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At least 973 records · Page 54Linked to original sources
PubMed2001

Early and delayed afterdepolarizations in rabbit heart Purkinje cells viewed by confocal microscopy.

We investigated action potentials and Ca(2+) transients in rabbit Purkinje myocytes using whole cell patch clamp recordings and a confocal microscope. Purkinje cells were loaded with 5 microM Fluo-3/AM for 30min. Action potentials were elicited by application of a stimulus delivered through the recording pipettes. When Purkinje cells were stimulated in 2.0mM Ca(2+), transverse XT line scans revealed a symmetrical 'U'-shaped Ca(2+) transient demonstrating that the transient was initiated at the cell periphery. When Purkinje cells were superfused with 1 microM isoprenaline, both early and delayed afterdepolarizations were induced. XT line scans of cells exhibiting early afterdepolarizations showed a second symmetrical 'U'-shaped transient. This Ca(2+) transient was initiated at the cell periphery suggesting reactivation of the Ca(2+) current. In contrast, in Purkinje cells exhibiting delayed afterdepolarizations and a corresponding transient inward current, XT line scans revealed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell. Immunofluorescence staining of Purkinje cells with an antibody to ryanodine receptors (RyRs) revealed that RyRs are located at regularly spaced intervals throughout the interior of Purkinje cells. These results suggest that, although RyRs are located throughout Purkinje cells, only peripheral RyRs are activated to produce transients, sparks and early afterdepolarizations. During delayed afterdepolarizations, we observed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell as well as large central increases in Ca(2+). Although the latter may result from central release, we cannot exclude the possibility that it reflects Ca(2+) diffusion from subsarcolemmal sites.

Action Potentials↗
PubMed1999

2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein as a dual-emission fluorescent indicator of intracellular pH suitable for argon laser confocal microscopy.

The widely used fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) serves as a pH-sensitive indicator in classical microscopy. Characteristics of BCECF were studied and a way of employing the probe in a confocal laser scanning microscope equipped with an argon laser at 488 nm was developed, based on the fact that the emission fluorescence spectra are pH-dependent with spectral maximum shift from 518 to 529 nm. Optical filters for the dual-emission ratio method were set to 506 and 529 nm. pH values measured inside a single cell of Saccharomyces cerevisiae were similar to those obtained with other pH estimation methods.

Argon↗
PubMed1998

Simultaneous detection of two GFP spectral mutants during in vivo confocal microscopy of migrating Dictyostelium cells.

A method is described that allows simultaneous measurement of two spectrally distinguishable green fluorescent protein (GFP) mutants with a confocal microscope. In contrast to previously described methods, neither UV excitation nor repetition of scans is required. Therefore the method is well-suited to the long-time observation of living cells in three-dimensional microscopy and time series recording, as demonstrated with GFP-expressing Dictyostelium discoideum cells.

Animals↗
PubMed2005

Multimodal in vivo optical imaging, including confocal microscopy, facilitates presurgical margin mapping for clinically complex lentigo maligna melanoma.

Knowledge of the accurate margins of a lentigo maligna melanoma (LMM) is crucial in the presurgical evaluation of the patient. Towards this end clinicians have utilized the Wood's lamp and dermoscopy to help delineate the borders of the LMM. However, many LMMs arise on photodamaged skin, making it difficult to determine the border of the LMM and separate it from the background lentiginous skin. We present a case of a patient with a recurrent LMM on the scalp that developed in a background of photodamage with diffuse melanocytic atypia and lentigines, making it virtually impossible to determine the precise margins of the LMM by clinical, Wood's lamp or dermoscopic examination. To avoid subjecting the patient to multiple staged excisions we attempted to determine the margins of the LMM by utilizing in vivo confocal laser scanning reflectance microscopy. Using this, it was apparent that there were increased numbers of atypical/dendritic intraepidermal melanocytes in all layers of the epidermis within the LMM. In contrast, skin not involved with the LMM, as viewed under confocal laser examination, had normal honeycomb architecture and no abnormal melanocytes. The confocally determined border was further confirmed by obtaining multiple punch biopsies that were evaluated by haematoxylin and eosin histology and immunohistochemistry. Based on this information, the presurgical margins were marked and the tumour excised accordingly. The excised tissue was examined with multiple-step sections and the margins were determined to be clear. There has been no evidence of tumour recurrence after 1 year. In conclusion, this case illustrates that confocal reflectance microscopy, in conjunction with other in vivo optical instruments, can be utilized to enhance the accuracy for the presurgical margin mapping of LMM.

