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At least 1,477 records · Page 82Linked to original sources
PubMed1995

Dystrophin localization at presynapse in rat retina revealed by immunoelectron microscopy.

PURPOSE: It has been accepted that retinal dystrophin is localized in the outer plexiform layer (OPL) of the retina, but no electron microscopic evidence has been reported until now. Using immunoelectron microscopy, the authors investigate whether retinal dystrophin localizes at the presynaptic or postsynaptic membrane of synaptic regions. METHODS: Monoclonal antibody raised against human dystrophin C-terminus was used. Immunoblotting analysis was used for testing dystrophin protein in retinal tissues. Tissue preparations were stained with the immunofluorescence or immunoperoxidase method and were observed with light and electron microscopy. RESULTS: Immunoblotting analysis showed a molecular weight of band 420 kDa in the sodium dodecyl sulfate--polyacrylamide gel electrophoresis of the retinal tissues. Immunoelectron microscopy disclosed that retinal dystrophin was localized at the presynaptic membrane of synaptic regions in the OPL. CONCLUSIONS: Rat retinal dystrophin localizes at the presynaptic membrane in the OPL, suggesting that it may play some role in the neurotransmitter release of the photoreceptor cell.

Amino Acid Sequence↗
PubMed1995

Comparative localization of mannose-6-phosphate receptor with 2,6sialyltransferase in HepG2 cells: an analysis by confocal double immunofluorescence microscopy.

Recent advances in confocal immunofluorescent microscopy have led to significant improvements in delineating membrane-bounded organelles. In this study using HepG2 cells we focused on two functionally distinct but closely apposed organelles that have been difficult to distinguish by conventional immunofluorescent microscopy, namely the Golgi apparatus, the trans Golgi network (TGN) and late endosomes. The following markers were used: for the Golgi apparatus beta 1,4galactosyltransferase (gal-T), for the TGN, 2, 6(N)sialytransferase (sia-T) and for late endosomes/TGN, the mannose-6-phosphate/insulin growth factor II receptor (CIMPR). In addition, that part of the TGN previously shown to contain CIMPR was also identified using antibodies to the gamma-chain of the HA-1 adaptor (Klumperman et al. J. Cell Biol. 121, 997-1010 (1993)). True colocalization of intracellular antigens was ascertained by double staining of gal-T using both monoclonal and polyclonal antibodies. As previously reported, our results revealed essentially complete colocalization of gal-T and sia-T in this cell line. While the compartments containing CIMPR appeared to overlap with those containing sia-T by conventional immunofluorescence, both compartments were clearly distinct by double-label confocal microscopy. Differences between these organelles became more evident following treatment with brefeldin A. Finally, HA-1 gamma-chain was also localized to structures that were close to but clearly different from the sia-T-containing compartment. Absence of colocalization of CIMPR or HA-1 gamma-chain with sia-T indicates that these markers are enriched in distinct domains of the trans Golgi network.

Adaptor Protein Complex gamma Subunits↗
PubMed1995

Site of localization of Mycoplasma pulmonis and Mycoplasma hominis in the genital tract of female mice demonstrated by culture and scanning and immuno-electron microscopy.

Thirty young adult mice, of strain BALB/c, treated previously with progesterone, were inoculated intravaginally (10 mice) or directly into the uterus (10 mice) with Mycoplasma pulmonis and 10 mice remained uninoculated. Ten mice not treated with the hormone were also inoculated intrauterinely with M. pulmonis. The same numbers of mice treated with oestradiol were inoculated in the same ways with M. hominis. Vaginal swab specimens were obtained from all mice 7, 14 and 28 days after inoculation and samples of genital tract tissue were collected from pairs of mice at the same time intervals. Large numbers of M. pulmonis and M. hominis organisms were isolated from the vagina throughout the course of the experiments and they were cultured also from the cervix and uterine horns. Mycoplasma-like bodies were demonstrated by scanning electron microscopy in the cervix and in the uterus, but neither mycoplasmal species was found attached to vaginal epithelium. The results of silver-enhanced immunogold labelling in conjunction with scanning microscopy provided assurance that the mycoplasma-like bodies were, in fact, mycoplasmas. The importance of hormone treatment was indicated by the diminished susceptibility of untreated mice to M. pulmonis and the almost complete insusceptibility to M. hominis, shown by culture and scanning electron microscopy.

