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PubMed1977

Scanning and transmission electron microscopy of the nasal mucosa.

We attempted to study the nasal mucosa of children (both normal and diseased) under light microscopy followed by both scanning and transmission electron microscopy. Combined methods of microscopy are valuable tools in establishing the diagnosis and lead to a better understanding of disease and its management. For this reason we found it necessary to study the ultrastructural details of the cells of the nasal mucosa. Through scanning electron microscopy we studied the functional aspects and the nature of the surface of these cells. Ultrastructural details of these cells were studied with the aid of transmission electron microscopy.

Adolescent↗
PubMed1996

[Complementarity of microscopies in the structural analysis of DNA minicircles associated to protein MC1].

Electron microscopy of DNA, either free or complexed with ligands, allows the analysis of local conformational variations along individual molecules. Electron microscopy is unique, in that it has the capacity to determine the average behaviour of a population of molecules observed individually, and can thus provide a better appreciation of variability within the series of molecules than biophysical or biochemical methods. Very encouraging results have been obtained by cryoelectron and near-field microscopies, especially atomic force microscopy, in parallel with traditional techniques for visualizing DNA molecules adsorbed onto a support film. Differences in sample processing procedures and image formation modes render these 3 types of microscopies complementary. The torsional stress of a DNA molecule together with a local curvature induced by the protein MC1 from archaebacteria, can be detected within minicircles comprising 207 base pairs.

Adenosine Triphosphatases↗
PubMed1998

New time-resolved techniques in two-photon microscopy.

Microscopy is traditionally a tool for determining biological structures. Many recent advances in optical microscopy involves the incorporation of spectroscopy techniques to monitor biochemical states of microscopic structures in living cells and tissues. By minimizing tissue photodamage, two-photon excitation microscopy provides a new opportunity to study the dynamics of biological systems on time scales from nanoseconds to hours. This review will focus on a number of these new methods: two-photon time-lapse microscopy, two-photon photoactivation, two-photon correlated spectroscopy, two-photon single particle tracking and two-photon lifetime microscopy.

Animals↗
PubMed1995

The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules.

Radiation damage is the main problem which prevents the determination of the structure of a single biological macromolecule at atomic resolution using any kind of microscopy. This is true whether neutrons, electrons or X-rays are used as the illumination. For neutrons, the cross-section for nuclear capture and the associated energy deposition and radiation damage could be reduced by using samples that are fully deuterated and 15N-labelled and by using fast neutrons, but single molecule biological microscopy is still not feasible. For naturally occurring biological material, electrons at present provide the most information for a given amount of radiation damage. Using phase contrast electron microscopy on biological molecules and macromolecular assemblies of approximately 10(5) molecular weight and above, there is in theory enough information present in the image to allow determination of the position and orientation of individual particles: the application of averaging methods can then be used to provide an atomic resolution structure. The images of approximately 10,000 particles are required. Below 10(5) molecular weight, some kind of crystal or other geometrically ordered aggregate is necessary to provide a sufficiently high combined molecular weight to allow for the alignment. In practice, the present quality of the best images still falls short of that attainable in theory and this means that a greater number of particles must be averaged and that the molecular weight limitation is somewhat larger than the predicted limit. For X-rays, the amount of damage per useful elastic scattering event is several hundred times greater than for electrons at all wavelengths and energies and therefore the requirements on specimen size and number of particles are correspondingly larger. Because of the lack of sufficiently bright neutron sources in the foreseeable future, electron microscopy in practice provides the greatest potential for immediate progress.

Biophysical Phenomena↗
PubMed2000

Surface microscopy of pigmented basal cell carcinoma.

OBJECTIVES: To describe the relevant morphologic features and to create a simple diagnostic method for pigmented basal cell carcinoma (BCC) using in vivo cutaneous surface microscopy (ie, dermoscopy, dermatoscopy, or oil epiluminescence microscopy). DESIGN: Pigmented skin lesions were photographed in vivo using immersion oil (surface microscopy). All pigmented skin lesions were excised and reviewed for histological diagnosis. Photographs of 142 pigmented BCCs, 142 invasive melanomas, and 142 benign pigmented skin lesions were randomly divided into 2 equally sized training and test sets. Images from the training set were scored for 45 surface microscopy features. From this a model was derived and tested on the independent test set. SETTING: All patients were recruited from the primary case and referral centers of the Sydney Melanoma Unit, Sydney, Australia, and the Skin and Cancer Unit, Skin and Cancer Associates, Plantation, Fla. PATIENTS: A random sample (selected from a larger database) of patients whose lesions were excised. MAIN OUTCOME MEASURES: Sensitivity and specificity of the model for diagnosis of pigmented BCCs. RESULTS: The following model was created. For a pigmented BCC to be diagnosed it must not have the negative feature of a pigment network and must have 1 or more of the following 6 positive features: large gray-blue ovoid nests, multiple gray-blue globules, maple leaflike areas, spoke wheel areas, ulceration, and arborizing "treelike" telangiectasia. On an independent test set the model had a sensitivity of 97% for the diagnosis of pigmented BCCs and a specificity of 93% for the invasive melanoma set and 92% for the benign pigmented skin lesion set. CONCLUSION: A robust surface microscopy method is described that allows the diagnosis of pigmented BCCs from invasive melanomas and benign pigmented skin lesions. Arch Dermatol. 2000;136:1012-1016