Aged↗
PubMed1995

Hepatocyte growth factor-induced calcium waves in hepatocytes as revealed with rapid scanning confocal microscopy.

Cytosolic Ca2+ transients induced by hepatocyte growth factor (HGF) were imaged in primary cultured rat hepatocytes using newly developed rapid scanning confocal microscopes and indo-1. HGF (40 ng/ml) increased cytosolic free Ca2+ concentration ([Ca2+]i) in about 60% of hepatocytes, in 45% of which the increases were oscillatory. In each of the oscillatory hepatocytes, the repetitive increases in [Ca2+]i originated from a specific same region adjacent to the cell membrane and propagated across the cell like waves. Phenylephrine (10 microM) also induced Ca2+ waves. The locus where HGF-induced Ca2+ waves and phenylephrine-induced Ca2+ waves were originated was the same, and there was a correlation in the peak height between HGF-induced Ca2+ waves and phenylephrine-induced Ca2+ waves in each cell, although the mechanisms of inositol 1,4,5-trisphosphate (ins(1,4,5)P3) formation induced by HGF should be different from those by phenylephrine. On the other hand, there was no correlation between sensitivity of each cell to HGF and that to phenylephrine which were measured as latent periods prior to Ca2+ rises after an addition of the agonists. These results suggested the following: the spatial patterns of Ca2+ waves were decided by a common mechanism, probably not the propagation of ins(1,4,5)P3 but the distribution of ins(1,4,5)P3-sensitive Ca2+ pools; sensitivities of each cell to the agonists did not mainly depend on the common mechanism.

Adrenergic alpha-Agonists↗
PubMed1995

Differences in features of calcium transients between the nucleus and the cytosol in cultured heart muscle cells: analyzed by confocal microscopy.

We analyzed spatio-temporal characteristics of Ca2+ transients in the cytosol and the nucleus of cultured neonatal rat heart cells using confocal imaging with Indo-1 and Fluo-3. In resting heart muscle cells, nuclear [Ca2+] was maintained lower than the cytosolic level. The rise in nuclear [Ca2+], during either E-C coupling or propagation of the Ca2+ wave, began at the edge of the nucleus in the immediate vicinity of the rise in global or localized cytosolic [Ca2+], and spread inwardly. The rise in [Ca2+] was slower and smaller in the nucleus than in the cytosol. The decay in [Ca2+] was also slower in the nucleus than the cytosol, thereby reversing the initial [Ca2+] gradient between them. Caffeine markedly enhanced the rise in nuclear [Ca2+] while maintaining inward spreading. The heterogeneity of nuclear Ca2+ transients during cellular contractilities suggests that influx of Ca2+ from perinuclear stores into the nucleus plays a predominant role in the nuclear [Ca2+] rise. The results also indicated that spatio-temporal characteristics of Ca2+ transients are quite different between the nucleus and the cytosol, thereby suggesting that they are differentially regulated in the nucleus and the cytosol.

Animals↗
PubMedDate not supplied

Corneal cell density measurement in vivo by scanning slit confocal microscopy: method and validation.