Animals↗
PubMed1994

Topology of the morphological domains of the chaperonin GroEL visualized by immuno-electron microscopy.

Electron microscopy of the tetradecameric double-ring complex of GroEL reveals a four-layered structure, indicating that the 58 kDa subunits are composed of two major morphological domains. We have used immuno-electron microscopy to assign these domains to the corresponding segments of the GroEL sequence. Upon chemical modification of GroEL with N-ethylmaleimide, protease treatment in the presence of ATP or ADP generates GroEL fragments of 15 kDa (N15; residues 1-141) and 40 kDa (C40; residues 153-531). As visualized by scanning transmission electron microscopy, affinity-purified antibodies directed against C40 recognize the outer layers, whereas antibodies against N15 interact with the equatorial portions of the GroEL double-ring. Thus, the two major domains of the subunits in the chaperonin complex are arranged in the order C40-N15:N15-C40. The single-ring chaperonin co-factor GroES interacts with the C40 domain while the ATP-binding site of GroEL is probably close to the junction between N15 and C40.

Antibodies↗
PubMed1993

Contributions of electron microscopy to the study of the hypertrophic scar and related lesions.

Prior to 1969, only one study of the hypertrophic scar had been done using electron microscopy, and that one used electron diffraction. Since that time, studies using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) have been integral in establishing not only the characteristics of this lesion but in formulating the reasons why the scar develops and how it resolves. The first SEM studies demonstrated a homogeneous, dense dermal matrix which supported the conclusion that the hypertrophic scar and keloid reflected excess collagen. These same studies were integral in identifying the collagen nodule as the basic anatomical unit of these lesions. SEM and TEM studies have been complimentary. The TEM studies revealed the first evidence of the phenomenon of occluded microvessels; but, their significance was not established until later quantitative studies. A hierarchy of fibroblasts was first demonstrated by TEM. Later, evidence came from several different investigators, through tissue culture and molecular differentiation, that the previously observed different electron microscopic features may reflect different fibroblast types of cells. Finally, the degenerative (or apoptotic) events, involving fibroblast-type cells and microvessels have been revealed by TEM studies, and supported by SEM observations. This phenomenon has been presented as a major factor in formation of the nodules, and, also, in the natural resolution of the hypertrophic scar and keloid.

Animals↗
PubMed1978

[Morphological virus diagnosis--electron microscopy study of animal viruses with negative contrast procedure].

Reported in this paper are results obtained in morphological virus diagnosis by using the negative contrast technique on the basis of electron microscopy. The availability of high-efficiency electron microscopy as well as of perfectly improved techniques of preparation, knowledge of the latest virus model concepts, and indivudual skills in diagnosis are essential conditions for the above approach. Parvoviridae, picornaviridae, and togaviridae are identifiable only in high particle concentrations and by group representation. Papovaviridae, adenoviridae, herpetoviridae, poxviridae, and reoviridae, on the other hand, can be safely identified even as single particles. The diagnosis of orthomyxoviridae, paramyxoviridae, rhabdoviridae, and retroviridae is facilitated by their own dimensions and their characteristic helico-symmmetrical nucleocapside. Coronaviridae are of highly conspicuous morphology but, nevertheless, pose problems in differential diagnosis. Substantive improvement of morphological virus diagnosis, in terms of minute details, may be achieved by means of the negative contrast method on the basis of immune electron microscopy. Advantages implied in that morphological method include less time-consuming and quite uninvolved practicability and good dependability of diagnosis for more efficient decision-making in research and practice.

Animals↗
PubMed1995

Imaging of single antigens, antibodies, and specific immunocomplex formation by scanning force microscopy.

The most sensitive analytical techniques available today for detecting immuno assay complexes are radio or enzyme immuno analytical techniques, by which quantities of 10(7)-10(8) analyte molecules can be detected. With the introduction of scanning force microscopy, a new method for detecting biological processes became available. Here, we examine the feasibility of using scanning force microscopy as a biosensitive tool. We demonstrate that single or multiple rabbit anti-human serum albumin molecules form complexes with preadsorbed single human serum albumin molecules on mica. However, no interaction is observed between human immunoglobulin G molecules and preadsorbed single albumin molecules; only separate antigens and antibodies are observed at random positions on the mica. This shows the ability of scanning force microscopy to act as a biosensor for detection of immunocomplexes, and to act as a very powerful tool to study molecule-surface interactions in general.

