Correlative microscopy of cerebellar Bergmann glial cells.

Double fluorescent labelling of rat cerebellar cortex using antibody to glial fibrillary acidic protein (GFAP) and Alexa fluor conjugates for secondary detection for confocal laser scanning microscope (CLSM), field emission scanning electron microscopy (FESEM) of Rhesus monkey cerebellar cortex, ultrathin sectioning and freeze-etching replica method for transmission electron microscopy of mouse cerebellar cortex have been examined in an attempt to obtain a new and more accurate view of three-dimensional image of Bergmann glial cells (BGC) and their topographic relations in the molecular layer. Intense immunopositive GFAP green staining was observed in the BGC and glial limiting layer. Secondary antibody conjugated with Alexa fluor 488 and Alexa fluor 668-1B4 stained in red capillary endothelial cells and microglial cells. BGC morphology revealed the existence of several cell types or subpopulations of BGC. Bergmann glial fibers, in palisade arrangement, branch and rebranch forming a complex glial network in the molecular layer. Field emission SEM and freeze-fracture SEM method show the SE-I image of high mass dense Bergmann glial cytoplasm ensheathing like a veil the Purkinje cell (PC) soma and dendritric arborization. Bergmann glial fibers appeared completely surrounding individual parallel fibers or parallel fiber bundles, terminal climbing fiber collaterals, basket and stellate cells and capillaries. Freeze-etching direct replicas showed the typical orthogonal arrangement of intramembrane particles, corresponding to the large repertoire of BGC receptors. The study reveals three-dimensional Bergmann glial cells heterogeneity and the complex network formed by Bergmann glial cells in the molecular layer.

Confocal laser scanning, conventional scanning and transmission electron microscopy of vertebrate cerebellar granule cells.

Confocal laser scanning microscopy of hamster cerebellar granular layer showed in montages of z-series the presence of small, medium and large granule cells. A granule cell Golgi cell ratio of 50/4 was observed surrounding glomerular regions. Field emission high resolution scanning electron microscopy of mouse cerebellar granular and molecular layers showed SE-I images of the outer and inner surfaces of nuclear and cytoplasmic compartments of chromium coated granule cells and the axo-spinodendritic synapses of parallel fibers with Purkinje cell dendrites. Conventional scanning electron microscopy of teleost fish cerebellar cortex showed three dimensional morphology of granule cell soma and processes and the synaptic relationship with mossy and climbing fibers, Golgi cell axonal ramifications and dendrites of stellate neurons, by means of SE-II and SE-III signal image mode, in sagittally and transversally cryofractured cerebellar cortex. SE-II images of the non-synaptic segments and synaptic varicosities of parallel fiber outer surface were characterized in the molecular layer. Ultrathin sections of transmission electron microscopy (TEM) revealed somato-somatic, dendro-somatic and dendro-dendritic like-desmosomal and like-hemidesmosomal junctions in human cerebellar granule cells. Freeze-etching replicas of mouse cerebellar cortex displayed granule cell intramembrane morphology, cytoplasmic fractured face and the Bergman glial cell cytoplasm completely surrounding the parallel fibers in the molecular layer. The mossy fiber-granule cell dendrite synaptic relationship was observed in sagittally and transversally cryofractured cerebellar cortex and correlated with TEM images. SE-II images of the climbing fiber synaptic connections with granule cell dendrites were obtained in teleost fish cerebellar cortex. One to one axo-dendritic synaptic contacts between Golgi cell axonal ramifications and granule cell dendrites were also seen. The above findings provide new vistas for future studies dealing with intracortical circuits and information processing in the cerebellar cortex.

High-resolution analysis of engineered type I collagen nanofibers by electron microscopy.