PURPOSE: Method and validation of a technique to quantify cell density in vivo in 6 corneal layers with a scanning slit confocal microscope (SSCM). METHOD: A confocal image of a small volume in a corneal layer is registered on videotape. Cells or nuclei according to a layer classification are counted manually using an unbiased frame. Surface cell density is calculated from an image on the screen, and volumetric density is obtained using stereological methods. RESULTS: Image distortion on the screen is less than 3%. The classification of a cell layer is verified by determining the position of the measurement volume in the cornea. Validation of density measurements is performed by comparing confocal results with those obtained by histology. The difference between the two methods varies from -24.1% (posterior stroma) to +16.4% (basal layer). Intersession and intrasession repeatability are 8.3 and 5.8%, respectively. The cell density (mean +/- SD) in 20 healthy controls in the superficial, basal and endothelial layers was 759 +/- 162, 5,817 +/- 632 and 2,743 +/- 285 cells.mm(-2) (surface), and in the anterior, mid and posterior stroma 28,616 +/- 3,081, 19,578 +/- 4,421 and 26,073 +/- 5,962 cells.mm(-3) (volumetric). These results are in accordance with those of other investigators. CONCLUSIONS: The SSCM can produce repeatable quantitative measurements of corneal cell density in conscious humans.

Adult↗
PubMed2003

Europium uptake and partitioning in oat (Avena sativa) roots as studied by laser-induced fluorescence spectroscopy and confocal microscopy profiling technique.

The uptake of Eu3+ by elongating oat roots was studied by fluorescence spectroscopy, fluorescence lifetime measurement, and a laser excitation time-resolved confocal fluorescence profiling technique. The results of this work indicated that initial uptake of Eu3+ was highest within the undifferentiated cells of the root tip just behind the root cap, a region of maximal cell growth and differentiation and with incomplete formation of the Casparian strip around the central vascular cylinder. Distribution of assimilated Eu3+ within the root's differentiation and elongation zone was nonuniform. Higher concentrations of Eu3+ were observed within the vascular cylinder, specifically in the phloem and developing xylem parenchyma. Elevated levels of the metal were also observed in the root hairs of the mature root zone. Fluorescence spectroscopic characteristics of the assimilated Eu3+ suggested that the Eu3+ exists as inner-sphere mononuclear complexes inside the root. This work also demonstrated the effectiveness of a time-resolved Eu3+ fluorescence spectroscopy and confocal fluorescence profiling techniques for the in vivo, real-time study of metal [Eu3+] accumulation by a functioning intact plant root. This approach can prove valuable for basic and applied studies in plant nutrition and environmental uptake of actinide radionuclides.

Avena↗
PubMed1995

Use of a new fluorescent probe, seminaphthofluorescein-calcein, for determination of intracellular pH by simultaneous dual-emission imaging laser scanning confocal microscopy.

A new pH indicator, seminaphthofluorescein (SNAFL)-calcein acetoxymethyl ester, was used for intracellular pH (pHi) measurement in living MDCK cells with a laser scanning confocal microscope (LSCM) equipped with an Argon/Krypton laser and dual-excitation and dual-emission (FITC/Texas Red) filter set. SNAFL-calcein excitation maxima are approximately 492/540 nm (acid/base) and emission maxima are approximately 535/625 nm (acid/base) with a pKa value at approximately 7.0. The absorption/emission spectra of SNAFL-calcein indicate that the ratio of emission intensities of its basic/acidic forms is pH dependent. With an Argon/Krypton LSCM, we were able to monitor the acidic and basic forms of this dye simultaneously using dual-excitation (488/568 nm) and dual-emission (525-614 nm/> or = 615 nm) wavelengths (lambda s). The simultaneous dual-excitation/emission LSCM system allows for efficient recording of pHi dynamics (time resolution approximately 1 sec) in living cells. We have analyzed emission stability of the dye at different temperatures (22 degrees C and 37 degrees C) and constant pH, and at the same temperature (22 degrees C) but various pHs (6.6, 7.0, and 7.4). Bleaching rate is slightly higher at 37 degree C than that at 22 degrees C. The basic form of the dye (lambda Em approximately 625 nm) has a slightly higher bleaching rate than the acidic form (lambda Em approximately 535 nm) in standard culture medium (pH 7.3) at either 22 degree C or 37 degrees C. The pHi in MDCK cells calculated from ratio images (535 nm/625 nm) was 7.19 +/- 0.03 (mean +/- SEM, n = 20). Calibration experiments show that the useful pH range of SNAFL-calcein appears to be between 6.2 and 7.8, as the dye is difficult to calibrate outside this pH range.