Adsorption↗
PubMed1979

Comparative scanning and transmission electron microscopy of the prostatic gland in different species (mouse, rat, dog, man).

Man, dog, rat and mouse prostatic glandular cells were compared by light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). In rodents and dogs, a merocrine secretion exists, and in these species the undulating surface represented a confluence of secretory granules. The prominent secretory blebs observed in dog and human benign prostatic hyperplasia (BPH) corresponded to apocrine secretions. Also variations in the amount of secretory activity were seen. Androgenic hormones were responsible for the differentiation of the prostatic epithelial cells during development. Therefore, an absence of microvilli as well as the appearance of ruffles, microplicae, and bare cells were seen in the prepuberal and castrated rat. In the elderly rat and in human BPH, a pleomorphism of the apical cell surface was evident.

Age Factors↗
PubMed1996

Quantitative imaging in electron and confocal microscopies for applications in biology.

Among the large number of topics related to the quantification of images in electron and confocal microscopies for applications in biology, we selected four subjects that we consider to be representative of some recent tendencies. The first is the quantification of three-dimensional data sets recorded routinely in scanning confocal microscopy. The second is the quantification of the textural and fractal appearance of images. The two other topics are related to image series, which are more and more often provided by imaging instruments. The first kind of series concerns electron energy-filtered images. We show that the parametric (modelling) approach can be complemented by non-parametric approaches (e.g., different variants of multivariate statistical techniques). The other kind of series consists of multiple mappings of a specimen. We describe several new tools for the study and quantification of the co-location, with potential application to multiple mappings in microanalysis or in fluorescence microscopy.

Image Processing, Computer-Assisted↗
PubMed1998

Structural studies of fibrinolysis by electron microscopy.

Fibrin is degraded by the fibrinolytic system in which a plasminogen activator converts plasminogen to plasmin, a serine protease that cleaves specific bonds in fibrin leading to solubilization. To elucidate further the biophysical processes involved in conversion of insoluble fibers to soluble fragments, fibrin was treated with either plasmin or the combination of plasminogen and plasminogen activator, and morphologic changes were observed using scanning electron microscopy. These changes were correlated with biochemical analysis and with characterization of released, soluble fragments by transmission electron microscopy. Initial changes in the fibrin matrix included creation of many free fiber ends and gaps in the continuity of fibers. With more extensive digestion, free fiber segments associated laterally, resulting in formation of thick fiber bundles. Supernatants of digesting clots, containing soluble derivatives, were negatively contrasted and examined by transmission electron microscopy. Large, complex fragments containing portions of multiple fibers were observed, as were pieces of individual fibers and smaller fragments previously identified. Some large fragments had sharply defined ends, indicating that they had been cleaved perpendicularly to the fiber direction. Other fibers showed splayed ends or a lacy meshwork of surrounding protofibrils. Longer times generated more small fragments whose molecular composition could be inferred from their appearance. These results indicate that fibrinolytic degradation results in larger pieces than previously identified and that plasmin digestion proceeds locally by transverse cutting across fibers rather than by progressive cleavage uniformly around the fiber.

Electrophoresis, Polyacrylamide Gel↗
PubMed2026

Advanced High-Resolution and Super-Resolution Microscopy Techniques for Investigating Chromatin Structure and Epigenetics.

Fluorescence microscopy has emerged as an indispensable tool for investigating chromatin structure and epigenetic states. This technique not only enables super-resolution imaging to unveil detailed localizations and structures but also offers precise quantitative capabilities for comparing variations across multiple conditions. In this chapter, we present a comprehensive guide to immunofluorescence methods, detailing two primary fixation protocols. Additionally, we describe the application of super-resolution spinning disc microscopy, emphasizing its utility in detecting nuclear and epigenetic markers.

Chromatin↗
PubMed1995

Epiluminescence microscopy. A useful tool for the diagnosis of pigmented skin lesions for formally trained dermatologists.

