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PubMed1987

Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature.

A survey of methods combining light microscopy and scanning electron microscopy is presented. A simple correlation is made when two preparations from adjacent parts of one specimen are investigated in two different microscopes. A more sophisticated method is the consecutive investigation of one specimen with two microscopes. A major problem in this method is the relocation of the area of interest. Several authors have presented solutions for this problem. It is preferable when one preparation is investigated in only one instrument, combining the two microscopical (LM and SEM) techniques, thus making relocation redundant.

Microscopy↗
PubMedDate not supplied

Three-dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy.

The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze-fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4-64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing-over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high-resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.

Animals↗
PubMed1985

A new approach for studying semithin sections of human pathological material: intermicroscopic correlation between light microscopy and scanning electron microscopy.

In order to obtain useful and complete information on the study of pathological material, we observed by scanning electron microscopy (SEM) the same semithin sections observed by light microscopy (LM). For this purpose, the specimen must have, at the same time, chromatic and electron dense characteristics. We thus developed different specimen preparation methods, subjecting the semithin sections to specific polychromatic staining with high atomic number (Z) elements, to monochromatic staining followed by routine contrasting with uranyl acetate and lead citrate, and to specific cytochemical and immunocytochemical procedures. The specimens were examined in sequence by LM, by SEM equipped with secondary electron, backscattered electron, transmitted electron detectors and by scanning transmission electron microscopy (S(T)EM).

Actins↗
PubMed2000

Complementary visualization of mitotic barley chromatin by field-emission scanning electron microscopy and scanning force microscopy.

The surface structure of mitotic barley chromatin was studied by field-emission scanning electron microscopy (FESEM) and scanning force microscopy (SFM). Different stages of the cell cycle were accessible after a cell suspension was dropped onto a glass surface, chemical fixed, and critically point dried. Imaging was carried out with metal-coated specimen or uncoated specimen (only for SFM). The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data. The experimentally determined volume of the residue chromatin during mitosis was within the range of 65-85 microm(3). A comparison with the theoretically calculated volume indicated a contribution of about 40% of internal cavities. Decondensation of chromosomes by proteinase K led to a drastic decrease in the chromosome volume, and a 3-D netlike architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious. Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing. We imaged intact chromosomes in liquid by SFM without any intermediate drying step. A granular surface was obvious but with an appreciably lower resolution. Under similar imaging conditions proteinase K-treated chromosomes exhibited low topographic contrast but were susceptible to plastic deformations.

Chromatin↗
PubMed1979

Arrays of hemispheric surface projections on Chlamydia psittaci and Chlamydia trachomatis observed by scanning electron microscopy.

Scanning microscopy of two strains of Chlamydia psittaci and four strains of Chlamydia trachomatis representative of the wide diversity in origin and behavior of members of the genus revealed patches of regular arrays of hemispheric projections on the surfaces of elementary bodies of all six strains. These distinctive and perhaps unique surface structure are probably present in all populations of chlamydiae.

Cell Membrane↗
PubMed1980

[Surface ultrastructure of the human uterine exfoliated endometrial cells--comparative studies of the same cell by light microscopy and scanning electron microscopy (author's transl)].

The preparations for observations of the same cells by light microscopy (LM) and scanning electron microscopy (SEM) were examined and the exfoliated endometrial cells of the uterus obtained by the newly introduced brush and cannula was evaluated. 1) The degree of shrinkage of the area was not significant between the air drying method and critical point drying method. In the air drying group, adhesion, entanglement and disappearance of microvilli (mv) and the adhesion of cilia, and the collapse of cells were noted morphologically. 2) Papanicolaou stain had no effect on the degree of shrinkage of the area and the shape of cilia or mv. 3) When the samples were transferred from ethanol to critical point drying, without immersion in isoamyl acetate, artifacts appeared in small granulated form. 4) The menopausal phase was divided into two groups which showed a shift of the maturation Index (MI) to center and had a slender mv and the other group which showed a shift to the left and granular mv. The nether surface of the cells showed a wrinkled and irregular undulations and did not show cilia or mv. 5) In adenomatous hyperplasia, the ratio of ciliated cell to no ciliated cell was four to one. It was two to one in cystic glandular hyperplasia. 6) The dense and short mv were observed in well-differentiated adenocarcinoma and poorly-differentiated adenocarcinoma showed neither mv nor cilia. Morphologic changes of mv and cilia in carcinomatous change of uterine endometrium commenced with a decrease in cilia, shifted to the shortening and disappearance disappearance of mv.

Adenocarcinoma↗
PubMed1975

The dendritic cells of the guinea pig popliteal lymph node: identification and classification of cells observed by scanning electron microscopy.

Scanning electrom microscopy of guinea pig popliteal lymph nodes revealed that 3 types of cells adhered to glass and possessed extensive cytoplasmic processes. Two of these types were considered to represent extremes of a macrophage population on the basis of possession of surface receptors for cytophilic Abs, complement and immunoglobulin, as well as their distribution, quantity and morphology. The third cell type was characterized by possession of large numbers of filamentous dendritic processes. On the basis of morphology, quantity, distribution and the absence of receptors, this cell was considered to represent a cell type clearly distinct from the other cells observed in the normal lymph node.