Basal Cell Carcinoma↗
PubMed1981

Application of electron microscopy in the interpretation of fine-needle aspiration biopsies.

A series of 31 fine-needle aspiration biopsies were studied ultrastructurally in order to evaluate the usefulness of electron microscopy in the diagnosis of aspiration biopsies. In seven cases, the electron microscopic findings were crucial to the diagnosis. In the remaining cases, electron microscopy helped to confirm the light microscopic diagnosis. In addition, in a number of cases the diagnosis rendered following electron microscopy was much more precise and specific than was possible with light microscopy alone. It is concluded that electron microscopy has a definite place in the diagnosis of fine-needle aspiration biopsies and should be increasingly utilized on a selective basis.

Adolescent↗
PubMed2007

Traditional and virtual microscopy compared experimentally in a classroom setting.

The technology known as virtual microscopy is now widely available to medical students. A number of medical school histology and pathology course directors, including those at the Eastern Virginia Medical School, are exploring the question of how best to make use of this new tool. The current study compared the efficacy of teaching and testing one unit of histology, bone and cartilage, using two technologies, namely, virtual microscopy and traditional microscopy. Additionally, the study examined whether low, moderate, high moderate, or high achieving students perform more effectively in any of the learning/testing formats. In a completely randomized block design, 96 first semester, first-year medical students were grouped by previous exam quartiles then subsequently randomly assigned to one of four groups. Using identical laboratory exercises with highly comparable slides for each experimental group, half of the subjects learned the exercise with traditional microscopy and half learned with virtual microscopy. Subjects were further randomly subdivided into virtual or actual testing groups. The authors found no significant differences in test scores when they examined effects by learning group or by testing group, nor were there significant interaction effects. Student performance evaluated by previous exam quartile was significant (P < 0.001). That is, students who had scored in a particular quartile on a previous test tended to score in the same quartile on the bone and cartilage test regardless of learning or testing method. In a short opinion survey, students were polled to evaluate their experience. Student preferences for both learning and testing method varied widely.

Bone and Bones↗
PubMed2006

Automated virtual microscopy of gastric biopsies.

BACKGROUND: Automated virtual microscopy of specimens from gastrointestinal biopsies is based on cytometric parameters of digitized histological sections. To our knowledge, cytometric parameters of gastritis and of adenocarcinoma have yet to be fully characterized. Our objective was to classify gastritis and adenocarcinoma based on cytometric parameters. We hypothesized that automated virtual microscopy using this novel classification can reliably diagnose gastritis and adenocarcinoma. METHODS: Routinely processed hematoxylin-and-eosin-stained histological sections from specimens that showed normal mucosa (14 cases), gastritis (35 cases), and adenocarcinoma (30 cases) diagnosed by conventional optical microscopy were scanned and digitized at high resolution. Thirty-eight cytometric parameters based on density and morphometry were applied to glands and superficial epithelium. Twelve cytometric parameters based on cytologic detail were applied to individual cells. RESULTS: Statistically significant differences in cytometric parameters for normal mucosa, gastritis, and adenocarcinoma were found. The most discriminatory parameter was the ratio of the total number of cells to the number of interstitial cells. These differences correctly classified adenocarcinoma at 100% accuracy and overall correctness was 86%. CONCLUSIONS: We describe a novel method of analyzing gastric mucosal histology based on cytometric parameters. Automated virtual microscopy can be used to classify gastric mucosa as normal, gastritis, or adenocarcinoma with reasonable accuracy. Further research is necessary to determine whether automated virtual microscopy can subclassify gastric mucosal histology in greater detail.

Adenocarcinoma↗
PubMed1995

Scanning electron microscopy of the human esophagus: application to Barrett's esophagus, a precancerous lesion.

