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PubMed2000

Melanocytes in nevi and melanomas synthesize basement membrane and basement membrane-like material. An immunohistochemical and electron microscopic study including immunoelectron microscopy.

Light microscopic studies have shown that nevus cell nests and melanoma nests are surrounded by basement membrane (BM) material containing type IV collagen and laminin. This study confirms this by electron microscopy and relates it to proteins which interact with the basement membrane. Nevi except for dysplastic and Spitz nevi, malignant melanomas, and melanoma metastases were studied by immunohistopathology, routine electron microscopy (EM), and immunoelectron microscopy. The lesions were incubated with monoclonal antibody (moAb) against type IV collagen, laminin, and the integrin alpha6 and studied by light microscopy. In addition, melanomas were studied by immuno-EM after incubation with a moAb against matrix metalloproteinase-2 (MMP-2). Nevus cell nests and melanoma nests are surrounded by BM material containing type IV collagen and laminin by immuno-EM. The BM material various in thickness and is amorphous. Type IV collagen, laminin, and MMP-2 are synthesized by melanoma cells as well as adjacent fibroblasts. Destruction or loss of the BM is not mandatory for melanoma invasion or even metastasis. Possibly the BM material is a protective wall for melanoma cells. Interactions between melanocytes and the extracellular matrix of which the BM is a part, can be traced back to the migration of melanocytes from the neural crest.

Antibodies, Monoclonal↗
PubMed1983

A new method of scanning electron microscopy for imaging biological tissues.

The scanning electron microscope (SEM) has proved of little value in the examination of material prepared for light microscopic histology. One of the chief reasons for this is that the secondary electron signal used for image formation in routine scanning microscopy derives from the surface of the specimen. In the case of histological material this surface is one which has been severely distorted by processing and cutting procedures. Light microscopy sections can be usefully studied in te SEM if the signal used to form the image derives from a considerable portion of the thickness of the section. Thus the backscattered electron (BSE) image has been successfully used in studying the distribution of dense material or densely staining components several micrometres deep to the surface of dried sections. Such sections are, however, usually mounted on low density (poorly BSE reflecting) non-transparent substrates such as beryllium or carbon, so that matching light microscopy of the same samples is not possible. We report here a method by which histological sections mounted on glass slides can be imaged in the SEM at a resolution higher than that obtained using conventional light microscopy. The method exploits the facts that the ordinary, cheap light microscope slide is strongly cathodoluminescent, yet the standard histological (7 micrometers) section is of such a mass thickness that it absorbs a significant proportion of electrons which energies (5-20 keV) usually used in biological SEM. Thus the measure of the glass cathodoluminescence signal is the measure of the electron flux passing through the specimen.

Animals↗
PubMed2001

The value of electron microscopy in the diagnosis of chronic renal allograft rejection.

The main causes of the late dysfunction of renal allografts are chronic rejection and chronic transplant nephropathy. Both are clinicopathologic entities, with a similar clinical presentation, but different histologic appearances. Chronic rejection is characterized by the presence of alloantigen-induced lesions (transplant arteriopathy and transplant glomerulopathy), and chronic transplant nephropathy by nonspecific sclerosing changes. The incidence of transplant arteriopathy and transplant glomerulopathy is relatively low. Electron microscopy (EM) may overcome the limitations in the histologic diagnosis of chronic rejection, because it verifies alloantigen-induced chronic microvasculopathy in the peritubular capillaries (transplant capillaropathy), and identifies transplant glomerulopathy more precisely than does light microscopy. To assess the value of EM in chronic rejection diagnosis, a retrospective search for transplant capillaropathy and transplant glomerulopathy was performed in a consecutive series of 91 biopsies performed > or = 6 months after implantation (median: 26 months, range 6-186) and the diagnoses were reclassified on the basis of the ultrastructural findings. The definitions used were: transplant capillaropathy: a peritubular capillary profile with seven or more circumferential basement membrane layers, or at least three profiles with five or six circumferential layers; ultrastructurally verified transplant glomerulopathy: thickening of the capillary wall in at least three loops in consequence of the widening of the subendothelial space by abnormal basement membrane material, and the formation of a new layer(s) of basal lamina; and chronic rejection: the presence of transplant capillaropathy and/or transplant glomerulopathy and/or transplant arteriopathy. Histologically, chronic transplant nephropathy, chronic rejection, chronic cyclosporine nephrotoxicity, glomerulonephritis, acute rejection, "suspicious" for acute rejection, and "others" were diagnosed in 37%, 34%, 21%, 19%, 57%, 30%, and 5% of the specimens, respectively. The results of EM increased the diagnosis of chronic rejection to 69% of the cases, and decreased chronic transplant nephropathy to 15%. The individual incidence of transplant capillaropathy and transplant glomerulopathy was 79% and 57%, respectively, and their cumulative incidence was 92%. Five biopsies exhibited merely transplant arteriopathy. A late dysfunction typically had more than one cause; the most frequent combination was chronic rejection and acute rejection. In conclusion, the EM search for transplant capillaropathy and transplant glomerulopathy doubled the frequency of the diagnosis of chronic rejection. Currently, the evaluation of renal allograft biopsies from recipients with a late dysfunction relies on standard light microscopy. Because light microscopy per se proved to be insensitive in the diagnosis of chronic rejection, incorporation of EM into the evaluation of late dysfunction biopsies is strongly recommended.

