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The epithelium of the rabbit vagina: a microtopographical study by light, transmission and scanning electron microscopy.

In order to obtain a more precise microtopographical surface map of the epithelium of the rabbit vaginal mucosa, investigations by light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) have been systematically correlated. The epithelium was examined from the "portio vaginalis cervicis uteri" down to the "vestibulum". This study shows that the upper 2/3 of the vagina--which, in this species, is very long, measuring 13-14 cm--is lined by a single epithelial layer of tall ciliated and microvillous cells closely resembling the endocervical epithelium with which it is continuous. Moreover, these ciliated and microvillous cells also cover mucosal infoldings in the upper part of the vagina, especially those on the ectocervix and in the fornices, and scattered vaginal crypts. In particular, the ciliated elements decrease in number below the fornices, so that in large areas of the middle part of the vagina only microvillous cells are recognizable. Prior to the squamo-columnar junction, however, the ciliated cells increase again. This study also reveals that in the rabbit the squamo-columnar junction is located at the level of the pubic symphysis and that a squamous pluristratified epithelium covers only the lower 1/3 of the inner surface of the vaginal wall. In the estrous, i.e., precoital rabbit, the microvillous cells show little sign of secretion, whereas after mating they exhibit remarkable secretory features. These seem to increase progressively with postcoital stages (5 h, 24 h and 10 days) in the form of extensive mucification. These secretions often come in contact with spermatozoa retained in the mucosal infoldings and crypts, and are similar to those occurring in the endometrium, where they clearly depend upon progesterone activity. These epithelial features, different from those of other mammals, including humans, suggest that the greater part of the rabbit vagina accomplishes functions other than serving for copulation and as a fetal passageway. The present findings support the view that the rabbit vagina also plays a role as a reservoir of spermatozoa and in maintaining their viability, like the endometrium and endocervix.

Animals↗

Choosing objective lenses: the importance of numerical aperture and magnification in digital optical microscopy.

Microscopic images are characterized by a number of microscope-specific parameters--numerical aperture (NA), magnification (M), and resolution (R)--and by parameters that also depend on the specimen--for example, contrast, signal-to-noise ratio, dynamic range, and integration time. In this article, issues associated with the microscope-specific parameters NA, M, and R are discussed with respect to both widefield and laser scanning confocal microscopies. Although most of the discussion points apply to optical microscopy in general, the main application considered is fluorescence microscopy.

Lenses↗

Scanning electron microscopy of elastic system fibers in the articular disc of the rat mandibular joint.

Elastic system fibers (ESFs), i.e., microfibrils (putative oxytalan fibers), elaunin and elastic (true elastin) fibers, in the rat mandibular joint were studied mainly using scanning electron microscopy (SEM) and light microscopy (LM) with the aid of image processing. The present quantitative analysis using LM showed that the articular disc and capsule, which are the sites that receive physical compressive force during mastication, contained more ESFs than the articular cartilage of the mandibular joint. In addition, oxytalan fibers were the principal ESFs in all the articular components (capsule, articular disc, supraossous layer of articular surfaces and articular cartilage). Subsequently, ESFs in the articular disc, which contained more thick ESFs, were closely examined by SEM using both collagen- and elastin-digestion methods. SEM showed networks of microfibrils beneath the articular surfaces (superior and inferior layers) in the thin central portion of the articular disc; the principal microfibrils ran at nearly right angles to the collagen fibers. The microfibrils were cemented with amorphous elastin, thickened and shifted towards interconnecting oblique fibers and many main ESF trunks, which were oriented in the direction of the layered wavy collagen fibers and parallel to the direction of applied force, to sustain the mechanical force. From the superior and inferior layers, the main ESFs shifted towards the middle portion of the disc, transitional zone (synovial osteochondral junction) and the other articular components, showing no specific directivity. Transmission mission electron microscopy revealed that the thick main ESFs in the elastic network were elaunin fibers. The present study indicated that ESFs unite, branch and therefore construct an extensive and complicated protective stretchable network, which is interposed with the less tensible collagen network in the mandibular articular disc.

Animals↗

Two-versus one photon excitation laser scanning microscopy: critical importance of excitation wavelength.

