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Results for “Confocal microscopy”

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At least 415 records · Page 23Linked to original sources
PubMed1998

Normal human corneal cell populations evaluated by in vivo scanning slit confocal microscopy.

PURPOSE: To analyze cellular populations in healthy human corneas. METHODS: The study group consisted of 58 eyes of 45 patients with normal corneas. The age distribution was 45 +/- 17 years (mean +/- SD; range, 20-84). Scanning slit confocal microscopy of the central corneas was performed. The images were analyzed visually for cell morphology, and the densities and areas of cells were measured. RESULTS: No statistically significant differences were measured in cell densities or cell areas of any corneal layer between female and male patients (p = 0.22-0.50) nor between right and left eyes (p = 0.16-0.45). The area of superficial epithelial cells was 913 +/- 326 microm2 (mean +/- SD; range, 518-2,112), and the superficial epithelial cell density was 1,213 +/- 370 cells/mm2 (mean +/- SD; range, 473-1,929). The area of basal epithelial cells was 177 +/- 19 microm2 (mean +/- SD; range, 138-242), and the basal epithelial cell density was 5,699 +/- 604 cells/mm2 (mean +/- SD; range, 4,135-7,267). The average apparent keratocyte density was 1,058 +/- 217 cells/mm2 (mean +/- SD; range, 604-1,599) in the anterior stroma, and 771 +/- 135 cells/mm2 (mean +/- SD; range, 493-1,145) in the posterior stroma. The difference in apparent keratocyte densities between the anterior and posterior stroma was statistically significant (p < 0.001). The average endothelial cell area was 334 +/- 51 microm2 (range, 273-553), and the cell density was 3,055 +/- 386 cells/mm2 (mean +/- SD; range, 1,809-3,668). The endothelial cell density had a negative, statistically significant correlation with age (r = -0.68, p < 0.001). The densities of the other corneal cell layers did not have a statistically significant correlation with age. CONCLUSION: In vivo scanning slit confocal microscopy is a useful tool for studying corneal cell populations. Central corneal cell densities were found to decrease significantly with age only in the endothelium. For the first time in human corneas using in vivo confocal microscopy, this study statistically confirms a higher apparent number of keratocytes in the anterior stroma than in the posterior stroma.

Adult↗
PubMed2003

In-vivo slit scanning confocal microscopy of normal corneas in Indian eyes.

OBJECTIVE: To study the cellular populations of healthy corneas of Indian eyes using confocal microscopy and to evaluate the correlation with age, gender and laterality. METHODS: The central corneas of 100 eyes of 50 healthy subjects were examined using an in-vivo slit scanning confocal microscope (Confoscan 2). Images were analysed for cell densities of the epithelium, stroma and endothelium. RESULTS: Good quality images enabling analysis of all cell layer populations were obtained in 74 eyes of 43 healthy subjects (22 males and 21 females) with a mean age of 31.89 +/- 13.47 (range 19-71 years). The basal epithelial cell density was 3601.38 +/- 408.19 cells/mm2 (range 3017.3-4231.1 cells/mm2). The mean keratocyte nuclei density in the anterior stroma was 1005.02 +/- 396.86 cells/mm2 (range 571.6-1249.6 cells/mm2) and in the posterior stroma was 654.32 +/- 147.09 cells/mm2 (range 402.6-1049.1 cells/mm2). Posterior keratocyte nuclei density was 30.76% less than the anterior stromal keratocyte nuclei density. The difference in keratocyte nuclei density was statistically significant (P=0.001). The mean endothelial cell density was 2818.1 +/- 361.03 cells/mm2 (range 2118.9-4434 cells/mm2) and the mean endothelial cell area was found to be 385.44 +/- 42.66 mm2 (range 268.9-489.2 mm2). Hexagonal cells formed 22.5-69.4% of the endothelial cell populations (mean 42.04 +/- 11.81%). Mean coefficient of cell size variation was 32.29 +/- 3.06 (range 27.2-39.2). No statistically significant differences were found in cell densities of any corneal layer either between female and male patients or between right and left eyes. Basal epithelial cell density, anterior stromal keratocyte nuclei and posterior stromal keratocyte nuclei density were unaffected by age (r=0.12, 0.07, -0.12 respectively) (P=0.001). There was a statistically significant negative correlation between mean endothelial cell density and increase in age (r=-0.42, P=0.001). Coefficient of cell size variation and age were positively correlated (r=0.73, P=0.001). CONCLUSION: In-vivo slit scanning confocal microscopy is useful for the study of corneal cell populations. Our study provides normative data of these cell populations.

