Electron Microscopy Of Tissue Sections Research Articles

Fluorescence In Resin Morphology (FIRM) Imaging Provides Histologic Context for Correlated Immunofluorescence and Electron Microscopy of Tissue Sections.

Fluorescence In Resin Morphology (FIRM) Imaging Provides Histologic Context for Correlated Immunofluorescence and Electron Microscopy of Tissue Sections.

Dynamic changes in basal lamina fenestrations in rat intestinal villous epithelium under high-fat diet condition.

The basal lamina of the villous epithelium in the small intestine has numerous fenestrations, which are produced by leukocytes for their intraepithelial migration. We previously showed that these fenestrations change due to the dynamics of migrating leukocytes in response to dietary conditions and suggested the possibility that this change is related to the regulation of the absorption of large-sized nutrients such as chylomicrons. The present study was, thus, designed to investigate structural changes in basal lamina fenestrations in response to a high-fat diet. The ultrastructure of the intestinal villi in the rat upper jejunum was investigated by electron microscopy of tissue sections in both the normal and the high-fat diet groups, and the fenestrations in the villous epithelium of rat upper jejunum were studied by scanning electron microscopy of osmium macerated/ ultrasonicated tissues. The present study showed that free cells adhering to the fenestrations increased in the upper jejunum two hours after feeding high-fat diet and the size of the fenestrations in this region also increased after feeding high-fat diet for 2 days. This enlargement of fenestrations may play an important role in increasing the efficiency of lipid absorption by facilitating the movement of chylomicrons from the intercellular space to the lamina propria.

Morphological and clinical aspects of scapular fasciocutaneous free flap transfer for treatment of venous insufficiency in the lower extremity.

We have recently shown that free scapular fasciocutaneous flaps transferred to the lower extremities of patients with chronic venous insufficiency and cutaneous ulcers have resulted in improvement in venous refilling times measured by photoplethysmography in the flap areas and that recurrent ulceration does not recur for up to 7 years. We hypothesized that the transferred flaps contained valves in their microvascular bed, which facilitated venous return, and using scanning electron microscopy of vascular corrosion casts and light and transmission electron microscopy of tissue sections prepared from human dorsal thoracic fascia, we showed that valves were most abundant in veins with a luminal diameter of 30-120 microm (59.3% of 905 valves). The depth of these valves increased with venous diameter, but the size of valve sinuses was not different for individual valves. Except for veins > 1,000 microm in diameter, there was no significant difference in the number of valves in different parts of an individual flap or between different flaps. Most valves were bicuspid; only in the vein Category 30-120 microm were unicuspid valves encountered. Valves were sometimes located in series in a short segment of a vein; occasionally they were found at the merging of two veins. Transmission electron microscopy showed that valve leaflets had collagen fibers that ascended toward the tip of the leaflet and were occasionally accompanied by elastic fibers. Myofibroblasts were regularly present in the valve leaflets. The present report reviews and updates these anatomic data about the human scapular region, focusing on venous valvular microstructure, and suggests that the high number of smaller-size valves contributes to improved hemodynamic of the leg and thus the clinical success of free scapular flaps used to treat cutaneous ulcerations in the lower extremity.

Müller glial cells of the tree shrew retina

The tree shrew is one of the few mammalian species whose retinae are strongly cone dominated, which is usually the case in reptilian and avian retinae. Müller cells of the tree shrew (Tupaia belangeri) retina were studied by transmission electron microscopy of tissue sections and freeze-fracture replicas, by immunolabeling of the intermediate filament protein vimentin in radial paraffin sections and in whole retinae, as well as by intracellular dye injection in slices of retinae. In addition, enzymatically isolated cells were stained by Pappenheim's panoptic staining method. The cells showed an ultrastructure that is similar to other mammalian Müller cells with two exceptions: Due to the extensive lateral fins of cone inner segments, the apical microvilli of Müller cells are arranged in peculiar palisades, and the basket-like Müller cell sheaths around neuronal somata in both nuclear layers consist of unusual multilayered membrane lamellae. Unlike Müller cells in other mammalian species studied thus far, but similar to reptilian and avian Müller cells, those of tree shrews commonly have two or more vitread processes rather than one main trunk. Müller cell densities range between some 13,000 mm-2 in the periphery and about 20,000 mm-2 in the retinal center. Neuron:(Müller)glial cell ratios were estimated to be 7.9:1 in the center and 6.2:1 in the periphery. For each Müller cell, about 1.5 (cone) photoreceptor cells, four or five interneurons of the inner nuclear layer, and about one cell of the ganglion cell layer were counted. This is a much lower number of neurons per Müller cell than in most other mammals studied.