In Barrett's esophagus, metaplastic columnar epithelium replaces the normal squamous epithelium. The importance of this lesion lies in the increased incidence of adenocarcinoma of the esophagus occurring in patients with Barrett's esophagus. We characterized the surface epithelial cells of Barrett's esophagus using quantitative scanning electron microscopy. Three distinct surface cell types, in addition to the globlet cell, were recognized in Barrett's epithelium: the gastric-like cell and the intestinal-like cell, both of which were similar to normal gastric and small intestinal surface cells, respectively, by quantitative scanning electron microscopy, and the variant cell which had a range of surface features. In four biopsy specimens from the squamo-Barrett's junction in three patients, we found the distinctive cell that had features intermediate between those of squamous and columnar epithelium. On the distinctive cell's surface there are two disparate structures not normally present on the same cell in the gastrointestinal tract: microvilli (a scanning electron microscopy feature of glandular epithelium) and intercellular ridges (a scanning electron microscopy feature of squamous epithelium). The surface characteristics of this cell were almost identical to those of cells found in the transformation zone of the uterine cervix, an area in which squamous epithelium physiologically replaces columnar epithelium. Scanning electron microscopy of Barrett's esophagus has increased our understanding of this precancerous lesion by showing striking cellular heterogeneity. It has also identified the distinctive cell which may represent an intermediate step in the development of Barrett's epithelium during which the surface characteristics of two different cell types, columnar and squamous, coexist in the same cell.

Barrett Esophagus↗
PubMed2004

Three-dimensional microscopy migrates to the web with "PowerUp Your Microscope".

"PowerUp Your Microscope" is a software package designed and realized for the optimization of 3D optical microscopy image quality using the Internet and inverse problems computational approaches. The package is mainly devoted to 3D microscopy users, being operative for wide-field, confocal, and multiphoton microscopy. It provides the microscopy community with an extremely easy and comparatively powerful access to advanced image restoration methods. The core of the computational section is the optical system modeling and inverse deconvolution implementation, which is strongly linked to Web-based software and technology. This project constitutes a real and effective migration to the Web, extending computational approaches to image restoration to the whole microscopy user community, regardless of their background.

Databases as Topic↗
PubMedDate not supplied

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 pallidum, the microbe that causes syphilis.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Detection of submicroscopic magnetite particles using reflectance mode confocal laser scanning microscopy.

Light microscopy and transmission electron microscopy work at such different scales that some components of cells may be too small to detect using light microscopy but too dispersed among cells within tissues to be discovered using electron microscopy. We have used reflectance mode confocal laser scanning microscopy to detect single-domain magnetite crystals in both live and resin-embedded preparations of magnetotactic bacteria. We show that reflections from bacterial cells are uniquely associated with the magnetite, which underpins the magnetotactic response of the bacteria. En bloc viewing shows that relatively large volumes of material can be searched with sufficient resolution to enable detection of submicroscopic particles. The techniques reported here may be of interest to others wishing to detect submicroscopic objects dispersed in large volumes of tissue.

Bacteria↗
PubMed2006

Applications of microscopy to genetic therapy of cystic fibrosis and other human diseases.

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging.

Adenoviridae↗
PubMed1978

Bovine olfactory and nasal respiratory epithelium surfaces. High-voltage and scanning electron microscopy, and cryo-ultramicrotomy.

High-voltage transmission electron microscopy and cryo-ultramicrotomy together with scanning electron microscopy and some conventional transmission electron microscopy of ultrathin sections have been applied to the mucous surfaces of bovine olfactory and respiratory epithelia. Distal segments of olfactory cilia tend to run in parallel and could be followed over distances up to about 30 micrometer using high-voltage electron microscopy. This technique and scanning electron microscopy showed that on average 12--13 of such cilia could be observed per nerve ending. After correction for obscured cilia this number becomes about 17. High-voltage micrographs and micrographs made from sections prepared with a cryo-ultramicrotome showed the presence of electron-lucent pockets inside the olfactory mucus. The latter technique also showed that the mucus itself is not fibrous, but rather a continuum varying in electron density. The mucus layer contains various granular structures. Ciliary and microvillar membranes appear thicker with cryo-ultramicrotomy than when the sections are prepared with conventional techniques. The cores of the axonemal microtubules in olfactory as well as in respiratory cilia are darkly stained with this technique. Vesicles present inside the nerve endings are also darkly stained. Dimensions and some other numerical values of interest in olfaction are presented.

Animals↗
PubMed1993

Remodelling of cardiomyocyte cytoarchitecture visualized by three-dimensional (3D) confocal microscopy.