Collagen nanofibers were generated at ambient temperature and pressure by electrospinning a 1 wt% solution of type I collagen and polyethylene oxide. Products were imaged with high-resolution scanning electron microscopy (HRSEM) at medium (approximately 30,000 x) and high magnifications (approximately 100,000 x) and with transmission electron microscopy (TEM). The capacity to produce collagen nanofibers may lead to the generation of extracellular matrix-based fabrics with applications in the fields of wound healing and tissue engineering.

Field emission scanning electron microscopy and freeze-fracture transmission electron microscopy of mouse cerebellar synaptic contacts.

Samples of albino mice were processed by the cryofracture method for scanning electron microscopy and examined with the field emission scanning electron microscope (FESEM). Freeze-etching direct replicas of mice cerebellar cortex were also studied with the transmission electron microscope (FFTEM), as a complementary technique for obtaining higher resolution, three-dimensional correlative images of cerebellar synaptic contacts. At the granular, Purkinje cells and molecular layers, the cryofracture method for FESEM selectively removed the neuroglial cell investment, facilitating the visualization of the outer and inner surfaces of cerebellar synaptic contacts. In addition, FFTEM showed the real extension of perisynaptic neuroglial investment. The outer surface of mossy fiber rosettes and their digitiform processes were seen at the granular layer, making flat and invaginated synaptic contacts with the granule cell dendrites. At the molecular layer, the longitudinal traject of parallel fibers or nonsynaptic segments and their synaptic varicosities were characterized. These latter established synaptic contacts with Purkinje dendritic spines. Fractured parallel fiber endings showed the SE-I images of clustered spheroidal synaptic vesicles and mitochondria and the surrounding cotton-like appearance of Bergmann glial cell cytoplasm. Climbing fibers showed a characteristic crossing-over bifurcation pattern in the white matter and in the three-layer structure of cerebellar cortex, formation of tendril collaterals in the granular layer, topographical relationship with Purkinje cell soma and retrograde collaterals in the molecular layer. The climbing fiber synaptic relationship with Purkinje dendritic spines was characterized, by means of FFTEM, by the presence of large synaptic endings and aggregation of intramembrane particles at the P and E faces of presynaptic endings, characteristic of excitatory synapses.

Engineered collagen-PEO nanofibers and fabrics.

Type I collagen-PEO fibers and non-woven fiber networks were produced by the electrospinning of a weak acid solution of purified collagen at ambient temperature and pressure. As determined by high-resolution SEM and TEM. fiber morphology was influenced by solution viscosity, conductivity, and flow rate. Uniform fibers with a diameter range of 100-150 nm were produced from a 2-wt% solution of collagen-PEO at a flow rate of 100 microl min(-1). Ultimate tensile strength and elastic modulus of the resulting non-woven fabrics was dependent upon the chosen weight ratio of the collagen-PEO blend. 1H NMR dipolar magnetization transfer analysis suggested that the superior mechanical properties, observed for collagen-PEO blends of weight ratio 1:1, were due to the maximization of intermolecular interactions between the PEO and collagen components. The process outlined herein provides a convenient, non-toxic, non-denaturing approach for the generation collagen-containing nanofibers and non-woven fabrics that have potential application in wound healing, tissue engineering, and as hemostatic agents.

Confocal laser scanning, conventional scanning and transmission electron microscopy of vertebrate cerebellar granule cells.