Adult↗
PubMed1997

Spatial distribution of dihydropyridine receptors in the plasma membrane of guinea pig cardiac myocytes investigated by correlative confocal microscopy and label-fracture electron microscopy.

Excitation-contraction coupling in cardiac muscle is thought to depend fundamentally on the spatial organization of sarcolemmal dihydropyridine receptors (L-type calcium channels) in relation to ryanodine receptors (calcium-release channels of the sarcoplasmic reticulum). In the present study, we have investigated the distribution of dihydropyridine receptors in the guinea pig myocyte plasma membrane by correlative immunoconfocal microscopy and label-fracture electron microscopy. Label-fracture, a method in freeze-fracture cytochemistry, permits immunogold localization of cell surface proteins in en face membrane views. Taken together, results from confocal microscopy and label-fracture replicas suggest that, in the peripheral plasma membrane, calcium channels are organized predominantly in the form of clusters. Confocal microscopy also suggests a similar organization in the transverse tubules. It is hypothesized that these clusters may lie adjacent to junctional sarcoplasmic reticulum, permitting the close coupling of influx of calcium through plasma membrane calcium channels to trigger release of calcium from the intracellular stores, as part of the mechanism of calcium-induced calcium release.

Animals↗
PubMed2001

Imaging of single fluorescent molecules using video-rate confocal microscopy.

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nipkow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled with fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecomposition lifetimes of the fluorophores were approximately 10 s for TMR and approximately 2 s for IC5 under the incident laser power of 0.5 W/mm(2). Both the fluorescence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm(2). 2D sliding movement of single kinesin molecules along microtubules on glass surface and 3D Brownian motion of individual kinesin molecules in viscous solution could be observed using this microscopy. These results indicated that this method could be applicable to the study of single molecular events in living cells at real time.

Animals↗
PubMed1996

Variation in quantitative measures of enamel prisms from different species as assessed using confocal microscopy.

This study presents a statistical analysis of variability in six measures of enamel prism and ameloblast size and spacing gathered using confocal microscopy, and applies the results to a consideration of appropriate sampling strategies for taxonomic analyses. Variability within individuals was examined within depth series. Individual variability was also assessed within a nested analysis of variation for prism measurements between micrographs, specimens and species. While sample depth was not often significantly associated with differences in prism and ameloblast measures, there was significant variation between micrographs taken from the same region of a tooth. The highest levels of variation were found between species, while variation between conspecific individuals was relatively small. These results demonstrate that data gathered from several micrographs are likely to be representative of a specimen, but that several micrographs of a single specimen will rarely illustrate the range of variation contained within a species. It is essential for systematic and taxonomic analyses that several micrographs be used to characterize an individual. It is also recommended that samples from several individuals be used to characterize species. While data from isolated specimens is often of great interest, taxonomic or systematic conclusions based on isolated individuals should be approached cautiously.

Animals↗
PubMedDate not supplied

Calcium imaging of murine thoracic aorta endothelium by confocal microscopy reveals inhomogeneous distribution of endothelial cells responding to vasodilator agents.

The aim of the study was to assess, in intact murine thoracic aorta in vitro, the distribution of endothelial cells responsive to endothelium-dependent vasodilators ACh, ATP, bradykinin and substance P, using laser line confocal microscopy in combination with two Ca2+ fluorescent dyes, Fluo-4 and Fura-red. We observed that 82 +/- 3% of endothelial cells responded to ATP, 33 +/- 5% to Ach, whereas less than 3% of them responded to bradykinin or substance P. In order to determine whether the findings of pharmacological tests agree with confocal microscopy data, endothelium-dependent vasodilators induced relaxation was evaluated using isometric tension measurement. We show a marked correlation between a higher number of activated endothelial cells, using confocal microscopy, and a greater degree of endothelium-dependent relaxation using isometric tension measurement (p = 0.00286). Our results suggest that endothelial cells responding to endothelium-dependent vasodilators are not homogeneously distributed in intact murine thoracic aorta. This could be due to nonhomogeneous distribution of surface receptors or to differences in post-receptor coupling mechanisms.

Acetylcholine↗
PubMedDate not supplied

[Clinical forms of acanthamoeba keratitis as viewed from the standpoint of biomicroscopy and confocal microscopy].