It is often anticipated that two-photon excitation (TPE) laser scanning microscopy should improve cell survival and tissue penetration relative to conventional one-photon excitation (OPE) confocal scanning laser microscopy (CLSM). However few studies have directly compared live cell imaging using one- vs two-photon laser scanning microscopy. We have used calcein-loaded in situ chondrocytes within cartilage as a model for quantitatively comparing these techniques. TPE reduced photo-bleaching and improved cell viability compared to OPE. Using improved detection sensitivity coupled with increased tissue penetration of the near infra-red TPE laser, it was possible to capture images deeper within the cartilage. However, the advantages of TPE vs OPE were strongly dependent on excitation wavelength. We conclude that optimising TPE conditions is essential for realizing the full benefits of this approach.

Animals↗

The amelocemental junction in young premolar teeth. A replica study by scanning electron microscopy.

To investigate the validity of traditional data on the frequency of various relationships of root cementum to coronal enamel at the amelocemental junction, the relationship in 50 young premolar teeth extracted on orthodontic indications was documented, using replica techniques for scanning electron microscopy (SEM), combined with polarization microscopy of thin ground sections (< 40 microns). Of 200 surfaces, 30 were excluded because of damage during specimen preparation. Replicas of the other 170 surfaces were photographed at 20x and 80x in SEM and classified as having an edge-to-edge relationship of enamel to cementum in 76% and an overlap of cementum onto the cervical enamel in 14%. What appeared to be a gap between enamel and cementum at low magnification (10%) was seen at high magnification to be a ditch or trough in the cementum: no gaps with exposed dentin were observed in SEM. Polarization microscopy of ground sections of the original specimens confirmed the SEM classification. In this material, the edge-to-edge relationship of enamel to cementum predominated: overlapping of cementum onto the enamel was less prevalent than previously reported. The methods applied in this study circumvented some major sources of artefact formation due to shrinkage during specimen preparation, which may previously have caused misinterpretation of 'gap' relationships.

Bicuspid↗

Acellular scanning electron microscopy of spicular renal amyloidosis.

Two human renal biopsies containing glomerular amyloid deposits organized into spicular formations (spicular amyloid) were studied by scanning electron microscopy following removal of the cellular components (acellular SEM). Following SEM studies, portions of the same acellular tissue were embedded in paraffin and plastic for light microscopy and transmission electron microscopy, respectively. Spicular deposits by acellular SEM appear as tapering conical formations interconnected by a delicate branching network of fibrils, which imparts a higher degree of organization than previously appreciated by two-dimensional LM and TEM. Silver stains of paraffin- and plastic-embedded acellular tissue showed persistence of argyrophilia in spicular deposits, while acellular TEM showed that the spicules appeared comprised "purely" of amyloid fibrils without visible contaminating material. We conclude that the argyrophilia of spicular amyloid is an inherent feature of the parallel organization of fibrils rather than a result of incorporation of glomerular basement membrane or cell components and that spicular amyloid deposits have a higher degree of organization than is apparent by two-dimensional studies.

Amyloid↗

Correlative study of adult respiratory distress syndrome by light, scanning, and transmission electron microscopy.

The sequential pulmonary changes occurring in the evolution of adult respiratory distress syndrome (ARDS) were studied in 35 patients by correlative light, scanning, and transmission electron microscopy. The causes of ARDS were diverse, the major ones being sepsis or aspiration. Patient survival ranged from 3 to 51 days. The acute stage in patients surviving 2 to 7 days was characterized by an exudative reaction with a predominance of hyaline membranes. This acute stage merged with and was replaced by a subacute reparative stage in patients surviving 7 to 14 days, which in turn was replaced by a chronic fibroproliferative stage complicated by interstitial pulmonary fibrosis and a deranged acinar architecture. Correlation of findings by scanning electron microscopy with those by light and transmission electron microscopy provided an added dimension to understanding of the evolving stages of ARDS and demonstrated that type 2 pneumocytes contributed to the fibroproliferative stage through organization of hyaline membranes and re-epithelialization of alveoli.

Adolescent↗

Identification of focal viral infections by confocal microscopy for subsequent ultrastructural analysis.