Scanning and transmission electron microscopy and high resolution intravital video-microscopy of capillaries in the mouse exocrine pancreas, with special emphasis on endothelial cells.

Capillaries in the mouse exocrine pancreas were studied by scanning electron microscopy of microvascular corrosion casts, transmission electron microscopy of tissue sections, and high resolution intravital video-microscopy. Two types of capillaries were discerned by corrosion casting. The first type was rather straight, had a constant diameter of 5-6 microns, and its surface showed multiple circumferential furrows. The frequency of such constrictions was less in the second type, which was more undulated and had a diameter of 7-9 microns. In the second type, these constrictions defined bulged areas of the capillary cast. Corresponding tissue sections also showed two types of capillaries, fenestrated and non-fenestrated capillaries. Microtubules were abundant in all capillary endothelial cells, whereas bundles of microfilaments were scarce. Microtubules were arranged along the long axis of endothelial cells as well as parallel to endothelial cell border regions. Endothelial cells were joined by intermediate junctions along cell borders running both circumferentially and longitudinally. Flow reversal in capillaries and spontaneous endothelial contractions were documented in vivo. Endothelial cells bulged into the lumen, either at their nuclear region or distant from it. Spontaneous contraction of pericytes was not observed. These results suggest that contraction of capillaries is carried out by endothelial cells, representing an autonomous flow regulatory device. Capillary contraction in exocrine pancreas may be influenced by blood-borne agents, probably by those released in Langerhans islets.

The cochlear glomeruli in the modiolus of the guinea pig.

The cochlear glomeruli were studied in guinea pigs using scanning electron microscopy of vascular corrosion casts and transmission electron microscopy of tissue sections. Two types of coiled vessels forming the cochlear glomeruli were found in the bony wall of the modiolus. First, upper glomeruli were seen situated in the bony wall next to the scala vestibuli; second, lower glomeruli were located in the osseous spiral lamina just above the spiral ganglion. Upper glomeruli gave rise to radiating arterioles which supplied capillaries of the stria the spiral lamina and limbus. Unlike the main supplying arteries, smooth muscle cells were not present in the walls of the arterioles forming the glomeruli and a peculiar layer of lamellar pericytes was found. The arterioles were strikingly longer than their parent vessels and no autonomic nerves were found in close spatial relationship. Hence, these findings indicate that the cochlear glomeruli serve as efficient devices for reducing cochlear blood pressure.

Carbonic anhydrase in the membrane of the endoplasmic reticulum of male rat liver.

We have prepared subcellular fractions of male rat liver homogenate by the method of Lewis and Tata [Lewis, J. A. & Tata, J. R. (1973) J. Cell Sci. 23, 447-459], further purifying the membranes of the microsomal fraction by exposure to 0.01% Triton X-100 and centrifugation. We determined the purity of the fractions with marker enzymes and measured carbonic anhydrase (CA; EC 4.2.1.1) activity in intact and solubilized particulates with 18O exchange between CO2/HCO3- and water. We measured the concentration of CA by titration with a sulfonamide inhibitor, ethoxzolamide, obtaining an average value of 3.8 mumol/mg of microsomal membrane protein. The equilibrium constant for binding ethoxzolamide was 0.49 x 10(-9) M. The Km for CO2 was 1.7 mM and the turnover number was 560,000 sec-1, characterizing this as a membrane-bound, high-activity isozyme of type IV. By electron microscopy of tissue sections after staining with a cobalt precipitation technique, CA was seen in small cytoplasmic vesicles in hepatocytes and in microsomal particles and membranes. There was a sulfonamide-resistant (isozyme type III) and a sulfonamide-sensitive (isozyme type II) CA in the cytosol but none in the rapidly sedimenting endoplasmic reticulum. We conclude that there is no CA normally within the matrix of the cell endoplasmic reticulum but that the CA type III found in the microsome may have been captured from the cytosol during resealing. Thus the adult male rat hepatocyte contains CA type IV in the membrane of the endoplasmic reticulum and CA type II and CA type III in the cytoplasm.