BACKGROUND AND DESIGN: Epiluminescence microscopy (ELM) is a noninvasive technique that, by employing the optical phenomenon of oil immersion, makes subsurface structures of the skin accessible for in vivo examination and thus provides additional criteria for the clinical diagnosis of pigmented skin lesions. At present, almost all studies about the value and clinical importance of ELM are based on data derived from ELM experts (ie, dermatologists specifically trained in this technique). In the present study, we attempt to determine whether the clinical diagnosis of pigmented skin lesions is significantly improved using ELM and whether ELM-trained individuals and dermatologists not trained in this technique profit equally from this technique. Randomly selected histologically proven pigmented skin lesion specimens, photographed with (ELM) and without oil immersion (surface microscopy) were presented by slide projection to six ELM experts and 13 ELM nonexperts (ie, dermatologists not formally trained in ELM) for diagnosis. To evaluate the diagnostic performance of ELM experts and nonexperts with and without the oil immersion technique (ie, ELM vs surface microscopy), the following parameters were obtained: intraobserver and interobserver agreement by kappa statistics and sensitivity and specificity of diagnostic performance. RESULTS: Our results show that by using the ELM technique the ELM experts reach a substantially better intraobserver agreement than nonexperts (median kappa, 0.56 vs 0.36). The interobserver agreement was markedly increased in the ELM experts group (average gain, 7%) but decreased in the ELM nonexperts group (average loss, 6%). The sensitivity of diagnosis was significantly increased in the ELM experts group (average gain, 10%), but decreased in the nonexperts group (average loss, 10%). Finally, the specificity of diagnosis was excellent in the ELM experts group, both with and without oil immersion (0.91) and was somewhat improved by ELM in the nonexperts group (0.77 vs 0.85). CONCLUSIONS: We conclude that the ELM technique increases sensitivity in formally trained dermatologists, but may decrease the diagnostic ability in dermatologists not formally trained in the ELM technique. Consequently, formal broad-based training in ELM should be offered to the dermatologic community.

Clinical Competence↗
PubMed2004

Use of in vivo confocal microscopy in malignant melanoma: an aid in diagnosis and assessment of surgical and nonsurgical therapeutic approaches.

BACKGROUND: Melanomas with poorly defined borders, lack of pigmentation, lentiginous extension, and location in cosmetically sensitive regions represent diagnostic and therapeutic challenges. Repeated surgical reexcisions are frequently required to achieve tumor-free margins. The use of reflectance mode confocal microscopy as an noninvasive method has shown to be a promising tool for diagnosing pigmented lesions in vivo. OBSERVATIONS: We report 3 clinical cases of melanoma: amelanotic melanoma (case 1), locally recurrent melanoma (case 2), and lentigo maligna melanoma (case 3). In case 1, in vivo confocal microscopy was instrumental in making the diagnosis and in monitoring the response to imiquimod therapy for in situ residual disease. It was also used to successfully delineate preoperative surgical margins in cases 2 and 3. CONCLUSION: As new methods for treating melanoma emerge and become more available, confocal microscopy can play a significant role by improving sensitivity in diagnosis, by increasing rates of successful initial excision, and by serving as a noninvasive means of monitoring therapy.

Aminoquinolines↗
PubMed2006

In vivo confocal microscopy of filtering blebs after trabeculectomy.

OBJECTIVES: To analyze filtering blebs after trabeculectomy by means of in vivo confocal microscopy and to correlate the images with clinical bleb appearance and function. METHOD: In vivo confocal microscopy using the Heidelberg Retina Tomograph/Rostock Cornea Module (Heidelberg Engineering, Heidelberg, Germany) was performed in 53 filtering blebs in 45 patients 6 days to 30 years postoperatively. RESULTS: In vivo confocal microscopic findings significantly correlated with good bleb function included the number of epithelial microcysts (P = .03), a large total stromal cyst area (P = .009), the absence of encapsulated stromal cysts (P = .002), minimal vascularization (P = .05), and the absence of tortuous conjunctival vessels (P = .01). In contrast, a hyperreflective condensed bleb stroma was significantly associated with bleb failure (P<.001). Bleb stroma mainly consisting of a rarified collagenlike network was significantly linked to trabeculectomy performed with mitomycin C (P = .001). Epithelial and stromal inflammation were observed at a median of 1 and 4 months after surgery, respectively. CONCLUSIONS: In vivo confocal microscopy using the Heidelberg Retina Tomograph/Rostock Cornea Module permits diagnostic imaging of filtering blebs and differentiation between good and insufficient bleb function. Moreover, the postoperative inflammatory reaction can be monitored directly for adapted postoperative anti-inflammatory treatment.

Adult↗
PubMed1997

Complementary advantages of fluorescence and SIMS microscopies in the study of cellular localization of two new antitumor drugs.