A correlative microscopy method for the ultrastructural analysis of focal viral tissue infections is presented. Using a confocal scanning laser microscope, foci of infection are identified in tissue sections prior to embedment; a variety of techniques can be employed for viral detection, including staining with standard histochemical reagents and fluorescently labeled antibodies. Areas of infection identified using confocal microscopy are excised from the tissue sections, embedded, and examined by transmission electron microscopy. Applications of this technique in both diagnostic and basic research settings are described.

Animals↗

Fixation of platelets for scanning and transmission electron microscopy.

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.

Blood Platelets↗

Epoxy-slide embedment of cytochemically stained tissues and cultured cells for light and electron microscopy.

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cast-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.

Animals↗

Quantitative analysis of immunofluorescent signals for dystrophin, beta-dystroglycan and myosin skeletal muscle by epifluorescence microscopy.

Quantitative analysis of signal intensities in immunostained sections has been performed in only a few studies owing to difficulties with quantifying amounts of antigen present. We determined correlations between fluorescent signal intensities and amounts of antigen in muscle cryosections by altering section thickness from 4 to 10 microm. Fluorescent signals of dystrophin. beta-dystroglycan and myosin were detected with monoclonal and/or polyclonal primary antibodies using routine procedures. Confocal laser microscopy demonstrated that these signals were distributed uniformly along the z-axis suggesting that the antibodies permeated well through the sections. Epifluorescence microscopy with microfluorometry demonstrated a positive correlation between the optical density of signals and section thickness. These findings suggest that immunofluorescent signals can be quantitated by epifluorescence microscopy.

Adult↗

Study of lichens with different state of hydration by the combination of low temperature scanning electron and confocal laser scanning microscopies.

The use of techniques such as low temperature scanning electron microscopy (LTSEM) and confocal laser scanning microscopy (CLSM) allows the study of lichen thalli in different states of hydration and also near the natural state. The spatial organization of desiccated thalli, with reduced, very compact algal layers, is different from that of hydrated ones. Sometimes, the observation with transmission electron microscopy (TEM) of photobiont pyrenoids from desiccated thalli reports pyrenoids with a central part of a weak stained matrix lacking pyrenoglobuli, named "empty zones". "Empty zones" are not distinguishable with LTSEM and do not present immunolabelling with rubisco antibody in TEM. These zones could be originated by an expansion process during rehydration produced in chemical fixation.

Cold Temperature↗

Scanning electron microscopy of mast cell degranulation.

Scanning and transmission electron microscopy were used to study the morphologic changes occurring in isolated mast cells after stimulation by compound 48/80. Two approaches were used to obtain cells for scanning electron microscopy. Cells incubated in suspension were collected on Millipore filters and air-dried from isopropyl alcohol. Other cells were allowed to attach to cover slips, stimulated, fix in situ, and dried from Freon at its critical point. Resting mast cells prepared by the latter method were covered by a tracery of shelflike ridges, whereas cells prepared by the first method had no ridges but presented a bumpy surface disclosing the underlying granules. Stimulation by compound 48/80 caused marked changes in the surface configuration of the cells, with loss of the regular pattern of ridges, extrusion of granules, and the appearance of openings leading to the interior of the cell. Correlation with transmission electron microscopy indicates that these openings lead to channels containing granules and lined by an intact membrane. These openings give extracellular cations access to granules within the confines of the cell, where the cations may exchange for histamine.

Animals↗

Scanning electron, light, and transmission electron microscopy of intestine of gnotobiotic calf.

Adjacent areas of upper, middle, and lower parts of the small intestine and spiral colon from a 48-hour-old gnotobiotic calf were compared by scanning electron microscopy (SEM), light microscopy (LM), and transmission electron microscopy (TEM). As visualized by all 3 methods, small intestinal histologic features, except for apical location of villous epithelial cell nuclei in sections of upper and middle parts of small intestine, were similar to those described for other species. The colonic surface visualized by SEM was composed of flattened ridges separated by furrows into which opened the crypts of Lieberkühn. The epithelial surfaces of the ridges and the furrows had an extensive microvillous covering and scattered goblet cell openings.

Animals↗

Test of canine sperm function in vitro using primary homologous oocytes with fluorescence and confocal microscopy.