Structure and permeability differ in subepithelial villus and peyer's patch follicle capillaries

Intravenously injected sheep antilaminin immunoglobulin G conjugated with horseradish peroxidase uniformly labeled the basement membrane of mouse villus epithelium but not that of Peyer's patch follicle domes, although laminin is abundant in both. This suggested differences in the permeability of capillaries underlying these epithelia. Therefore, the fine structure of mouse subepithelial villus and follicle capillaries and their permeability to selected macromolecules was compared. Fenestrae, abundant in villus capillaries, were extremely rare in dome capillaries as assessed by electron microscopy of tissue sections and freeze-fracture replicas. Two minutes after IV-injected horseradish peroxidase and 9 minutes after IV-injected hemoglobin, reaction product decorated the pericapillary space of 97% and 95%, respectively, of capillary profiles in the upper half of villi but only 28% and 2%, respectively, of capillary profiles in the upper half of patch domes. Reaction product was intense surrounding most villus capillaries but, when present, was faint in dome capillary adventitia. These results indicate that most subepithelial capillaries in mouse Peyer's patch domes, unlike those in villi, generally lack endothelial fenestrae, and the dome capillary network is less permeable to some macromolecules than that of the villus.

Structure and Permeability Differ in Subepithelial Villus and Peyer’s Patch Follicle Capillaries

Intravenously injected sheep antilaminin immunoglobulin G conjugated with horseradish peroxidase uniformly labeled the basement membrane of mouse villus epithelium but not that of Peyer's patch follicle domes, although laminin is abundant in both. This suggested differences in the permeability of capillaries underlying these epithelia. Therefore, the fine structure of mouse subepithelial villus and follicle capillaries and their permeability to selected macromolecules was compared. Fenestrae, abundant in villus capillaries, were extremely rare in dome capillaries as assessed by electron microscopy of tissue sections and freeze-fracture replicas. Two minutes after IV-injected horseradish peroxidase and 9 minutes after IV-injected hemoglobin, reaction product decorated the pericapillary space of 97% and 95%, respectively, of capillary profiles in the upper half of villi but only 28% and 2%, respectively, of capillary profiles in the upper half of patch domes. Reaction product was intense surrounding most villus capillaries but, when present, was faint in dome capillary adventitia. These results indicate that most subepithelial capillaries in mouse Peyer's patch domes, unlike those in villi, generally lack endothelial fenestrae, and the dome capillary network is less permeable to some macromolecules than that of the villus.

The Immunoglobulin E- and Calcium-Dependent Release of Histamine and Eicosanoids from Human Dispersed Mastocytosis Spleen Cells

The clinical features of systemic mastocytosis have been ascribed to mast cell-dependent mediators, but there have been no studies of their release from isolated cells. We have investigated the release of histamine and eicosanoids from isolated spleen cells obtained from tissue of a mastocytosis patient undergoing therapeutic splenectomy. Dispersed cell preparations contained lymphocytes 65.9%, monocytes/macrophages 22.3%, neutrophils 9.9%, mast cells 1.1%, and eosinophils 0.8%; upon challenge with 0.1-3.0 microM A23187 they released histamine much greater than PGD2 greater than TXB2 greater than LTB4 greater than LTC4 approximately equal to LTD4 greater than LTE4. With immunological activation of passively sensitized cells, histamine and PGD2 release had similar dose-response characteristics, but TXB2, LTC4, LTD4, and LTE4 release differed in reaching maximum at 50 micrograms/ml and declining at 125 micrograms/ml anti-human IgE. Percoll centrifugation separated most of the histamine-containing cells to the middle of the gradient, but they were refractory to release with 0.3 microM A23187 or 50 micrograms/ml anti-IgE. Spontaneous release of histamine from these cells was not abnormally high (1.3%-4.5%). Electron microscopy of tissue sections revealed large numbers of mast cells with empty granules. It is possible that the refractory cells observed are such mast cells where intracellular histamine is no longer granule-associated. Most net histamine and PGD2 release was confined to cells at the bottom of the gradients (1.078-1.09 g/ml), although some release of PGD2 occurred near the top (1.05-1.058 g/ml). There was a significant correlation between the net release of histamine and PGD2 with both immunological (r = 0.92; n = 16) and A23187 (r = 0.97, n = 14) activation. These studies provide evidence for a link between PGD2 and histamine release in mastocytosis spleen cells.