Confocal laser scanning microscopy of hamster cerebellar granular layer showed in montages of z-series the presence of small, medium and large granule cells. A granule cell Golgi cell ratio of 50/4 was observed surrounding glomerular regions. Field emission high resolution scanning electron microscopy of mouse cerebellar granular and molecular layers showed SE-I images of the outer and inner surfaces of nuclear and cytoplasmic compartments of chromium coated granule cells and the axo-spinodendritic synapses of parallel fibers with Purkinje cell dendrites. Conventional scanning electron microscopy of teleost fish cerebellar cortex showed three dimensional morphology of granule cell soma and processes and the synaptic relationship with mossy and climbing fibers, Golgi cell axonal ramifications and dendrites of stellate neurons, by means of SE-II and SE-III signal image mode, in sagittally and transversally cryofractured cerebellar cortex. SE-II images of the non-synaptic segments and synaptic varicosities of parallel fiber outer surface were characterized in the molecular layer. Ultrathin sections of transmission electron microscopy (TEM) revealed somato-somatic, dendro-somatic and dendro-dendritic like-desmosomal and like-hemidesmosomal junctions in human cerebellar granule cells. Freeze-etching replicas of mouse cerebellar cortex displayed granule cell intramembrane morphology, cytoplasmic fractured face and the Bergman glial cell cytoplasm completely surrounding the parallel fibers in the molecular layer. The mossy fiber-granule cell dendrite synaptic relationship was observed in sagittally and transversally cryofractured cerebellar cortex and correlated with TEM images. SE-II images of the climbing fiber synaptic connections with granule cell dendrites were obtained in teleost fish cerebellar cortex. One to one axo-dendritic synaptic contacts between Golgi cell axonal ramifications and granule cell dendrites were also seen. The above findings provide new vistas for future studies dealing with intracortical circuits and information processing in the cerebellar cortex.

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy.

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin metal coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the "flower-like" structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.

High resolution (SE-I) scanning electron microscopy features of primate cerebellar cortex.

This paper provides an exploration into the outer and inner surfaces of primate cerebellar neurons using secondary electron-I (SE-I) topographic contrast. SE-I enriched, chromium coated, cryofractured cerebellum staged within the condenser/objective lens stage of SEMs, equipped with high brightness LaB6 and field emission emitter, generated quality images of intact and fractured nerve cells studied at intermediate and high magnifications. Granule and Golgi cell surfaces revealed smooth, accurately delineated profiles of the true cell surface features, which lacked the SE-III dominated brilliance of conventional gold or gold-palladium decorated images. Fractured non synaptic segments of parallel fibers in the molecular layer showed interconnected anastomotic networks of ER tubules, vesicles and cisterns, whereas cross fractured presynaptic "en passant" endings of these fibers exhibited spheroidal synaptic vesicles and SE-I edge brightness contrast delineated their limiting plasma membranes. Parallel fiber fractured synaptic endings showed a homogeneous extravesicular material surrounding the synaptic vesicles. The neuroglial cytoplasm ensheathing nerve processes exhibited a smooth discontinuous surface. The high mass density surface of the myelin sheath showed a mixed population of globular structures, 10-30 nm, corresponding to protein and phospholipid microdomains.

Proteoglycan ultracytochemistry and conventional and high resolution scanning electron microscopy of vertebrate cerebellar parallel fiber presynaptic endings.

The parallel fiber "en passant" synaptic endings of mouse cerebellar molecular layer have shown by means of transmission electron microscopy, the presence of an electron dense extravesicular material in samples perfused with Alcian blue. This alcianophilic material was digested in cerebellar tissue previously treated with testicular hyaluronidase, suggesting the presence of hyaluronic acid or chondroitin 4- or 6-sulphate. Freeze-fractured Rhesus monkey cerebellar cortex prepared for conventional scanning electron microscopy also revealed the presence in fractured synaptic varicosities of parallel fibers of a high mass density material, in which the synaptic vesicles are embedded. Examination of cryofractured primate cerebellar cortex coated with thin chromium films, 1-2 nm thick, in the high resolution field emission scanning electron microscope showed the SE-I topographic contrast of an extravesicular material deposited in axoplasmic matrix of fractured parallel synaptic endings. The precise localization of this material corresponds to that observed in transmission electron microscopy and conventional freeze-fracture scanning electron microscopy. These electron microscopic findings tend to agree with the omnipresence in several vertebrates of a presynaptic axoplasmic material, which seems to be proteoglycan in nature.

Energy-filtering transmission electron microscopy as a tool for structural and compositional analysis of isolated ferritin particles.