Adolescent↗
PubMed2005

Optical sectioning microscopy.

Confocal scanning microscopy, a form of optical sectioning microscopy, has radically transformed optical imaging in biology. These devices provide a powerful means to eliminate from images the background caused by out-of-focus light and scatter. Confocal techniques can also improve the resolution of a light microscope image beyond what is achievable with widefield fluorescence microscopy. The quality of the images obtained, however, depends on the user's familiarity with the optical and fluorescence concepts that underlie this approach. We describe the core concepts of confocal microscopes and important variables that adversely affect confocal images. We also discuss data-processing methods for confocal microscopy and computational optical sectioning techniques that can perform optical sectioning without a confocal microscope.

Animals↗
PubMed2002

Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell.

Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes such as green fluorescent proteins, allow fluorescence resonance energy transfer (FRET) to be used to study protein interactions in living specimens. Intensity-based FRET microscopy is limited by spectral bleed-through and fluorophore concentration. Fluorescence lifetime imaging (FLIM) microscopy and lifetime measurements are independent of change in fluorophore concentration or excitation intensity, and the combination of FRET and FLIM provides high spatial (nanometre) and temporal (nanoseconds) resolution. Because only the donor fluorophore lifetime is measured, spectral bleed-through is not an issue in FRET-FLIM imaging. In this paper we describe the development of a nanosecond FRET-FLIM microscopy instrumentation to acquire the time-resolved images of donor in the presence and the absence of the acceptor. Software was developed to process the acquired images for single and double exponential decays. Measurement of donor lifetime in two different conditions allowed us to calculate accurately the distance between the interacting proteins. We used this approach to quantify the dimerization of the transcription factor CAATT/enhancer binding protein alpha in living pituitary cells. The one- and two-component analysis of the donor molecule lifetime in the presence of acceptor demonstrates the distance distribution between interacting proteins.

CCAAT-Enhancer-Binding Protein-alpha↗
PubMed1997

X-ray microscopy and imaging of Escherichia coli, LPS and DNA.

Ultrastructural examination by transmission and scanning electron microscopy involves a series of specialized preparation steps which may introduce artefacts in the micrographs. X-ray microscopy can take instant images of specimens but is mostly restricted to a few synchrotron X-ray sources. We have utilized a bench-top nanosecond laser-plasma to produce a single-shot source of nanosecond X-rays tuned for maximum contrast with carbon-rich material. To examine the ultrastructure by absorption profiles, we utilized a laser-produced plasma generated by a single-shot laser (1.06 microns wavelength, 5 x 10(12) W cm-2 intensity) focused on to a silicon target as an X-ray source for high-resolution X-ray microscopy. This approach eliminates the specimen preparation steps. Whole hydrated cells of Escherichia coli and purified preparations of lipopolysaccharide (LPS) and chromosomal DNA (cDNA) were streaked onto poly(methyl methacrylate) (PMMA)-coated grids (resist). This resist was exposed to X-rays under vacuum at a distance of 2.5 cm from the target disc. The silicon plasma produced by a 10-ns burst of laser energy (at 20J) radiates strong emission lines in the region of 300 eV. The X-rays penetrate the sample and their absorption profile is transferred on to the resist where PMMA acts as a negative to generate an image. By atomic force microscopy imaging of this photoresist we have visualized layers around cells of E.coli, darker areas inside the cell probably corresponding to cDNA, and preliminary images of LPS and DNA molecules. This technique has resolution at the 100 A level, produces images similar to the space-filling models of macromolecules and may be of great value in the study of the ultrastructure of hydrated live biological specimens.

DNA, Bacterial↗
PubMed1998

Measurement accuracy in confocal microscopy.