Localization of a nuclear protein during development in the pea seedling

The subcellular localization of a nuclear antigenic protein has been accomplished through the application of immunogold methodology for immunocytochemistry. Protein extracted from internode tissue of pea seedlings was used for the production of a monoclonal antibody that was applied to tissue sections for electron microscopy. As second antibody, goat anti-mouse IgG conjugated with colloidal gold served as label. The 30 kDa nuclear protein was localized in internode tissue only in the chromatin of differentiating and mature companion cells and parenchyma cells of the phloem. By contrast, virtually all of the meristematic, undifferentiated, cells in the apical dome exhibited nuclear labeling. In young, differentiating internode tissue immediately behind the apex nuclear labeling was found in all cell types except pith parenchyma. The changing localization of this 30 kDa protein with the progression of cell differentiation suggests the possibility that this protein may, in some way, be related to regulatory events of development.

Movement and Intracellular Location of Sonchus Yellow Net Virus within Infected Nicotiana edwardsonii

Summary Systemic movement of Sonchus yellow net virus to leaves and roots was first detected by ELISA 24 h after mechanical inoculation. Thereafter, virus levels rose to a maximum 10 days after inoculation; the highest levels were between 4·0 and 7·3 µg/g tissue, in leaves which were not yet fully expanded. Electron microscopy of tissue sections revealed that when the virus content of tissues was greatest, virtually all leaf and root cells were infected. Most of the virions were in the perinuclear space; only a few were scattered in the cytoplasm. Nuclei contained large viroplasms associated with viral nucleocapsids. Between 10 and 20 days after inoculation, levels of virus antigen and viral RNA fell to about 20% of their maximum. By 20 days after inoculation, no more than 10% of cells contained virus particles and almost all the virions were in the cytoplasm. These results suggest that this virus spreads systemically until most or all cells are infected. The plants then undergo a recovery phase during which virus disappears from the nuclei of infected cells and vesiculates into the cytoplasm.

Synapsin I (Protein I), a nerve terminal-specific phosphoprotein. II. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes.

Synapsin I (protein I) is a major neuron-specific endogenous substrate for cAMP-dependent and Ca/calmodulin-dependent protein kinases that is widely distributed in synapses of the central and peripheral nervous system (De Camilli, P., R. Cameron, and P. Greengard, 1983, J. Cell Biol. 96:1337-1354). We have now carried out a detailed analysis of the ultrastructural localization of synapsin I in the synapse. For this purpose we have developed a novel immunocytochemical technique that involves the labeling of isolated synaptosomes immobilized in a thin agarose gel. Special fixation conditions were designed to maximize accessibility of synapsin I to marker molecules. Immunoferritin and immunoperoxidase studies of this preparation indicated that synapsin I is localized in the presynaptic compartment and that it is present in close to 100% of all nerve endings. Immunoferritin labeling also indicated that, inside the nerve ending, synapsin I is specifically associated with the cytoplasmic surface of synaptic vesicles. In agreement with these immunoferritin results, the labeling produced by immunoperoxidase was compatible with a specific association of synapsin I with synaptic vesicle membranes. However, at variance with the very specific distribution of immunoferritin, immunoperoxidase reaction product was also found on other membranes of the terminals, presumably as a result of its diffusion over a short distance from the synaptic vesicles. Anti-synapsin I immunoperoxidase staining of tissue sections for electron microscopy produced an uneven labeling of terminals of the neuropile, in agreement with results of a previous study (Bloom, F. E., T. Ueda, E. Battenberg, and P. Greengard, 1979, Proc. Natl. Acad. Sci. USA. 76:5982-5986). A comparison with results obtained in isolated synapses indicates that the limited labeling of nerve endings in tissue sections results from limited and uneven penetration by marker molecules. The specific association of synapsin 1 with synaptic vesicle membranes in the great majority of nerve terminals suggests a prominent role for this phosphoprotein in the regulation of synaptic vesicle function.