In vitro capacitated canine spermatozoa can interact with both mature and immature homologous oocytes in vitro, but it is difficult to determine whether spermatozoa have actually penetrated the oocyte or have simply bound to the zona pellucida. The aim of the first part of this study was to determine accurately the location of spermatozoa in relation to the oocyte by comparing observations made using confocal and fluorescence microscopy. The fluorescence technique had a sensitivity of 66%, and correctly identified 98% of the spermatozoa that had penetrated or bound oocytes and could discriminate between penetrated and bound spermatozoa with a sensitivity and specificity of 97% and 100%, respectively. This finding demonstrates that evaluation of the assay by fluorescence microscopy is reliable for detecting the presence and location of spermatozoa in relation to homologous oocytes. The aim of the second part of the study was to attempt to simplify the assay using fluorescence microscopy and immature oocytes. No significant difference was found between sperm interaction with mature and immature oocytes, which demonstrates that culturing oocytes before the assay has no benefit, and that the assay can be performed quickly and easily using non-cultured oocytes to provide rapid evidence of the fertilizing potential of spermatozoa.

Animals↗

The influence of the buffer on maintenance of tissue lipid in specimens for scanning electron microscopy.

Preparations of tissues and cells for scanning electron microscopy (SEM) fixed with glutaraldehyde (G CHO) buffered in either Na-cacodylate or Piperazine-N-N' bis (20-ethanol sulfonic acid) (PIPES) differ in their morphology. The influence of the nature of these buffers in the tissue fixing and washing solutions on appearance in scanning electron microscopy is described. Biochemical determinations of lipid retention were performed on frog and chick embryos fixed for 24 hours with 3% G CHO in either 0.03 and 0.1 M PIPES (ph. 7.3) or 0.1 M Na-cacodylate (ph. 7.3). Embryonic tissue was chosen for its relatively high lipid content and delicacy which may be expected to enhance the sensitivity at which buffer effects become apparent. The comparable small and uniform sizes of the embryos minimize differences in fixation quality due to penetration. Lipid, recovered from homogenized tissue after treatment with chloroform/methanol/water (1:2.1:1, v/v) was considered as retained. The extraction results, which showed a significant reduction of lipid losses when PIPES buffer was used, are taken to account for the morphological differences observed in SEM and transmission electron microscopy (TEM).

Animals↗

Salmonella 3, 10:r: toxicity in rabbit ileum and liver by light and electron microscopy.

Salmonella 3, 10:r:- (a monophasic variety of otherwise diphasic serotypes such as S. weltevreden and S. simi) Cell_free filtrate, when introduded into rabbit ligated ileal loops causes fluid exsorption, as studied 18-hr after treatment. Light microscopic histology of treated ileum shows denudation of the columnar epithelium at several places, thereby allowing the passage of the toxic principle into circulation. An important target organ, liver shows extensive centrilobular necrosis, as observed by light microscopy. Transmission electron microscopy of ileum reveals opening of membrane junctions between the adjacent cells of epithelial lining of the treated ileum at places, and focal devitalization including formation of intra-cellular membranous inclusinos. Electron microscopy of liver shows extensive damage and swelling of cytoplasmic membranes. However, the areas of darkly staining lamellae of granulated endoplasmic reticulum are also seen in stacks as will as dispersed. These studies stress that Salmonella toxic substances can cause extensive damage to intestine and liver both.

Animals↗

Electron microscopy of bone response to titanium cylindrical screw-type endosseous dental implants.

This study investigated the undecalcified bone and enveloping tissues supporting commercially pure titanium one-stage and two-stage endosseous dental implants placed into the mandibles of adult mongrel dogs. Correlative light microscopy, transmission electron microscopy, and high-voltage transmission electron microscopy demonstrated a dynamic bone interface to the implants. Mineralized tissue was routinely observed within 20 to 50 nm from the implant interface, separated from the implant interface only by an electron-dense deposit. The densely mineralized collagen fiber matrix was oriented parallel to the implant interface, as were osteocytes that were found close to the interface. Osteocytic projections progressed through canaliculi, often directly to the implant interface. Unmineralized areas that appeared to have the potential for mineralization were also identified.

Alveolar Process↗