Low light level fluorescence microscopy studies have been carried out on MCF7-P human mammary tumor cells to localize the intracellular distribution of two new anticancer drugs, Pazelliptine and Intoplicine, which are currently under clinical evaluation. These two molecules are thought to act at the nuclear level, through DNA topoisomerase interactions. Because fluorescence of these compounds appears strongly quenched by intercalation in double strand DNA, secondary ion mass spectrometry (SIMS) imaging was used to check the presence of the drugs in the nuclear compartment. In spite of chemical structure similitudes, pazelliptine and intoplicine appear to be distributed in quite different ways within the cells. Incubation for 1 and 24 hours also allowed us to bring to light strong differences in the distribution kinetics. Pazelliptine quickly enters into the nucleoli but is no longer present in the nucleus after 24 hours incubation. Intoplicine was not detected by fluorescence in the nucleus, however SIMS microscopy allowed us to show its accumulation within this cellular compartment as a function of time of exposure. This study shows the complementarity of fluorescence and SIMS microscopies.

Antineoplastic Agents↗
PubMed1997

Preparation of single-celled marine dinoflagellates for electron microscopy.

Electron microscopy has been used successfully to study and identify single-celled marine dinoflagellates including parasitic ones and others, such as those that cause red tide. Delicate cells can be preserved for scanning electron microscopy with a combined glutaraldehydeosmium tetroxide mixture that is adjusted for the osmolality of the medium. The protocol allows resolution of fine morphological features. Preservation for transmission electron microscopy can be accomplished with a standard glutaraldehyde fixation and osmium-tetroxide post-fixation in a suitable buffer, but again, the osmolality of the mixture must be adjusted. The protocol allows ultrastructural resolution of vesiculated cells and has been modified for small sample sizes.

Animals↗
PubMed1997

The contribution of quantitative confocal laser scanning microscopy in cartilage research: chondrocyte insulin-like growth factor-1 receptors in health and pathology.

The use of immunohistochemical detection techniques and fluorescent molecular probes in light and fluorescence microscopy allows accurate and specific analysis of a great variety of cell and tissue components. However, when staining yields only low intensity levels, serious problems may arise with discrimination of specific signals against background staining. This problem is often inherent with articular cartilage research. Application of confocal laser scanning microscopy (CLSM) can circumvent these problems. The CLSM collects images that are almost free of out-of-focus signals, which results in improved spatial resolution and discrimination as compared with conventional microscopy. Moreover, CLSM allows optical sectioning of specimens and three-dimensional reconstruction of the microscopical object. Quantitative evaluation of microscopical images is hampered by out-of-focus signals because they interfere with specific signals in the image. Interference of these nonspecific signals can be diminished by application of CLSM; in CLSM only one single point in microscopical objects is illuminated at any time and this point is then imaged into the pinhole at the entrance of the photo-detector and subsequently digitized. The present review is a discussion of the present state of the art in digital imaging with the use of CLSM in cartilage research. This discussion includes aspects such as sensitivity, specificity, spatial resolution and accuracy of quantitative analysis in microscopical immunofluorescent objects.

Animals↗
PubMed1999

Imaging of collagen type III in fluid by atomic force microscopy.

Type III collagen is a component of the basement membrane of endothelial cells, and may play a role in the interaction between hemostatic system proteins and the basement membrane of blood vessels. To begin to investigate these structural interactions, we have imaged type III collagen in solution by atomic force microscopy. A 20 microg/ml solution of type III collagen in bicarbonate buffer (pH 9.5) from calf skin was deposited onto a freshly cleaved mica substrate. Atomic force microscopy images were acquired using a fluid cell and tapping mode with oxide-sharpened silicon nitride probes 2, 3, and 4 hours after deposition of the collagen onto the mica. Two-hour preparations displayed fibrillar networks with well-defined sites of nucleation and lateral growth. At 3 and 4 hour polymerizations, more mature fibrils of increasing lengths, diameters, and complexity were observed. Fibrils appeared to be aligning and twisting (helical formation) to form a mature fibril with a higher mass per unit area. Interestingly, the mature fibrils appeared larger centrally with tapered ends displaying declining slopes. These observations compare favorably with those previously published on collagen type I assembly [Gale et al. (1995) Biophys. J. 68:2124-2128]. High resolution atomic force microscopy images of type III collagen in solution should provide a template for observation of the interactions between basement membrane components and hemostatic system proteins present in cardiovascular disease.

Animals↗