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PubMed2001

Free-floating cryostat sections for immunoelectron microscopy: Bridging the gap from light to electron microscopy.

Frozen skin sections are routinely used for light microscopic immunohistochemical study of the skin basement membrane zone for two reasons: some skin basement membrane zone proteins are labile to routine chemical fixation, and skin is not amenable to vibratome sectioning. However, inherent limitations of conventional frozen sections, including compromised morphology and a requirement for glass slide-mounting, usually limit immunohistochemical study to the light microscopy level. In the present study, we introduce use of unfixed, free-floating cryostat sections for characterization of immunolocalizations of selected skin basement membrane proteins at both the light and electron microscopy level. The new procedure employs free-floating cryostat sections that can be processed as routine tissue specimens and can be subjected to a variety of special staining procedures including immunohistochemistry. Especially useful is the ease of progressive processing of the same tissue specimen from light microscopy to electron microscopy. In this regard, the method renders itself useful when results of immunolabeling experiments need to be elucidated quickly at histological and ultrastructural levels as required for diagnostic and accelerated investigative strategies.

Antigens, CD↗

PubMed2000

Combining laser scanning confocal microscopy and electron microscopy to determine sites of synaptic contact between two identified neurons.

Here we report a double labelling method for correlative confocal and electron microscopy (EM) which allows selective characterisation of structural relationships between two single identified neurons in the same preparation. Using the lobster stomatogastric nervous system, we labelled pairs of identified, synaptically-connected neurons by intracellular injection of Lucifer Yellow (LY) in one neuron and a mixture of Rhodamine (Rdh) and Horseradish Peroxidase (HRP) in its partner. First, whole-mounts of LY- and Rdh-stained neurons were visualized using laser scanning confocal microscopy (LSCM) in order to isolate neuropilar regions of possible synaptic contact. Second, after conventional treatment for electron microscopy (LY was revealed with immunogold and HRP with DAB), areas of close appositions were viewed in EM. This technique allowed us to determine all the regions of close contact between two cells, and then to use electron microscopy to determine the presence or absence of synaptic contact within each of these restricted areas. These techniques enabled us to show that there were few areas of apposition and that only an extremely small proportion of these areas was in fact regions of synaptic contact between the two labelled neurons.

Animals↗

PubMed2001

Confocal laser scanning microscopy and scanning electron microscopy of tissue Ti-implant interfaces.

Microscopic inspection of heterogenous three-dimensional (3D) objects such as oral implants, or implants in general, is conventionally performed either on ground sections of methyl-metacrylate-embedded material, at the cellular level by histologic analysis of the peri-implant tissue by light microscopy (LM), or at the supramolecular level by transmission electron microscopy (TEM). Alternatively, the architecture of the tissue/implant interface is visualized by scanning electron microscopy (SEM). The two approaches exclude each other because of the sample preparation.We elaborate conditions for the non-invasive analysis of tissue/implant interfaces by confocal laser scanning microscopy (CLSM) in buffer, hoping to obtain a 3D view of fluorescently labeled tissue constituents at the tissue implant interface and, through subsequent SEM, of the metal surface. The use of water-immersion objectives, originally developed for high LM under physiological conditions is essential. In an exploratory approach, the tissue/Ti-interfaces of two retrieved dental implants were analyzed. One was a step-cylinder used for orthodontic anchoring and the other was an endosseous step-screw implant retrieved after infection-related loosening prior to load. The adhering tissue fragments were fluorescently triple-labeled for actin, fibronectin, and sm-alpha-actin. Optical sections for fluorescent images and for the laser reflection map were registered concomitantly. This approach allowed the labeled structures to be located on the metal surface. Subsequently, the same implants were prepared for SEM of the tissue/implant interface, and upon removal of the adhering structures, of the underlying metal surface. Thus, specific proteins can be identified and their spatial architecture as well as that of the underlying metal surface can be visualized for one and the same implant. The immediate visualization after fluorescence labeling in buffer by means of water immersion objective lenses proved most critical.

Actins↗

PubMed1976

The renal vascular lesions of severe and malignant hypertension: a light-immunofluorescent microscopy, transmission and scanning electron-microscopy study.

1. Arterial and glomerular lesions were studied in seventy-eight specimens from patients with severe or malignant primary or secondary hypertension by transmission and scanning electron microscopy and light-microscopy and immunofluorescent microscopy. 2. The lesions were classified as compensatory, decompensated and reparative. The arterial lesions of primary and secondary hypertension were qualitatively similar. Each of the microscopy techniques contributed to a better understanding of the changes.