Contacts between receptors and electrophysiologically identified neurones in the retina of the larval tiger salamander

1. Following the intracellular recording of bipolar and horizontal cell responses, each unit was injected with horseradish peroxidase and a histochemical staining used to identify it at the level of the light and electron microscopes.2. Centre-depolarizing bipolar cells made contact with rods and cones at basal and ribbon junctions, the latter being fewer. Centre-hyperpolarizing bipolar cells made the same types of contacts with the receptors, but ribbon junctions predominated.3. It appears, therefore, that there is no fixed relationship between the sign of synaptic transmission from receptors to bipolar cells and the junctional features revealed by present methods for electron microscopy of tissue sections. Accordingly, the reason for the existence of more than one type of contact between receptors and bipolar cells remains to be determined.4. Light microscopy of the peroxidase-injected horizontal cells gave further support to the notion that type B responses are recorded from the cell body, and type A responses from the axon terminal, of a single type of horizontal cell. Electron microscopy showed that the processes (dendrites) originating from the cell body make ribbon and distal junctions with rods and cones, just as shown before for the axon terminals.5. As a result of these observations, it is possible to exclude one of two alternative hypotheses previously proposed to account for the properties of the surround responses recorded from the horizontal cell bodies.

Quantitation of damage to the alveolar epithelium by means of type 2 cell proliferation.

The purpose of this study was to determine whether the amount of alveolar epithelial tissue damaged during exposure of NO2 could be quantified by measuring the proliferative response to Type 2 cells. To accomplishe this, we used tissues from previously published experiments in which rats had been exposed to NO2 and the proliferative response to Type 2 cells had been measured during a 5-day period. The proportion of alveolar epithelium damaged was determined by stereologic examination with electron microscopy of tissue sections from those rats exposed to NO2 for 24 hours. These values were then compared with the total proliferative response to Type 2 cells for the 5 days of exposure. The study demonstrated that increasing tissue damage is assocaited with a greater proliferative response to Type 2 cells. The high degree of correlation (r = 0.93) indicates that the proliferative response of Type 2 cells can be used as an indirect means to quantify acute damage to the alveolar epithelium.

Mitochondrial alterations associated with avian reticuloendotheliosis virus (strain T) pathogenicity

This report indicates that reticuloendotheliosis virus (REV) induces morphological, physical and enzymatic alterations in chick target organ mitochondria. Succinoxidase activity of infected mitochondria is decreased at 5 days post-REV-infection, but ADP:O ratios are unchanged. Calcium accumulation is decreased from 100 nMoles/mg of protein in controls to 50 nMoles/mg of protein in infected mitochondria. Mitochondria from infected tissue exhibit a buoyant density of 1.193 gm/cc, significantly greater than the buoyant density of 1.167 gm/cc for control mitochondria. Mitochondria isolated from REV-infected livers were effective in transmitting reticuloendotheliosis when inoculated intraperitoneally into day old chicks. Electron microscopy of tissue sections indicates that infected cell mitochondria are enlarged with a less dense matrix relative to controls. Purified mitochondria from infected livers contained up to 16 particles having the size and shape of unenveloped(naked) C-type RNA tumor virus.