Animals↗
PubMed1997

Confocal light microscopy and scanning electron microscopy of the human eye lens.

The potential of confocal light microscopy (CLM) for in vivo observation of pathology in the anterior pole of the eye lenses was evaluated by performing an in vitro study of human lenses comparing this type of microscopy with scanning electron microscopy (SEM). In vitro CLM showed high resolution images of the epithelium which would enable early detection of pathology and easily allows cell counting and estimating cell size. Superficial lens fibres are well visualised and low and high frequency bands as well as vacuolar elements were easily detected. SEM observations fully supported the CLM observations. This study shows that CLM has the potential to become a useful tool for detecting lens changes, after suitable adaptation for clinical use.

Adult↗
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↗
PubMed2001

Specific surface area of snow samples determined by CH4 adsorption at 77 K and estimated by optical microscopy and scanning electron microscopy.

Snow is a divided medium that can adsorb atmospheric trace gases. Evaluating the impact of the snow cover on atmospheric chemistry therefore requires the knowledge of the specific surface area (SSA) of snow. This paper compares the results of three methods used to measure or estimate the SSA of four snow samples: CH4 adsorption at 77 K, optical microscopy (OM), and scanning electron microscopy (SEM, used only on two samples). Within error bars, CH4 adsorption and OM yield similar results on three of the four snow samples. Values for the 4th sample are within a factor of 2. For both samples where CH4 adsorption, OM, and SEM are used, all three methods yield similar results, but CH4 adsorption always has a better accuracy and a much better precision. Thus, despite its ease of use, estimates from OM images are often not accurate enough to monitor the evolution of snow SSA. The main sources of error in the OM method are the difficulty to determine snow crystal thicknesses and to take into account the topography of the snow crystal surface. The combination of CH4 adsorption and OM or SEM can provide useful information on the evolution of both the SSA and the shape of snow crystals. This will be useful to evaluate the respective contributions of adsorption/desorption and sublimation/condensation processes to the impact of the snow cover on atmospheric chemistry.

Adsorption↗
PubMed2001

Retinopathy of Great Pyrenees dogs: fluorescein angiography, light microscopy and transmitting and scanning electron microscopy.

OBJECTIVE: Investigation of the pathogenesis of Great Pyrenees retinopathy. ANIMALS: One male and two female puppies of parents who were affected with Great Pyrenees retinopathy and one 4-year-old affected adult male Great Pyrenees dog. PROCEDURE: The puppies were examined daily from 7 weeks of age by indirect ophthalmoscopy and their fundi were photographed until the lesions were static. Fluorescein angiography was completed at 7 weeks of age, within 24 h of detection of ophthalmoscopic lesions, and then weekly. The eyes of a 4-year-old and two 20-week-old puppies were examined with light microscopy, and transmitting and scanning electron microscopy. RESULTS: Blocked choroidal fluorescence was detected at 7 weeks of age. The blocked fluorescence enlarged, when the characteristic serous retinal detachments developed at 11 weeks of age. The detachments enlarged in size and number until the puppies were approximately 20 weeks old. Fluorescein pooling confirmed the presence of transient retinal pigment epithelial detachments. Leakage of dye into serous retinal detachments was not detected in this study. Light microscopy and transmitting and scanning electron microscopy confirmed the presence of multifocal serous retinal detachments with focal retinal degeneration that extended to the inner nuclear layer in a 4-year-old dog. The retinal detachments were accompanied by hypertrophy, hyperplasia, increased pigmentation, and vacuolation of the retinal pigment epithelium. CONCLUSIONS AND CLINICAL RELEVANCE: Great Pyrenees retinopathy is manifested by multifocal serous retinal and retinal pigment epithelial detachments. These detachments are similar to those noted with central serous retinopathy of humans. The sudden development of multifocal retinal and retinal pigment epithelial detachments, and the serous nature of these detachments, supports a theory that they develop secondary to focal secretion and absorption defects in retinal pigment epithelial cells. Given the age of the puppies when the blocked choroidal fluorescence was noted and maturation of the dog retina at 8 weeks postpartum, this retinopathy is considered to be a retinal pigment epithelial dysplasia. This unique inherited retinopathy offers an opportunity to study retinal pigment epithelial secretion.

Animals↗
PubMed1983

Microcirculation of the pancreas: a correlative study of intravital microscopy with scanning electron microscopy of vascular corrosion casts.

The microcirculation of the pancreas in anesthetized rats was examined by intravital microscopy. Scanning electron microscopic observation of vascular corrosion casts of the same parts of the pancreas as viewed light microscopically in vivo, allowed detailed analysis of microcirculation in particular vessels. Blood vessels with flowing blood cells were clearly observed under incident ultraviolet illumination following systemic injection of an intravascular fluorescent tracer. The islets of Langerhans were easily identified by their brighter appearance than the exocrine portions under incident ultraviolet illumination. Blood leaving the islets passed through the insulo-acinar portal vessels into the capillary network around the acini. The periductular plexus received blood from the periacinar capillary network via either venules or capillaries as well as directly from the inter- or intralobular arteries. The periductular plexus also received blood from the islets located close to the ductules. The results obtained show blood in the pancreas flowing in a manner in which it carries insular secretions in high concentrations through short and direct vascular routes from the islets to the pancreatic exocrine tissues, including their duct system.