Structural and compositional analysis of isolated horse-spleen ferritin particles was performed by energy filtering transmission electron microscopy (EFTEM). Ferritin particles were collected in ultrathin (2 nm thick) chromium films and analyzed without any additional stain by electron energy-loss spectroscopy (EELS) for iron and carbon and by electron-spectroscopic imaging (ESI) for carbon. The ultrastructure of the proteinaceous shell of the ferritin particle, as obtained by the carbon net-intensity electron spectroscopical and carbon concentration-distribution images, was qualitatively compared to the structure as acquired by a negative-staining procedure. Quantitative analysis of the number of carbon atoms in the ferritin-shell proteins was carried out through an ESI-acquisition protocol and processing procedure with calibrated attenuation filters in the optical path to the TV camera. This procedure included images acquired with calibrated attenuation filters for the compensation of shading and the non-linear performance of the TV camera used in the analytical part of the procedure. A new ¿ESI-Spectra¿ program is proposed that allows element-related spectra to be generated at any place and with any frame size in a contrast-sensitive or other type of image present on the computer monitor screen.

Analysis of high quality monatomic chromium films used in biological high resolution scanning electron microscopy.

During the recent employment of field emission (FE) in-lens scanning electron microscopes (SEMs), refractory metal deposition technology has co-evolved to provide enhanced contrast of 1-10 nm hydrocarbon based biological structures imaged at high magnifications (> 200,000 times). Pioneer development employing the Penning sputter system in a high vacuum chamber proved that imaging of chromium (Cr) coated biological specimens contained enriched secondary electron (SE)-(I) contrasts. Single nanometer size fibrillar and particulate ectodomains within the context of complex biological membranes were accurately imaged without significant enlargement using the high resolution SE-I mode (HRSEM). This paper reports the transmission electron microscopy (TEM) testing of ultrathin (0.5-2.0 nm) Cr films deposited by planar magnetron sputter coating (PMSC). Essential parameters necessary to reproduce quality sputtered films of refractory metals used in HRSEM studies were described for the vacuum system and target operation conditions (current, voltage, and target distance). HRSEM imaging of biological specimens is presented to assess contrast attained from ultrathin fine grain Cr films deposited by PMSC. High magnification images were recorded to illustrate high quality contrasts attainable by HRSEM at low (1-5 kV) and high (10-30 kV) voltages. Dispersed molecules on formvar coated grids were sputter coated with a 1 nm thick Cr film before employing scanning transmission (STEM)/SEM modes of the FESEM to establish non-decorative image accuracy in the transmitted electron mode.

Conventional and high resolution scanning electron microscopy and cryofracture techniques as tools for tracing cerebellar short intracortical circuits.

The present paper shows the potential contribution of conventional and high resolution scanning electron microscopy (SEM) to trace short intracortical circuits in cryofractured fish, primate and human cerebelli. Conventional SEM slicing technique allowed us to identify afferent mossy and climbing fibers and their synaptic relationship in the granular layer. SEM freeze-fracture method exposed the mossy glomerular synapses and the axo-dendritic connections of climbing fibers. At the Purkinje cell layer, the cryofracture process removed the satellite Bergmann glial cell layer, displaying a partial view of the supra- and infra-ganglionic plexuses of Purkinje cells and the ascending pathways of climbing fibers. High resolution SEM (HRSEM) showed the specimen specific secondary electron (SE-I) image of axosomatic synapses on Golgi cell surface. At the molecular layer, the outer surface of parallel fiber synaptic varicosities were distinguished, establishing the cruciform en passant synaptic contact with the Purkinje cell dendritic spines. HRSEM showed the fractured parallel fiber synaptic varicosities containing spheroidal synaptic vesicles embedded in a high dense extravesicular material. Conventional SEM and gold-palladium coating are useful to trace intracortical circuits. With HRSEM and chromium coating, it is possible to study the outer and inner surfaces of synaptic connections.

Conventional and high resolution field emission scanning electron microscopy of vertebrate cerebellar parallel fiber-Purkinje spine synapses.