Animals↗
PubMed1994

In vivo human corneal confocal microscopy of identical fields of subepithelial nerve plexus, basal epithelial, and wing cells at different times.

A technique is described to obtain time-lapse reflected light confocal images of cells in the basal epithelium and adjacent wing cell layer from the in vivo human cornea. The technique is based on the sequential relocation of the unique patterns of the subepithelial nerve plexuses immediately posterior to Bowman's membrane. The patterns of individual subepithelial nerve plexuses, as well as perforation points where nerves traverse Bowman's membrane, serve as fixed landmarks. A real-time, scanning slit, confocal microscope is used to obtain reflected light images of a subepithelial nerve plexus, and the anterior and adjacent fields of basal and wing cells in the in vivo human cornea. All of the photographs are obtained from single video frames without the necessity of frame averaging or digital image processing. The instrument and relocation technique are prerequisites for applying a time-lapse observation technique to investigate the dynamics of basal cell proliferation and differentiation in the living eye.

Adult↗
PubMed1991

Subcellular distribution of daunorubicin in P-glycoprotein-positive and -negative drug-resistant cell lines using laser-assisted confocal microscopy.

Four well defined multidrug-resistant cell lines and their drug-sensitive counterparts were examined for intracellular distribution of daunorubicin (DNR) by laser-assisted confocal fluorescence microscopy: P-glycoprotein-negative HL-60/AR cells, and P-glycoprotein-positive P388/ADR, KBV-1, and MCF-7/ADR cells. Both drug sensitive cell lines (HL-60/S, P388/S, KB3-1, and MCF-7/S) and drug-resistant cell lines (HL-60/AR, P388/ADR, KBV-1, and MCF-7/ADR) exposed to DNR showed a similar rapid distribution of drug from the plasma membrane to the perinuclear region within the first 2 min. From 2-10 min, the drug sensitive HL-60/S, P388/S, and MCF-7/S cells redistributed drug to the nucleus and to the cytoplasm in a diffuse pattern. In contrast, drug-resistant HL-60/AR, P388/ADR, and MCF-7/ADR redistributed DNR from the perinuclear region into vesicles distinct from nuclear structures, thereby assuming a "punctate" pattern. This latter redistribution could be inhibited by glucose deprivation (indicating energy dependence), or by lowering the temperature of the medium below 18 degrees C. The differences in distribution between sensitive and resistant cells did not appear to be a function of intracellular DNR content, nor the result of drug cytotoxicity. Drug-sensitive KB3-1 and -resistant KBV-1 cells did not fully follow this pattern in that they demonstrated an intracellular DNR distribution intermediate between HL-60/S and HL-60/AR cells with both "punctate" and nuclear/cytoplasmic uptake sometimes in the same cell. These data indicate that the intracellular distribution of DNR is an important determinant of drug resistance regardless of the overexpression of P-glycoprotein. The intracellular movement of drug requires the presence of glucose and a temperature above 18 degrees C, implicating energy-dependent processes and vesicle fusion in the distribution process. This intracellular transport of DNR away from the nucleus in multidrug-resistant cells may protect putative cell targets such as DNA against drug toxicity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Human fibroblast alterations induced by low power laser irradiation at the single cell level using confocal microscopy.

Low power laser irradiation is regarded to have a significant role in triggering cellular proliferation and in treating diseases of diverse etiologies. The present work contributes to the understanding of the mechanisms of action by studying low power laser effects in human fibroblasts. Confocal laser scanning microscopy is used for irradiation and observation of the same area of interest allowing the imaging of laser effects at the single cell level and in real time. Coverslip cultures were placed in a small incubation chamber for in vivo microscopic observation. Laser stimulation of the cells was performed using the 647 nm line of the confocal laser through the objective lens of the microscope. Mitochondrial membrane potential (delta psi(m)), intracellular pH, calcium alterations and generation of reactive oxygen species (ROS) were monitored using specific fluorescent vital probes. The induced effects were quantified using digital image processing techniques. After laser irradiation, a gradual alkalinization of the cytosolic pH and an increase in mitochondrial membrane potential were observed. Recurrent spikes of intracellular calcium concentration were also triggered by laser. Reactive oxygen species were generated as a result of biostimulation. No such effects were monitored in microscopic fields other than the irradiated ones.