Basement Membrane↗

PubMed1997

Spatial distribution of dihydropyridine receptors in the plasma membrane of guinea pig cardiac myocytes investigated by correlative confocal microscopy and label-fracture electron microscopy.

Excitation-contraction coupling in cardiac muscle is thought to depend fundamentally on the spatial organization of sarcolemmal dihydropyridine receptors (L-type calcium channels) in relation to ryanodine receptors (calcium-release channels of the sarcoplasmic reticulum). In the present study, we have investigated the distribution of dihydropyridine receptors in the guinea pig myocyte plasma membrane by correlative immunoconfocal microscopy and label-fracture electron microscopy. Label-fracture, a method in freeze-fracture cytochemistry, permits immunogold localization of cell surface proteins in en face membrane views. Taken together, results from confocal microscopy and label-fracture replicas suggest that, in the peripheral plasma membrane, calcium channels are organized predominantly in the form of clusters. Confocal microscopy also suggests a similar organization in the transverse tubules. It is hypothesized that these clusters may lie adjacent to junctional sarcoplasmic reticulum, permitting the close coupling of influx of calcium through plasma membrane calcium channels to trigger release of calcium from the intracellular stores, as part of the mechanism of calcium-induced calcium release.

Animals↗

PubMed1976

Study on development of Agaricus bisporus by fluorescent microscopy and scanning electron microscopy.

Two strains of Agaricus bisporus have been investigated by fluorescent microscopy and scanning electron microscopy. Somatic nuclei, stained with auramin O and examined by fluorescent microscopy, appear to be randomly distributed, divide asynchronously, and assume a nonclassical or "two-track" configuration during mitotic metaphase. A similar configuration has been observed for nuclei during meiosis, but early meiosis in A. bisporus appears to be classical, usually with nine pairs of chromosomes evident during prophase I. Scanning electron microscopy has been used to document developmental stages in the formation and germination of basidiospores. Two-spored basidia were predominant, but occasionally one- or three-spored forms were observed. Four-spored basidia were absent, and uninucleated basidiospores were exceedingly rare to absent.

Basidiomycota↗

PubMed1995

Using laser scanning confocal microscopy as a guide for electron microscopic study: a simple method for correlation of light and electron microscopy.

Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large or have fine branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of light microscopic appearance and electron microscopic detail is very time-consuming. We report here a simple method combining laser scanning confocal microscopy and electron microscopy for study of the synaptic relationships of the neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Neurons were labeled intracellularly with neurobiotin or biocytin, two widely used stains. The tissue was then sectioned on a vibratome and processed with both streptavidin-nanogold (for electron microscopic study) and streptavidin-Cy3 (for confocal microscopic study) and embedded in epon/araldite. Interesting areas of the labeled neuron were imaged in the epon/araldite blocks with laser scanning confocal microscopy and then thin-sectioned at the indicated depth for electron microscopic study. This method provides an easy, reliable way to correlate three-dimensional light microscopic information with electron microscopic detail, and can be very useful in studies of synaptic connections.

Animals↗

PubMed2003

Comparison of wide-field/deconvolution and confocal microscopy in bioengineering. Interest of multi-photon microscopy in the study of articular cartilage.

The increase in lateral and spatial resolutions is one of the major targets of research and development in the field of optical microscopies applied to living tissue. The optical geometry of Confocal Laser Scanning Microscopy (CLSM) demonstrates its undeniable advantage on conventional fluorescence microscopy by segregating the planes outside the focussing plane. The methodological and technological advances of the last five years have been fast evolving, especially with regard to the optimisation of CLSM and deconvolution process. The limited analysis in thick tissue have given rise to the development of other techniques, multi-photon excitation microscopy in particular. In this paper, we have applied these techniques on major biological applications in bioengineering (endothelial cell, chondrocyte in 3D-culture, human cartilage) and discussed the technical limitations and perspectives.

Actins↗

PubMed1990

Comparison of Scheimpflug-photography, specular microscopy and scanning electron microscopy to detect corneal changes in toxicity studies in rats.

With an increasing number of in-vivo methods to examine the eyes of laboratory animals, the rat has become an important animal model in experimental eye research. Specular microscopy is a clinical tool to examine the corneal endothelium in-vivo. To evaluate the versatility of this method for small animal eyes, we studied both corneal endothelial cell-count and corneal thickness in normal rats as well as those with diabetic, naphthalene and UV-B cataract. As a reference scanning electron microscopy (SEM) of the corneal endothelium was performed. For cell-counts the correlation coefficient between both methods was found to be sufficient. The comparison of corneal thickness measurement (SEM-values) with specular microscopy and with Scheimpflugbiometry failed to show a satisfactory correlation. The study proves that specular microscopy is a useful tool to document changes also in the endothelium of the rat-cornea.