The break-down and reassembly of myofibrils in long-term cultures of adult rat cardiomyocytes was investigated by a novel combination of confocal laser scanning microscopy and three-dimensional image reconstruction, referred to as FTCS, to visualize the morphological changes these cells undergo in culture. FTCS is discussed as an alternative imaging mode to low-magnification scanning electron microscopy. The three-dimensional shape of the cells are correlated with the assembly state of myofibrils in different stages. Based on immunofluorescence and confocal laser scanning microscopy it was shown that myofibrils are degraded within a few days after plating and that newly assembled myofibrils are predominantly confined to the continuous area in the perinuclear region close to the membrane in contact with the substratum. The localization of myofibrils along the cell's vertical axis has been investigated both by optical sectioning using confocal light microscopy and by physical sectioning followed by transmission electron microscopy. Based on the distribution of myofibrillar proteins we propose a model of myofibrillar growth locating the putative assembly sites to a region concentric around the nuclei. We provide evidence that the cell shape is dominated by the myofibrillar apparatus.

Animals↗
PubMed1982

Scanning electron microscopy of Giemsa-stained chromosomes and surface-spread chromosomes.

Giemsa-stained chromosomes as prepared for light microscopy, and including G-banded, C-banded, and FPG-stained chromosomes, were examined by scanning electron microscopy. Although suitable for light microscopy, these chromosomes were too flat for a close examination of their fine structure by scanning electron microscopy. The surface of Giemsa-positive regions was rough and bright, whereas that of unstained or poorly stained regions was smoother and less bright. Giemsa-staining, therefore, seems to produce the bulkiness of the chromosomes. On topographical examination by scanning electron microscopy, the transparent chromosomes as observed with the light microscope proved to be "footprints". Stereographical examinations of surface-spread chromosomes showed that minimally stretched chromosomes were composed of a mass of nodular and twisted looping fibers with an average diameter of about 300 A. The substructure of these chromosome fibers was not determined. The kinetochore region was discernible as a constriction in the mass of the chromosome fibers, and was distinguishable from gaps by the presence of several chromosome fibers parallel to the axis of the chromatid. The organization of the chromosome fibers, however, was disordered rather than regular.

Azure Stains↗
PubMed1997

Sensitivity of microscopy versus enzyme immunoassay in the laboratory diagnosis of giardiasis.

The substitution of enzyme immunoassay (EIA) techniques for microscopy as a screening tool for Giardia lamblia infection was assessed. Paired stool samples obtained within a ten-day period from 366 patients with persistent diarrhea were examined by microscopy. In addition, two commercially available Giardia lamblia-specific EIAs were performed. Compared with microscopy, EIA for copro-antigen detection was more sensitive, based on examination of either one or two stool samples. Repeated examinations increased the number of cases detected, more so for microscopy than EIA. The negative predictive values of the two EIAs performed on the first stool sample were 98.7% and 97.8%. The results show that EIA for detection of copro-antigens in a single stool sample may be almost as sensitive for identifying Giardia infection as repeated microscopy on two sequential stool samples.

Adolescent↗
PubMed2004

Combining in situ reverse transcriptase polymerase chain reaction, optical microscopy, and X-ray photoelectron spectroscopy to investigate mineral surface-associated microbial activities.

A study was undertaken to investigate expression of a gene encoding a c-type cytochrome in cells of the dissimilatory metal reducing bacterium (DMRB) Geobacter sulfurreducens during association with poorly crystalline and crystalline solid-phase Fe(III)-oxides. The gene encoding OmcC (outer membrane c-type cytochrome) was used as a target for PCR-based molecular detection and visualization of omcC gene expression by individual cells and aggregates of cells of G. sulfurreducens associated with ferrihydrite and hematite mineral particles. Expression of omcC was demonstrated in individual bacterial cells associated with these Fe-oxide surfaces by in situ RT-PCR (IS-RT PCR) and epifluorescence microscopy. Epifluorescence microscopy also permitted visualization of total DAPI-stained cells in the same field of view to assess the fraction of the cell population expressing omcC. By combining reflected differential interference contrast (DIC) microscopy and epifluorescence microscopy, it was possible to determine the spatial relationship between cells expressing omcC and the mineral surface. Introduction of the fluorescently labeled lectin concanavalin A revealed extracellular polymeric substances (EPS) extending between aggregations of bacterial cells and the mineral surface. The results indicate that EPS mediates an association between cells of G. sulfurreducens and ferrihydrite particles, but that direct cell contact with the mineral surface is not required for expression of omcC. XPS analysis revealed forms of reduced Fe associated with areas of the mineral surface where EPS-mediated bacterial associations occurred. The results demonstrate that by combining molecular biology, reflectance microscopy, and XPS, chemical transformations at a mineral surface can be related to the expression of specific genes by individual bacterial cells and cell aggregates associated with the mineral surface. The approach should be useful in establishing involvement of specific gene products in a wide variety of surface chemical processes.

Bacterial Proteins↗