Clinical cases of 60 patients with acanthamebic keratitis examined by biomicroscopy and of 22 patients largely examined by confocal microscopy are generalized. Acanthamebic keratitis is a slowly progressing infectious lesion of the cornea, which is caused by acanthamebas freely residing in soil and water. Contaminated contact lenses are the key risk factor. The main clinical features of acanthamebic keratitis are defined; they are presence of risk factors; a unilateral lesion in young, healthy and immune-competent persons; a typical clinical pattern of surface keratitis mainly of the ring shape; corneal neuritis without corneal neovascularization but with a severe pain in the eye; and a slow chronic clinical course, i.e. lasting for several weeks and months. Confocal microscopy is the most effective and fast diagnostic tool because it ensures the detection of acanthamebic cysts and trophozoids in all strata of the corneal stroma. The authors isolate, within the clinical course of acanthamebic keratitis, 5 stages; they are surface epithelial keratitis; surface epithelial punctate keratitis; stromal ring-shaped keratitis; ulcerous keratitis; and keratoscleritis.

Acanthamoeba↗
PubMed1995

Confocal microscopy reveals local SR calcium release in voltage-clamped cardiac cells.

Confocal microscopy during voltage-clamp depolarization of mammalian heart cells revealed distinct local [Ca2+]i-transients that resulted from Ca2+ released through sarcoplasmic reticulum (SR) release channel(s). When voltage was varied, the latency to occurrence and the relative probability of local [Ca2+]i-transients varied as predicted if SR Ca(2+)-release is linked tightly to Ca2+ flux through co-associated L-type Ca(2+)-channels.

Animals↗
PubMed2004

Correction of image instability in confocal microscopy using image realignment. Effects on the analysis of intracellular calcium.

Using confocal microscopy, we have examined the increases in [Ca(2+)](i) evoked by sodium channel toxins in cells labelled with the fluorescent dye INDO-1. We describe a new image analysis method that improves the detection of region-specific, toxin-induced patterns of change of intracellular calcium. This method is based on correction of global image motion followed by calculation of the strength of correlation between calcium changes in "seed" or reference pixels chosen to represent different regions of cells and those in other regions of the image. When the selected "seed" pixel was chosen to be in either varicosities or neurites, correlations were detected in the same regions of other cells as well as in the soma, indicating specific but spatially distinct patterns of behaviour. Control images (without changes in [Ca(2+)](i)) did not reveal significant interpixel correlations. The ability to recognize correlated patterns of calcium change in different regions of cells was greatly improved by correction for global motion.

Animals↗
PubMed2001

Diffuse lamellar keratitis after laser in situ keratomileusis imaged by confocal microscopy.

OBJECTIVE: To report the confocal microscopic findings of two different cases of diffuse lamellar keratitis (DLK) after primary laser in situ keratomileusis (LASIK) and LASIK flap relifting. DESIGN: Observational case report of two cases. METHODS: Two cases of DLK with different clinical appearances after primary LASIK and LASIK flap relift were selected. A third case of uneventful LASIK was selected as control case. Examination with a white-light tandem slit-scanning confocal microscope was performed in addition to routinely applied slit-lamp biomicroscopy. RESULTS: In both cases, confocal microscopic examination showed infiltration of cells considered most likely to be mononuclear cells and granulocytes into the flap interface. Under treatment with topical steroids, the inflammation was reduced in both cases, to a minimum within 1 week, without loss of visual acuity. At this stage, only remnants of the granulocytelike cells, but not active inflammation, were imaged by confocal microscopic examination. CONCLUSIONS: Our findings confirm the clinical observation that DLK is an inflammation confined to the flap interface. Confocal in vivo microscopy could be a useful noninvasive tool to study the course of DLK in humans.

Adult↗
PubMed2001

Confocal microscopy of the human cornea in vivo.

In vivo, scanning-slit, confocal microscopy offers improved resolution and has resulted in new discoveries of corneal pathology at the cellular level. The ability to provide high resolution, real-time images of the full thickness of the living human cornea gives the clinician and the researcher an important new tool.

Cornea↗
PubMed1999

Application of laser scanning confocal microscopy in the analysis of particle-induced pulmonary fibrosis.