Purkinje dendrite-parallel fiber spine synapses of human, teleost fishes, Rhesus monkey and mouse cerebellar cortex have been studied by means of conventional scanning electron microscopy (SEM) using ethanol-cryofracturing technique and by high resolution field emission scanning electron microscopy (HRFESEM) for studying the outer and inner surface morphology of pre- and postsynaptic endings. Transmission electron microscopy of mouse cerebellar cortex either by means of ultrathin sections and freeze-etching replicas have been complementarily used for proper identification and comparative observations. Normal teleost fishes showed short neck and neckless Purkinje spines with exhibited spread or extended postsynaptic densities. In pathological human cerebellum, the ethanol cryofracturing technique exposed the outer surface of edematous flat and invaginated Purkinje spine synapses. In fractured presynaptic endings HRFESEM showed in Rhesus monkey cerebellar cortex a homogeneous extravesicular material binded to the synaptic vesicles and joining them to the presynaptic membrane. HRFESEM partially resolved the synaptic cleft as currently observed in high magnification transmission electron microscopy. Round subunits, 25-35 nm in diameter, were observed associated to postsynaptic membrane, apparently corresponding to the localization and distribution of E face postsynaptic intramembrane particles, which suggest that such subunits correspond to the domains of neurotransmitter postsynaptic receptors.

Conventional and high resolution scanning electron microscopy of outer and inner surface features of cerebellar nerve cells.

This paper provides an exploration into the inner and outer surfaces of vertebrate cerebellar neurons utilizing secondary electron (SE-I and SE-II) topographic contrasts. SE-I enriched chromium coated, cryofractured Rhesus monkey cerebellum staged within the condenser/objective stage of SEMs equipped with high brightness LaB6 and field emission emitters generated quality images of intact and fractured nerve cells studied at intermediate and high magnifications. These images were compared with conventional SE-III images of gold-palladium teleost fish cerebellar neurons and transmission electron micrographs of mouse cerebellar nerve cells obtained either by thin-sections or freeze-etch replicas. Chromium coated Rhesus monkey granule and Golgi cell surfaces revealed smooth, accurately delineated profiles of true cell surface features, which lacked the SE-III dominated brilliance of conventional teleost fish gold or gold-palladium, decorated neurons. Chromium coated fractured nerve processes showed the outer smooth surface of interconnected anastomotic networks or ER tubules, vesicles and cisterns. Cross fractured presynaptic endings of parallel fibers in the molecular layer exhibited spheroidal synaptic vesicles and SE-I edge brightness contrast delineated their limiting plasma membranes. Fractured synaptic endings showed a homogeneous extravesicular material surrounding the synaptic vesicles. The presynaptic dense projections appeared as columnar shaped structures. The postsynaptic membrane and associated postsynaptic density showed a discontinuous surface formed by round subunits 25-35 nm in diameter. The neuroglial cytoplasm ensheathing nerve process exhibited a smooth discontinuous surface. The surface of the myelin sheath showed a mixed population of globular structures 10-30 nm in diameter, apparently corresponding, according to freeze-etch images, to protein and phospholipid microdomains.

Human immunodeficiency virus type-1 (HIV-1) infection of endothelial cells in vitro: a virological, ultrastructural and immuno-cytochemical approach.

In an attempt to better understand the role of endothelial cells during HIV-1 infection, we report a virological and ultrastructural study on isolated endothelial cells from human adipose tissue, infected by HIV-1 in vitro. Supernatants from cultures showed the presence of p24 antigen and reverse transcriptase activity starting two days after HIV inoculation. A significant decrease of viral rescue was observed in cycloheximide treated cells confirming a de novo synthesis of viral products. SEM analysis individualized several surface slender projections and interdispersed virus-like particles in the infected cells. Furthermore, transmission electron microscopy (TEM) results showed cellular aspects of HIV phagocytosis and virus budding, suggesting that endothelial cells may represent a CD4 negative cell target of HIV-1 infection.