Animals↗

PubMed1997

Evaluation of particulate embolic materials with MR imaging, scanning electron microscopy, and phase-contrast microscopy.

PURPOSE: To analyze the properties and embolic effect of microfibrillar collagen (MFC), Gelfoam powder, and polyvinyl alcohol (PVA) materials that are used in embolization procedures in the head and neck. METHODS: The shape and surface of these embolic agents were examined with scanning electron microscopy and phase-contrast microscopy. The mean number of areas of T2-weighted high signal intensity was measured on MR images in a rat embolization model to estimate the embolic effect. RESULTS: By scanning electron microscopy and phase-contrast microscopy, MFC appears fibriform and has various sizes and an irregular surface. Gelfoam is of uniform size and has a smooth surface. PVA materials are granulated and have a rough surface. MFC is somewhat suspendable and its shape changes moderately after suspension. Gelfoam is very suspendable and its shape changes rapidly. PVA showed only mild swelling. The embolic effect of MFC was the lowest of the materials examined. Large PVA particles (250 to 500 microns) showed a lesser embolic effect than Gelfoam or small PVA particles (50 to 150 microns) or medium-sized PVA particles (150 to 250 microns). No significant differences were observed among the embolic effects of Gelfoam, small PVA particles (50 to 150 microns), and medium PVA particles (150 to 250 microns). CONCLUSIONS: MFC and large PVA particles (250 to 500 microns) should be used for embolization of vascular anatomy involving potentially dangerous anastomoses. Gelfoam, PVA particles of 150- to 250-micron diameter, and PVA particles of 50- to 150-micron diameter are adequate for embolization involving homogeneous and peripheral anatomy.

Animals↗

PubMed1998

Structure and blood supply of intrinsic lymph nodes in the wall of the rabbit urinary bladder--studies with light microscopy, electron microscopy, and vascular corrosion casting.

The urinary bladder is especially subject to infection by virtue of its direct connection to the external urethral opening, and it is natural to anticipate the presence of a well-developed immunological mechanism to respond to this potential threat. The present study describes small, very highly vascular lymph nodes located in the wall of the rabbit bladder, which may be involved in a local response to foreign antigens. The vasculature and structure of these lymph nodes was described using a combination of vascular corrosion casting, ink injection, and light and electron microscopy. The distal abdominal aorta was cannulated, and after clearing the bladder vasculature with buffered saline, one of the following procedures was used: 1) the bladder was perfuse-fixed in preparation for light and electron microscopy; 2) the bladder vasculature was filled with India ink for vessel tracing; or 3) vascular corrosion casts of the vasculature were prepared by infusing resin comprised of a mixture of Mercox, methyl methacrylate monomer, and catalyst. The resulting casts were cleaned with KOH, formic acid, and water in preparation for scanning electron microscopy. Vascular casts and India ink injections revealed the presence of a number of isolated capillary tufts consisting of clusters of one to five "glomeruli," closely associated with the major vesicular vessels along the lateral walls of the bladder, and supplied by tertiary branches of these vessels. Light and electron microscopy showed that the capillary tufts represented the blood supply to small, ovoid lymph nodes located near the serosal surface of the bladder wall and usually restricted to the basal half of the bladder. These nodes were encapsulated and exhibited subcapsular sinuses, numerous small blood vessels, a limited number of high endothelial cells, and, occasionally, nerves and a follicular substructure. The nodes contained abundant lymphocytes, stellate stromal cells, macrophages, and eosinophils, but lacked the obvious cortical and medullary organization and germinal centers often seen in larger lymph nodes. Vascular corrosion casts, vascular ink injections, and microscopic examination confirmed the presence of small, highly vascular lymph nodes closely associated with the main vesicular vessels along the lateral walls of the rabbit bladder. A follicular substructure of the nodes appears to correspond with the "glomerular" capillary arrangement within the nodes as seen with corrosion casts. The rich blood supply may be indicative of the high metabolic demand of lymphatic tissue, and may be altered in response to the level of activity of the node. The close association between the lymphatic tissue and the rich blood supply to the nodes may allow a rapid mobilization of lymphocytes during a local immune response to foreign agents.