Confocal laser scanning microscopy provides optical serial sections through thick biological samples, making it possible to perform both three-dimensional visualization and three-dimensional quantitative analysis. On human lymphocytes, we measured geometrical features, cell contents in DNA and in cyclin A and CDK1 proteins, localization and colocalization of these two proteins. Cells were acquired at a vertical sampling step of 0.5 micron, which gives sufficient information about cell labelling. For the purpose of obtaining fast and reliable data at a reduced time cost, we examined various possibilities to simplify acquisition. For example, it might be possible to increase the vertical sampling step to 2.0 microns while preserving an acceptable accuracy of measurements. Further limiting the acquisition to the central sections appeared to give only rough estimations about the whole cells. Finally, we compared confocal microscopy to conventional two-dimensional epifluorescence microscopy. Confocal microscopy appeared slightly less accurate as regards content estimation, but was an invaluable tool when investigating three-dimensional structures and, more especially, localization of proteins.

Cells, Cultured↗
PubMed2002

A new high-aperture glycerol immersion objective lens and its application to 3D-fluorescence microscopy.

High-resolution light microscopy of glycerol-mounted biological specimens is performed almost exclusively with oil immersion lenses. The reason is that the index of refraction of the oil and the cover slip of approximately 1.51 is close to that of approximately 1.45 of the glycerol mountant, so that refractive index mismatch-induced spherical aberrations are tolerable to some extent. Here we report the application of novel cover glass-corrected glycerol immersion lenses of high numerical aperture (NA) and the avoidance of these aberrations. The new lenses feature a semi-aperture angle of 68.5 degrees, which is slightly larger than that of the diffraction-limited 1.4 NA oil immersion lenses. The glycerol lenses are corrected for a quartz cover glass of 220 microm thickness and for a 80% glycerol-water immersion solution. Featuring an aberration correction collar, the lens can adapt to glycerol concentrations ranging between 72% and 88%, to slight variations of the temperature, and to the cover glass thickness. As the refractive index mismatch-induced aberrations are particularly important to quantitative confocal fluorescence microscopy, we investigated the axial sectioning ability and the axial chromatic aberrations in such a microscope as well as the image brightness as a function of the penetration depth. Whereas there is a significant decrease in image brightness associated with oil immersion, this decrease is absent with the glycerol immersion system. In addition, we show directly the compression of the optic axis in the case of oil immersion and its absence in the glycerol system. The unique advantages of these new lenses in high-resolution microscopy with two coherently used opposing lenses, such as 4 Pi-microscopy, are discussed.

Animals↗
PubMed2003

Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over-expression of enhanced green fluorescent protein-tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge-coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K+ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.

Animals↗
PubMed2001

Imaging posterior polymorphous corneal dystrophy by in vivo confocal microscopy.

To identify features of posterior polymorphous dystrophy (PPMD) by in vivo confocal microscopy, the corneas of a female patient with PPMD were exam ned using slit-lamp biomicroscopy and slit-scanning in vivo confocal microscopy. Characteristic endothelial vesicular and band lesions were seen clinically and easily identified using in vivo confocal microscopy. However endothelial pleomorphism, an increased density and reflectance of posterior stromal keratocytes, and prominence of corneal nerves were also delineated. In vivo confocal microscopy enhances clinicopathological diagnosis and follow up of corneal dystrophies with subtle clinical presentations, such as PPMD.

Corneal Dystrophies, Hereditary↗
PubMed2001

Microstructural assessment of rare corneal dystrophies using real-time in vivo confocal microscopy.

PURPOSE: To analyse and describe three cases of rare corneal dystrophy and highlight their in vivo microstructural features. METHODS: Subject 1 was diagnosed with a posterior stromal fleck corneal dystrophy Two of her three children were also affected. Subjects 2 and 3 exhibited an almost identical clinical appearance on biomicroscopic examination, such that both clinically were diagnosed as having pre-Descemet's dystrophies. All subjects underwent in vivo confocal microscopy and approximately 300 sequential digital images were obtained and analysed for each cornea. RESULTS: In vivo confocal microscopy of subject 1 demonstrated an abnormal appearance of numerous large ovoid particles, measuring 50-70 microm in diameter in the mid and posterior stroma as well as smaller hyperreflective dot-like intracellular deposits, of less than 1 microm diameter. Despite the near-identical clinical appearance, subjects 2 and 3 could be clearly differentiated by in vivo confocal microscopy. Subject 2 exhibited small, irregular, optically dense particles, mainly in the anterior stroma, whereas subject 3 possessed classical involvement of the stroma immediately adjacent to Descemet's membrane, with numerous regular small, hyperreflective particles. CONCLUSIONS: The ability of in vivo confocal microscopy to localize and accurately measure various elements in different corneal layers may help to resolve whether abnormalities are intra- or extracellular, and aid clearer differentiation of rare corneal disorders.