Animals↗
PubMedDate not supplied

Giant cell carcinoma of the lung: comparative studies of the same cancer cells by light microscopy and scanning electron microscopy.

Surface ultrastructures of giant cell carcinoma cells were studied utilizing a technique developed by the authors which permits observation of the same cells by both light and scanning electron microscopy. Although some general similarity was shown to exist between giant cell carcinoma and squamous cell carcinoma cells, the following distinguishing features of giant cell carcinoma cells were demonstrated: (1) absence of keratinization, (2) full, bulging cellular edges and (3) presence of deep invaginations or small hollows. The features which distinguished giant cell carcinoma from adenocarcinoma cells were: (1) presence of many flattened cells, (2) folded cell surface, (3) absence of tight clusters of cancer cells and (4) few microvilli. Furthermore, the characteristic granularity observed in small cell carcinoma was not observed in giant cell carcinoma cells. Characteristically, cells of giant cell carcinoma are extremely large and flattened, engulf numerous leukocytes and have a bulging cellular edge.

Biopsy, Needle↗
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↗
PubMed1994

Human cumulus cell complexes studied in vitro by light microscopy and scanning electron microscopy.

Various researchers describe the morphology of cumulus cells (CC) in vitro, but few have investigated their behaviour on plastic. Knowledge concerning the behaviour of human CC could be useful in improving the success of in vitro fertilisation procedures. This study aimed to describe the morphology and behaviour of CC in vitro and to investigate movement on a collagen-coated substrate. Following collection some cumulus were mechanically dissected from those surrounding the oocyte. Cumulus aggregates were cultured over 24 h using Earle's medium supplemented with 8% albumin. Substrata were plastic coverslips coated with collagens I, IV, or mixed collagens. Cumulus cultured over corresponding time periods on uncoated coverslips served as controls. Specimens were fixed and prepared for scanning electron microscopy. Over 24 h the controls began exhibiting the morphological features associated with cell movement: cell surface protrusions changed from blebs to microridges, lamellipodia and leading lamellae; cell shape altered from rounded and upright, to flattened. Extracellular matrix (ECM) transformed from a thick, sheet-like substance to a thin, fibrous material. By 24 h, cells contacting ECM remained rounded showing few features of movement. Collagens enhanced attachment of CC as a monolayer on the substrate. Cell morphology varied according to the collagen type used. On mixed collagens, cells attached rapidly, appearing to be predominantly non-motile. On collagen type I there was less attachment of cells but increased motility. On collagen type IV there was decreased attachment and the cells remained spherical. In conclusion, collagens enhance the settling of cumulus cells on a plastic substrate and the cells exhibit some specificity in attaching to collagens.

Adult↗
PubMed2006

A history of scanning electron microscopy developments: towards "wet-STEM" imaging.

A recently developed imaging mode called "wet-STEM" and new developments in environmental scanning electron microscopy (ESEM) allows the observation of nano-objects suspended in a liquid phase, with a few manometers resolution and a good signal to noise ratio. The idea behind this technique is simply to perform STEM-in-SEM, that is SEM in transmission mode, in an environmental SEM. The purpose of the present contribution is to highlight the main advances that contributed to development of the wet-STEM technique. Although simple in principle, the wet-STEM imaging mode would have been limited before high brightness electron sources became available, and needed some progresses and improvements in ESEM. This new technique extends the scope of SEM as a high-resolution microscope, relatively cheap and widely available imaging tool, for a wider variety of samples.

Equipment Design↗
PubMed1981

Scanning electron microscopy of cyclophosphamide-induced hyperplasia of the rat urinary bladder.

Urinary bladder damage caused by cyclophosphamide in male F344 rats was studied by light microscopy, scanning electron microscopy, and autoradiography. Cyclophosphamide was injected intraperitoneally at doses of 50, 75, 100, 150, and 200 mg. per kg. of body weight, and rats were killed at several intervals following treatment. Extensive necrosis of the bladder was followed by regenerative hyperplasia of the epithelium. A dose response was evident in the number and size of lesions induced and the time of regeneration and repair. Scanning electron microscopy disclosed pleomorphic microvilli on the luminal surface of cells during the early phases of hyperplasia. The greatest number of cells with pleomorphic microvilli occurred 2, 5 and 7 to 8 days following injection of 75, 100, and 200 mg. per kg. of cyclophosphamide, respectively. The hyperplastic lesions also had cells with short, uniform microvilli and ropy or leafy microridges on their surfaces. Autoradiographs of the urinary bladders showed marked labeling of epithelial cells in the early phase of hyperplasia. Of the few rats surviving beyond 1 year after a single injection, none had bladder lesions at any of the dose levels. The changes in the epithelium observed by scanning electron microscopy following cyclophosphamide injection were compared to those observed during bladder carcinogenesis.

Animals↗