture allowed direct correlation of detectable genetic aberrations with histology, and comparison of malignant components with adjacent histologically benign or LMP components.

Abstract: Interactions among discrete charges in the emission current of a photoelectron imaging system can limit its spatial resolution, significantly affecting the performance and design of emission microscopes employing high current densities. Under intense laser illumination a sample can emit photocurrent densities exceeding tens of amperes/cm2. This study describes Monte Carlo estimations of electron trajectory aberrations produced by discrete electron space charge interactions in a photoelectron emission microscope. The results also apply to photoelectron sources that might be used in other kinds of devices. A model of the emission process was used to assemble bunches of about 500 randomly positioned electrons whose individual trajectories were computed. Trajectory distortions of one member in each bunch that were due to the coulomb forces exerted by its neighbors were used to evaluate aberrations for a variety of voltages, current densities, and instrument configurations. Aberrations estimated in this way are smaller than those predicted by earlier theory or obtained in pulsed imaging experiments. Here we discuss the reasons for these differences, offer suggestions for improved instruments, and present experimental images.

Abstract: Here we present our observations of an anomalous desorption of copper oxides from a thin copper film during annealing as observed in an in situ transmission electron microscope. The epitaxial cuprous oxide that formed in air was observed to disappear when the specimen was annealed at 300°C and 10−8 torr. However, removal of the cuprous oxide that formed during oxidation in the microscope required both heating at 300°C and simultaneous exposure to methanol vapor.

Abstract: The purpose of this study was to determine whether a polymer could be formed from relatively innocuous monomeric ingredients and, if so, if it might serve as a suitable embedding medium for maximizing antigen retention. Such a polymer, HACH, was made up from a mixture of 2-hydroxyhexanedial and carbohydrazide. It polymerized spontaneously at room temperature within 24 hr. Preservation of protein antigenicity and subsequent immunocytochemical localization were demonstrated by three methods. To determine whether protein antigens were retained up to the polymerization stage, we studied hemoagglutination of red blood cells using antibodies directed against their protein antigens. In these trials, HACH-treated cells exhibited the same agglutination responses as control, untreated cells. Second, a guinea pig antibody was used to immunodecorate insulin in β cells of the islets of Langerhans. The number of gold particles, indicating sites where the antibody was bound, was several-fold greater in HACH- than in Lowicryl K4M-embedded pancreatic β cells. To assess the limit of detection of protein antigens in thin sections, an example of a protein present in mitochondria, lipoamide dehydrogenase, was also studied. An indirect procedure for immunodecoration, employing rabbit immunoglobulin G followed by gold-tagged secondary antibody, indicated that the enzyme was present at several sites within cross-sectioned mitochondria. The results suggest that the HACH polymer will be useful for the localization of antigens that are present in relatively few copies.