Adult↗
PubMed2002

Quantification of fetomaternal hemorrhage by fluorescence microscopy is equivalent to flow cytometry.

BACKGROUND: The quantification of fetal cells in the maternal circulation remains an important goal to determine the amount of anti-D necessary to prevent active immunization of a D- mother giving birth to a D+ baby. Underestimation of fetomaternal hemorrhage (FMH) results in inefficient anti-D prophylaxis and maternal immunization; overestimation of FMH results in higher doses of passively transferred anti-D, higher costs, and the risk of disease transmission. Thus, a reliable method to quantitatively assess FMH is necessary. STUDY DESIGN AND METHODS: Serial dilutions of artificial FMH were quantitatively measured by three different methods: flow cytometry, fluorescence microscopy (each after anti-D staining), and by the Kleihauer-Betke test. The accuracy and precision of the three methods were compared by statistical analysis. RESULTS: Fluorescence microscopy and flow cytometry were comparably accurate and precise in quantifying FMH. In contrast, the accuracy of the Kleihauer-Betke test was poor, resulting in substantial overestimation of FMH in the samples with lower fetal cell concentrations. CONCLUSION: Anti-D flow cytometry and fluorescence microscopy for detection of fetal cells offer equally reliable and precise methods in contrast to the Kleihauer-Betke test. Fluorescence microscopy may be established as standard to quantify FMH in clinical practice because it is comparable to flow cytometry; in addition, it is time saving and is less expensive.

Adult↗
PubMed1996

[Confocal in vivo microscopy after 15 mm sclerocorneoplasty à chaud in necrotizing keratitis].

BACKGROUND: Confocal microscopy allows an in-vivo visualization of corneal structures in frontal optical sections. By means of this method we show morphological changes in a corneal transplant after sclerocorneoplasty à chaud because of a necortizing keratitis. CASE REPORT AND METHOD: We report on a patient who had to undergo a 15 mm diameter sclerocorneoplasty à chaud because of a necrotizing keratitis of his right functionally last eye in October 1993. Five months after transplantation there was a circular vascularization of the scleral rim and a progressive crystalline deposition in the periphery of the cornea. The main part of the donor cornea remained clear with a vision of 0.6. After one year the patient had developed a mature complicated cataract and got a phacoemulsification with a tunnel in the transplanted cornea and an implantation of a posterior chamber lens. In May 1995 the visual acuity was 0.9. We examined the epithelium, stroma and endothelium of the graft by means of in-vivo confocal slit scanning microscopy. RESULTS: All layers of the cornea are demonstrated. There were crystalline needle-like structures in the peripheral stroma and round cystic bodies in the deep stroma. Nerves were visible one year after transplantation. The endothelial cells showed a mild polymorphism and still a density of 2200 cells/mm2. CONCLUSIONS: Confocal microscopy is a useful non invasive technique for routine examinations after keratoplasty. Microscopical changes in the tissue are also visible in case of absent postoperative complications. With growing knowledge the confocal microscopy will give more insight into processes of wound healing.

Aged↗
PubMed1993

Analysis of banded human chromosomes and in situ hybridization patterns by scanning force microscopy.

Scanning force microscopy was used to analyze banded human chromosomes and in situ hybridization patterns of biotinylated DNA probes. In standard human GTG-banded metaphase chromosome preparations (where GTG is G-banding with trypsin-Giemsa), chromosomal morphology and banding patterns were well preserved during the scanning procedure. The smallest identifiable features were in the range of about 100 nm and are similar to the typical structures seen by electron microscopy. In addition, in situ hybridization of human DNA probes of known chromosomal localization was used to map specific hybridization signals. Imaging of the precipitated crystals at the hybridization site clearly demonstrates the superior resolution of scanning force microscopy compared to conventional microscopy.

Chromosome Banding↗
PubMed1999

Continuous detection of extracellular ATP on living cells by using atomic force microscopy.