Ultrastructure of coronary arterial endothelium in atherosclerotic swine suggests lipid retro-endocytosis.

During experimental atherosclerosis, arterial endothelial cells show characteristic ultrastructural changes including the appearance of increased numbers of plasmalemmal and cytoplasmic vesicles. These structures have been shown by tracer studies to participate in the transcellular transport of low-density lipoprotein cholesterol and beta-very-low-density lipoprotein cholesterol from the arterial lumen into the abluminal extracellular matrix. Although this probably represents the major lipid transport pathway, other forms of transport may exist. We document the presence of apparent lipid structures averaging approximately 300 nm in diameter at or near the luminal surface of coronary arterial endothelium of atherosclerotic miniature swine. The structures exhibited a particulate nature with subunits of a heterogenous size distribution. The appearance of the endothelial plasmalemma adjacent and subjacent to these structures suggests exocytosis. We hypothesize that this previously unreported morphology may represent an in vivo structural correlate for the lipoprotein retro-endocytosis pathway which has been recently identified using biochemical methods in smooth muscle cells and fibroblasts in vitro.

Conventional and high resolution scanning electron microscopy of biological sectioned material.

Intracellular structures of embedded biological tissues (rat kidney, myocardium and small intestine) were observed by conventional-scanning electron microscopy (C-SEM) and high-resolution scanning electron microscopy (HR-SEM) after glass knife sectioning. C-SEM of semi-thin sections of material processed the same way as conventional transmission electron microscopy (TEM) provided strong backscattered electron (BSE)-dependent, two-dimensional secondary electron images (SEI(-)) which precisely integrated and further extended previous light microscopy (LM) observation of the same specimen. In addition, the three-dimensional (3-D) arrangement of intracellular organelles was appreciated using a mixture of acetone-soluble acrylic resin in place of epoxy resin embedding. Since the identification of such structures was hampered by the use of conventional fixations we introduced osmium maceration as a preliminary step to remove excess cytoplasmic matrix from the specimen. Consequently, semi-thin sections for LM and thin sections for TEM were obtained by sectioning of the tissue blocks. After resin removal, the sections were successfully observed in 3-D under a C-SEM. Finally, the deembedded, osmium treated sections proved to be smooth enough to facilitate deposition of continuous, ultra-thin (1 nm) chromium films and, therefore, HR-SEM studies of macromolecular cell membrane structures.

Correlative light, transmission, and high resolution (SE-I) scanning electron microscopy studies of rhesus adrenocortical vascular morphology.

A detailed correlative morphologic description using light microscopy (LM), transmission electron microscopy (TEM) and high resolution SE-I scanning electron microscopy (SEM) was conducted on the capillary endothelium of the zona-fasciculata (Z-F) in juvenile male rhesus monkeys. The glucocorticoid synthesis and release phenomena, associated with stress stimulated release of adrenocorticotropic hormone (ACTH) via the hypothalamic-pituitary axis, intimately involves capillaries of the Z-F. A comprehensive study of all the ultrastructural features implicated in the transendothelial uptake of steroidogenic precursors and release of glucocorticoids in perfused rhesus adrenals has not previously been made. This report presents correlative images of transendothelial openings that include previously described single diaphragmed fenestrae and plasmalemma vesicles, and double diaphragmed transendothelial channels. New observations of endothelial cell pockets, tight junctional complexes and membrane filled ghost sacs were recorded from perfused rhesus adrenal. Membranous ghosts associated with adrenocortical endothelium were reported in a previous TEM study of perfused rat, however the potential argument existed that ghosts were artifactual. Their role as steroid hormone releasing structures remains an open question, yet their structural characteristics appear justified based on imaging of identical profiles observed in perfused rhesus adrenocortical specimens. These structural features are considered for the potential of gating and sorting of metabolites, and release of glucocorticoids in response to ACTH stimulated stress events.