Animals↗

PubMed1988

Innervation of the C cells of chicken ultimobranchial glands studied by immunohistochemistry, fluorescence microscopy, and electron microscopy.

Innervation of the ultimobranchial glands in the chicken was investigated by immunohistochemistry, fluorescence microscopy and electron microscopy. The nerve fibers distributed in ultimobranchial glands were clearly visualized by immunoperoxidase staining with antiserum to neurofilament triplet proteins (200K-, 150K- and 68K-dalton) extracted from chicken peripheral nerves. The ultimobranchial glands received numerous nerve fibers originating from both the recurrent laryngeal nerves and direct vagal branches. The left and right sides of the ultimobranchial region were asymmetrical. The left ultimobranchial gland had intimate contact with the vagus nerve trunk, especially with the distal vagal ganglion, but was somewhat separated from the recurrent nerve. The right gland touched the recurrent nerve, the medial edge being frequently penetrated by the nerve, but the gland was separated from the vagal trunk. The left gland was innervated mainly by the branches from the distal vagal ganglion, whereas the right gland received mostly the branches from the recurrent nerve. The carotid body was located cranially near to the ultimobranchial gland. Large nerve bundles in the ultimobranchial gland ran toward and entered into the carotid body. By fluorescence microscopy, nerve fibers in ultimobranchial glands were observed associated with blood vessels. Only a few fluorescent nerve fibers were present in close proximity to C cell groups; the C cells of ultimobranchial glands may receive very few adrenergic sympathetic fibers. By electron microscopy, numerous axons ensheathed with Schwann cell cytoplasm were in close contact with the surfaces of C cells. In addition, naked axons regarded as axon terminals or "en passant" synapses came into direct contact with C cells. The morphology of these axon terminals and synaptic endings suggest that ultimobranchial C cells of chickens are supplied mainly with cholinergic efferent type fibers. In the region where large nerve bundles and complex ramifications of nerve fibers were present, Schwann cell perikarya investing the axons were closely juxtaposed with C cells; long cytoplasmic processes of Schwann cells encompassed large portions of the cell surface. All of these features suggest that C-cell activity, i.e., secretion of hormones and catecholamines, may be regulated by nerve stimuli.

Animals↗

PubMed1986

The Kupffer cell in experimental extrahepatic cholestasis in the rat--a light microscopy, immunohistochemical and electron microscopy study.

Kupffer cell phagocytic function is reduced in the presence of obstructive jaundice. To investigate possible mechanisms we report a study of the rat liver in extrahepatic cholestasis, using light microscopy, immunohistochemistry and electron microscopy, Immunohistochemistry was performed with monoclonal antibodies specific for rat Kupffer cells ED 1, ED 2 and ED 3 and monoclonal antibodies directed against class II antigens of the rat major histocompatibility complex Ox 3 and Ox 6. Extrahepatic cholestasis was produced by bile duct ligation. In bile duct ligated animals light microscopy showed proliferation of bile ductules and an increase in sinusoidal cells. Immunohistochemistry with ED 1, ED 2 and ED 3 demonstrated a marked increase in the number of positive cells, but few of these cells were positive with Ox 3 and Ox 6, whereas the proliferating bile ductules were strongly positive. Electron microscopy revealed two homogeneous granular substances within the sinusoidal lumen and loss of the space of Disse. Despite a reduction in Kupffer cell phagocytic function in obstructive jaundice there is an increase in Kupffer cells, but these cells appear to be in an inactivated state as few express class II antigens on their surface. Furthermore the granular substance within the space of Disse may interfere with function.

Animals↗

PubMed1985

Structural organization of an active, chromosomal nucleolar organizer region (NOR) identified by light microscopy, and subsequent TEM and STEM electron microscopy.

The three-dimensional arrangement of the chromatin components within the nucleolar organizer regions (NORs) from living oocyte nuclei was investigated. As a suitable cell system we chose vitellogenic oocytes of the orthopteran insect Acheta. This cell type is particularly attractive for analysis of nucleolar chromatin, since structural and functional aspects of NORs during early oogenic stages (including the association of NORs with amplified rDNA copies) are particularly well known (Lima-de-Faria 1974). In the course of the present study we first identified putative chromosomal NORs in isolated nuclei of mid-diplotene oocytes according to morphological characteristics using differential interference contrast (DIC) or phase contrast light microscopy. The presence of actively transcribing chromosomal NORs during this late stage of Acheta oogenesis obviously had been overlooked by previous investigators, probably due to difficulties of chromosome visualization. For a more detailed ultrastructural analysis, NORs were gently sedimented from opened nuclei and processed for sectioning using a modified "end-embedding" procedure (Mott and Callan 1975; Spring and Franke 1981). A small number of thick and thin sections could be made from individual NORs. Sections were analyzed by light microscopy, conventional transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). Whereas the structural connection of NORs to the chromosome axis and also the general arrangement of active nucleolar genes within the NOR complex could be seen with TEM, the visualization of individual nucleolar genes and the organization of transcription complexes was only possible using bright field STEM of thick sections at low temperature.