Atomic force microscopy is a powerful technique used to investigate the surface of living cells under physiological conditions. The resolution of the instrument is mainly limited by the softness of living cells and the interactions with the scanning tip (cantilever). Atomic force microscopy, in combination with myosin-functionalized cantilevers, was used in the detection of ATP concentrations in solution and on living cells. Functionally active tips were used to scan the surface of cells in culture and to show that the CFTR+ cell line (S9) had a basal surface ATP concentration that could be detected with atomic force microscopy (n = 10). ATP-dependent signals were not detectable in cells scanned with noncoated or heat-inactivated enzyme-coated tips (n = 9). Enzymatically active tips may serve as a model for future development of atomic force microscopy biosensors that can simultaneously detect topographical and biologically important compounds at the surface microenvironment of living cells.

Adenosine Triphosphate↗
PubMed2000

Comparison of blood-film microscopy, the OptiMAL dipstick, Rhodamine-123 fluorescence staining and PCR, for monitoring antimalarial treatment.

In an attempt to see if the OptiMAL dipstick (Flow Inc., Portland, OR) can be used to monitor antimalarial treatment, a pilot study of 17 patients with Plasmodium falciparum malaria, admitted to the Hospital for Tropical Diseases in London, U.K., was conducted. Sequential, follow-up, blood specimens were obtained from day 1 to day 3, 4 or 5 post-admission. Thin and thick films prepared from these samples were examined for the presence of malarial parasites, and the intensities of parasitaemia were estimated. In addition, each specimen was tested with the OptiMAL dipstick, Rhodamine-123 fluorescence staining and, on specimens collected on day 1 and the last follow-up before discharge, by a PCR-based test. The results showed that OptiMAL has good sensitivity for the initial diagnosis of P. falciparum malaria and also mirrors the decline in viability of the parasites on treatment, giving the potential to follow the efficacy of drug treatment. The results of the PCR-based tests were still positive when the blood film and OptiMAL result were negative. The OptiMAL dipstick compared well with blood-film microscopy for monitoring antimalarial treatment and could be a useful replacement for microscopy to monitor treatment in places where facilities for microscopy are either lacking or inadequate. In developed countries it could be a useful adjunct to blood-film microscopy, and it might permit a reduction in the duration of hospitalization and give an early warning of treatment failure.

Humans↗
PubMed1993

Lamin B distribution and association with peripheral chromatin revealed by optical sectioning and electron microscopy tomography.

We have used a combination of immunogold staining, optical sectioning light microscopy, intermediate voltage electron microscopy, and EM tomography to examine the distribution of lamin B over the nuclear envelope of CHO cells. Apparent inconsistencies between previously published light and electron microscopy studies of nuclear lamin staining were resolved. At light microscopy resolution, an apparent open fibrillar network is visualized. Colocalization of lamin B and nuclear pores demonstrates that these apparent fibrils, separated by roughly 0.5 micron, are anti-correlated with the surface distribution of nuclear pores; pore clusters lie between or adjacent to regions of heavy lamin B staining. Examination at higher, EM resolution reveals that this apparent lamin B network does not correspond to an actual network of widely spaced, discrete bundles of lamin filaments. Rather it reflects a quantitative variation in lamin staining over a roughly 0.5-micron size scale, superimposed on a more continuous but still complex distribution of lamin filaments, spatially heterogeneous on a 0.1-0.2-micron size scale. Interestingly, lamin B staining at this higher resolution is highly correlated to the underlying chromatin distribution. Heavy concentrations of lamin B directly "cap" the surface of envelope associated, large-scale chromatin domains.

Animals↗
PubMed2002

Interfibrillar collagen bonding exists in matrix produced by chondrocytes in culture: evidence by electron microscopy.

Interfibrillar bonding of collagen fibrils in tissue grown from rabbit chondrocytes in culture was examined by a variety of electron microscopy techniques. Interfibrillar bonding is expected to increase tissue strength and may be a desirable feature in engineered cartilage and other soft tissues. The apparent bonding evident by scanning electron microscopy, using standard chemical fixation processing, is suspected to be artifact due to drying. The goal of this article was to establish the existence of interfibrillar bonding, apart from any processing artifacts. Specimens prepared by transmission electron microscopy, scanning electron microscopy (SEM) after notching and fixing under load, and cryo-SEM all showed evidence of bonding, supporting the existence of bonding in the unprocessed tissue. Exclusion from the bond space of gold particles labeled to decorin further supported the existence of natural bonds. Artifactual bonding may still be occurring with some of the methods used, but interfibrillar bonds exist in natural tissue. The bond distance was estimated to be 7-14 nm. Demonstration of the existence of these bonds supports further study of their mechanism and effect on tissue properties.

Animals↗