Laser scanning confocal microscopy (LSCM) allows us to simultaneously quantitate the degree of lung fibrosis and distinguish various pathological lesions of intact lung tissue. Lucifer Yellow has been shown an ideal fluorescent stain to examine the connective tissue matrix components of embedded lung tissue with LSCM. We evaluated the use of LSCM in quantitating lung fibrosis and compared this procedure with the more traditional method of assessing fibrosis by measuring hydroxyproline, a biochemical assay of collagen. CD/VAF rats were intratracheally dosed with silica (highly fibrogenic), Fe2O3 (non-fibrogenic), and saline (vehicle control) at a high dose of 10-mg/100 g body weight. At 60 days post-instillation, the left lung was dissolved in 6 M HCl and assayed for hydroxyproline. Silica induced increases of 58% and 94% in hydroxyproline content over the Fe2O3 and control groups, respectively. The right lung lobes were fixed, sectioned into blocks, dehydrated, stained with Lucifer Yellow (0.1 mg/ml), and embedded in Spurr plastic. Using LSCM and ImageSpace software, the tissue areas of ten random scans from ten blocks of tissue for each of the three groups were measured, and three-dimensional reconstructions of random areas of lung were generated. The silica group showed increases of 57% and 60% in the lung areas stained by Lucifer Yellow over the Fe2O3 and control groups, respectively. Regression analysis of hydroxyproline vs. lung tissue area demonstrated a significant positive correlation (p < 0.05) with a correlation coefficient of 0.91. Histological analysis of right lung tissue revealed a marked degree of granulomatous interstitial pneumonitis for the silica group, which was absent in the Fe2O3 and control groups. No significant differences (p < 0.05) in hydroxyproline content and measured tissue area were observed between the Fe2O3 and control groups. LSCM, and its associated advanced image analysis and three-dimensional capabilities, is an alternative method to both quickly quantitate and examine fibrotic lung disease without physical disruption of the tissue specimen.

Animals↗
PubMed2007

In vivo confocal microscopy in hydroxychloroquine-induced keratopathy.

BACKGROUND: Vortex keratopathy, arising as a side effect of several medications, is characterized by golden-brown deposits in the cornea. METHODS: A 41-year-old woman treated for sarcoidosis with hydroxychloroquine therapy and suffering from vortex keratopathy was examined by in vivo confocal microscopy. Scans of both corneas were performed. RESULTS: By slit lamp examination, the left but not the right eye showed a golden-brown deposit throughout the cornea. In vivo confocal microscopy revealed the presence of highly reflective, dot-like intracellular inclusions concentrated in the basal epithelial layer. They were also detected within the anterior and posterior stroma, but not within the endothelium. In regions of the anterior stroma, devoid of inclusions, hyperreflective ramified keratocytes were observed, forming an extended interconnecting network. CONCLUSION: In addition to the granular deposits, in vivo confocal microscopy revealed hyperreflective, possibly phagocytic keratocytes.

Adult↗
PubMed2006

Epithelial dendritic cell distribution in normal and inflamed human cornea: in vivo confocal microscopy study.

PURPOSE: To evaluate dendritic cell (DCs) density, distribution, and morphology in central corneal and limbal epithelium in normal subjects and patients with immune-mediated corneal inflammation using in vivo confocal microscopy (IVCM). DESIGN: Comparative case-controlled, observational confocal microscopy study. METHODS: A total of 135 eyes of 135 patients were investigated. Group 1 (normal eyes) included 45 eyes of 45 healthy volunteers, group 2 photorefractive keratectomy (PRK-treated eyes) included 45 myopic eyes of 45 patients treated with PRK, and group 3 (inflamed eyes) comprised 45 eyes of 45 patients affected by immune-mediated corneal inflammation. The central cornea and limbus were examined for epithelial dendritic-shaped cells using laser scanning IVCM. DCs density was calculated using image analysis software. RESULTS: Cells with a branching dendritic morphology were visualized in the basal epithelial layer, basal lamina, and subbasal nerve plexus, in the central cornea, and in the basal layer and basal membrane of the limbal epithelium. The limbal epithelium demonstrated DCs in 93.3%, 89%, and 97.7% of eyes in group 1, 2, and 3, respectively (P = ns). Central epithelial DCs were observed in 20.0% and 13.3% of eyes in group 1 and 2 (P = ns), while in 93.3% of eyes in group 3 (P < .001). DCs were found to be significantly higher at the limbus compared with central cornea in each group (P < .001). Cell densities observed in group 3 were significantly greater than groups 1 and 2, at both locations (P < .05). CONCLUSIONS: Laser scanning IVCM is a useful method for evaluating epithelial DCs distribution at the limbus and central cornea in both healthy and diseased eyes.