Comparação electrono-microscópica dos tecidos de plantas infetadas por diferentes estirpes do vírus Y da batata que ocorrem no Estado de São Paulo

Exames, ao microscópio electrônico, de secções ultrafinas de tecidos, principalmente foliar, de plantas de diversas espécies, infetadas por uma das 17 estirpes do vírus Y da batata que ocorrem no Estado de São Paulo, mostraram: a)Ocorrência de inclusões citoplasmáticas, lamelares, do tipo descrito por Edwardson, e ocasional presença¹, no citoplasma, de partículas similares àquelas encontradas in vitroe em tecidos de plantas infetadas por qualquer uma das estirpes estudadas; b)aparecimento esporádico de agregados de partículas filamentosas e grossas (ca. 50-60 m¼ de diâmetro), no citoplasma de tecidos de plantas infetadas pelas estirpes Yc e YeI; c)ocorrência sistemática de inclusões citoplasmáticas, constituídas por membranas, empilhadas ou dispostas concêntricamente, consideradas como estrutura de Golgi hipertrofiada, em tecidos de plantas infetadas pelas estirpes Yp e Ynt.

THE ROLE OF FIXATION IN ELECTRON STAINING

SYNOPSISObservations made on the influence of the fixative on staining of thin tissue sections for electron microscopy permit the following statements:After acrolein fixation it is possible to demonstrate basic proteins of the chromatin by silver impregnation. The staining appears to be mediated by the product of the reaction between the fixative and the imidazole groups of histidine.In the absence of reduced osmium (fixation by formol or acrolein or preliminary oxidation of sections of osmium‐fixed tissue) conventional lead methods result in a selective staining of the ribonucleoproteins. Under the same conditions, a selective staining of the plasma membrane (or an associated substance) can be achieved by phosphotungstic acid.After formol or acrolein fixation, uranyl acetate (at pH 4.5 to 5) stains the desoxyribonucleoproteins more intensely than the ribonucleoproteins. The strongest differentiation between the nucleoproteins is found to occur after formol‐osmium fixation.

A modified procedure for lead staining of thin sections.

Meta l impregnat ion or ta in , of tissue sections for electron microscopy has become the accepted practice in recent years. Salts of metals of h igh atomic weight such as u ran ium, chromium, thor ium, lead, or tungsten, among many tested, have been found suitable (6, 1). Lead , as prepar ted by Watson (6), is now widely used, hu t this solution is extremely unstable in air and becomes covered by a meta l l ic-appear film which imparts to the s ta ined section a well known deposit of e lectron-opaque particles and crystals, reducing considerably the percentage of clean areas suitable for micrography (Fig. l) Several procedures and devices have been described to lessen these contaminat ions. Peachey (5), using lead hydroxide, or subacetate, found tha t if the solution is kept in a syringe with the protect cap filled with sodium to absorb the CO2 of the atmosphere, con tamina t ion is reduced. More recently, a fairly unwieldy appara tus has been suggested for the same purpose (4). Tests performed in our laboratory in which s t a in ing was a t tempted in a chamber under cont inuous ni t rogen flow and in the presence of a ba r ium chloride t rap were not completely successful, which is to say tha t con tamina t ion cont inued to be a problem. A n u m b e r of o ther var iat ions have been recommended. Lever (2), e.g., described a method for prepar a s t a in ing solution by adding potassium to a lead solution. To dissolve some of the occasionally formed crystals of contamina t ion the sections are rinsed afterwards for a few seconds in a weak potassium solution; this step is very critical because the ta in ing is also weakened by the alkali and uni form staining is difficult to achieve. These various shortcomings of current ly avai lable methods have st imulated us to search for a lead salt tha t would not be affected by the components of the atmosphere. Among several heavy metal salts tested, the above-ment ioned lead hydroxide of Watson seemed to be the most effective in ta in . Exper iments with the commercial ly available product showed that this salt would give no impregna t ion when dissolved in water, or in Institute. Dr. Millonig's present address is Biological

Double Staining of Tissue Sections for Electron Microscopy with Heavy Metals (KMnO<sub>4</sub> and Pb (CH<sub>3</sub> COO)<sub>2</sub> )

Double Staining of Tissue Sections for Electron Microscopy with Heavy Metals (KMnO<sub>4</sub> and Pb (CH<sub>3</sub> COO)<sub>2</sub> )