Animals↗

PubMed1977

[Examination of gallstones on their surface and in fractured cross sections by incident light microscopy and scanning electron microscopy (author's transl)].

Operatively removed gallstones were examined on their surfaces and in fractured cross sections by incident light microscopy and scanning electron microscopy. In addition, micro-bore samples and X-ray crystallography were done. Five gallstone types consisting of three basic structural layers are differentiable by incident light microscopy. The three layers consist of a central nucleus which is always present, a radially structured middle layer, and a fine crystalline outer shell, the presence or absence of the latter two layers differentiating the stone types. Two crystal structures could be differentiated by electron microscopy; a flat and a globular type. The nucleus is always of the globular crystalline type, while the outer layers are flat crystalline. From this we were lead to believe that the conditions in vivo, under which the different layers of a gallstone are built, change. The micro-bore samples lead us to believe that calcium is only secondarily layed down in the gallstone framework. White crystalline deposits, which were formed several seconds after fracture, were discovered on the fractured gallstone cross sections.

Cholelithiasis↗

PubMed1983

Light microscopy, electron microscopy, and X-ray diffraction analysis of glycerinated collagen fibers.

Light microscopy, transmission electron microscopy (freeze-fracture replicas and thin sections), and X-ray diffraction techniques were used to investigate the structure of rat tail tendon collagen fibers subjected to one of the following treatments: water, phosphate buffer, glutaraldehyde, glutaraldehyde followed by glycerol, glycerol, and glycerol followed by phosphate buffer. As seen by light microscopy, only treatment with glycerol induces a remarkable swelling of the native (untreated) collagen specimens. Replicas and thin sections show that this swelling is due to an expansion of the interfibrillar space, and to a dissociation of the collagen fibrils into microfibrils. X-Ray diffraction analysis reveals great disorder in the glycerol-swollen collagen fibers. However, this does not appreciably involve the microfibrillar and molecular structure. Light and electron microscopy as well as X-ray diffraction techniques show that the collagen fiber swelling induced by glycerol is an almost completely reversible process.

Animals↗

PubMed1989

Temporal artery biopsies. Correlation of light microscopy and immunofluorescence microscopy.

Immunopathologic studies are done routinely on biopsy specimens from tissues affected by many autoimmune diseases. To evaluate the role of direct immunofluorescence microscopy (DIFM) in identifying temporal arteritis, the authors reviewed all temporal artery biopsies done over a 30-month period (100 consecutive biopsies). The DIFM, using antibodies to IgG, IgM, IgA, complement, and fibrinogen, had a diagnostic sensitivity rate of 93% and a specificity rate of 87% compared with light microscopy. In biopsy specimens showing arteritis by light microscopy, IgG was demonstrated by DIFM in 85% of cases, IgM in 69%, and IgA in 15%. In one patient, a DIFM staining pattern highly suspicious of temporal arteritis identified a patient with features of clinical temporal arteritis despite negative findings by light microscopy. The demonstration of immunoglobulin by DIFM supports the possible role of humoral immunity in the pathogenesis of temporal arteritis.

Aged↗

PubMed1983

Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.

Pt K2 rat kangaroo epithelial cells and Rat-1 fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin, and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute significantly to the material contrast. By comparison with fluorescence micrographs obtained on the same individual cells, actin-containing stress fibers, keratin filaments, and vimentin filaments were identified in the photoelectron micrographs. The apparent volume occupied by the cytoskeletal network in the cells as judged from the photoelectron micrographs is much less than it appears to be from the fluorescence micrographs because the higher resolution of photoelectron microscopy shows the fibers closer to their true dimensions. Photoelectron microscopy is a surface technique, and the images highlight the exposed cytoskeletal structures and suppress those extending along the substrate below the nuclei. The results reported here show marked improvement in image quality of photoelectron micrographs and that this technique has the potential of contributing to higher resolution studies of cytoskeletal structures.

Actins↗