Adult↗
PubMed2001

Three-dimensional coherent transfer function for reflection confocal microscopy in the presence of refractive-index mismatch.

The three-dimensional (3-D) coherent transfer function for reflection confocal microscopy of high-numerical-aperture objectives is derived and calculated in the presence of refractive-index mismatch when a laser beam is focused into a medium of refractive index different from its immersion medium. This aberrated coherent transfer function is then used to estimate the readout efficiency of 3-D data bits recorded in a thick medium. It is shown that the readout efficiency of confocal microscopy for 3-D bit data storage is decreased with the focal depth of an objective in a recording medium. However, a high readout efficiency can be maintained if the tube length of a reading objective is linearly altered to compensate for the spherical aberration caused by the refractive-index mismatch.

Microscopy, Confocal↗
PubMed2000

A laser scanning confocal microscopy method. Simultaneous detection of intracellular Ca2+ and apoptosis using Fluo-3 and Hoechst 33342.

OBJECTIVE: To develop a simple and direct method to simultaneously determine apoptotic cells from a treated population of cells and detect the changes of intracellular Ca2+ in these apoptotic cells, in particular single ones, by confocal microscopy. STUDY DESIGN: MGC-803 cells treated with As2O3 were used as the double-staining cell model with Hoechst 33342 as a DNA probe and Fluo-3AM as a Ca2+ indicator. MGC-803 cell apoptosis induced by As2O3 was first demonstrated by DNA ladder in gel electrophoresis. Based on the difference in DNA stainability with Hoechst 33342 and corresponding fluorescence intensity between live and apoptotic cells, apoptotic cells and the changes in intracellular Ca2+ were detected at the same time by confocal microscopy. No necrotic cells in the group treated with As2O3 were found by the trypan blue exclusion test. RESULTS: The results from confocal microscope detection showed that intact and apoptotic cells were successfully recognized and the changes of intracellular Ca2+ in apoptotic and intact cells were simultaneously detected in the same sample. CONCLUSION: We provided a useful method to exactly detect changes in intracellular Ca2+ in apoptotic cells, especially in single ones, by confocal microscopy and to exclude the artifact effect of necrotic and intact cells.

Aniline Compounds↗
PubMed2002

Detection of dysplasia with near real time confocal microscopy.

The use of high resolution, in vivo confocal imaging may offer a clinical tool to detect early neoplasia and reduce the incidence and mortality of cancer. Our laboratory is currently examining the feasibility of using confocal microscopy for non-invasive diagnosis of dysplasia and early carcinoma in epithelial tissue. We are performing a series of ex vivo studies investigating the optical properties of normal and abnormal biopsies to quantify the diagnostic capability of this technology to discriminate between normal and pre-cancerous tissue. These studies use a near real time reflectance confocal microscope to acquire images at various depths throughout the epithelium. To date, we have completed a twenty-five patient study of cervical biopsies and have acquired images from six patients of an approved twenty-two patient study in the oral cavity. The cervical study has shown a distinct difference between normal and dysplastic tissue which can be used diagnostically, while initial results from the oral cavity are promising even with increased keratin scattering. In conclusion, our examination of normal and precancerous biopsies has demonstrated the confocal microscope's ability to image sub-cellular morphology at a resolution making accurate diagnosis possible and supporting this technique's potential for in vivo assessment of dysplasia.

Biopsy↗
PubMed1998

Simultaneous detection of cell volume and intracellular pH in isolated rat duodenal cells by confocal microscopy and BCECF.

The combination of confocal laser scan microscopy and the pH-sensitive fluorescent dye BCECF allowed us to record simultaneously intracellular pH, cell viability and relative cell volume. pH was measured by using the pH-sensitive excitation wavelength at 488 nm and the pH-independent excitation wavelength at 442 nm to obtain ratio images. Cell volume was traced by measuring fluorescence dye concentration at 442 nm. Isolated villus tip rat duodenal enterocytes were exposed to 20 mM NH4Cl, sodium free, or 1 mM amiloride buffer. Sodium free buffer and amiloride buffer acidified the cells. Cell volume did not change in sodium free buffer, or NH4Cl exposure, but amiloride led to an increase in cell volume of 20%. After acidification of the duodenal cells, amiloride buffer increased cell volume by almost 50%. These studies revealed that cell volume regulation during pH changes in short-living cells could easily be detected by confocal microscopy and BCECF.

Animals↗