Cells, Cultured↗
PubMed1997

Four-dimensional imaging of living chondrocytes in cartilage using confocal microscopy: a pragmatic approach.

Regulation of cell volume is a fundamental cellular homeostatic mechanism in the face of osmotic stress. In normal articular cartilage, chondrocytes are exposed to a changing osmotic environment. We present a comprehensive protocol for studying the volume regulatory behavior of chondrocytes within intact cartilage tissue using confocal laser-scanning microscopy. Our data acquisition regime optimizes both signal-to-noise and cell viability during time-lapsed three-dimensional (3-D) (x, y, z, t) imaging. The porcine cartilage is treated as an integrated component of the imaging system, and we demonstrate methods for the direct assessment of tissue-induced axial attenuation and image distortion. Parameterized functions describing these two components of image degradation are used to correct experimental data. The current study also highlights the problems associated with the analysis and visualization of four-dimensional (4-D) images. We have devised two new types of data reconstruction. The first compresses each 3-D time point into a single quantitative view, termed a coordinate view. From these reconstructions we are able to simultaneously view and extract cell measurements. A second type, a 4-D reconstruction, uses color to represent relative changes in cell volume, again while maintaining the morphological and spatial information. Both these approaches of image analysis and visualization have been implemented to study the morphology, spatial distribution, and dynamic volume behavior of chondrocytes after osmotic perturbation. We have mapped chondrocyte shape, arrangement, and absolute volume in situ, which vary significantly from the tissue surface through to the underlying bone. Despite the rigid nature of the extracellular matrix, cartilage cells are osmotically sensitive and respond to stimulation of volume regulatory mechanisms. The combined techniques of confocal laser-scanning microscopy and vital cell labeling have enabled us to study, for the first time, the response of chondrocytes in situ to changes in interstitial osmotic pressure.

Animals↗
PubMed2005

Thickness distribution of fresh eardrums of cat obtained with confocal microscopy.

The aim of this study was to measure the spatial thickness distribution of the cat tympanic membrane (TM), a very thin, virtually transparent and delicate biological membrane. Axial fluorescence images taken perpendicular through isolated TM were recorded for five different cats using confocal laser scanning microscopy. Thickness was measured on the cross-section of the membranes in the axial images. A correction for focal shift due to refractive-index mismatch was applied. Similar thickness distributions were obtained in all measured samples (n = 9). The pars tensa had a rather constant thickness in the central region between the annulus and manubrium. The thickness increased steeply toward the peripheral rim. Thickness was smallest in the inferior region, with values ranging between 5.5 and 9 microm in the central part and up to 50 microm near the annulus. More superiorly, thickness was slightly higher, up to 20 microm, between the annulus and manubrium. The anterior part was thicker than the posterior side. These findings are strongly different from a current value in the literature. Our data allow a more precise representation of the eardrum in mathematical models, which are a prerequisite for a better understanding of middle-ear mechanics. The optical sectioning technique of the confocal microscope did not result in any preparation artifacts and was therefore also used to quantify shrinkage due to preparation of histological sections of TMs.

Animals↗
PubMed1990

Human cranial bone structure and the healing of cranial bone grafts: a study using backscattered electron imaging and confocal microscopy.

This study explored the microstructure of human cranial bone at different ages, and the survival, remodelling and modelling of cranial bone grafts. A combination of reflection and fluorescence confocal optical microscopy and scanning electron microscopy in the backscattered electron imaging mode was employed to examine highly polished block faces of plastic-embedded bone fragments as harvested for grafting, or recovered after a period in situ as a graft. The methods enabled remarkably detailed information on bone content, maturation and turnover to be gleaned from tiny scraps of bone. Microfractures in the harvested bone were repaired at the graft site, with welding of old and new bone indicating revascularization. Human cranial bone grafts successfully stimulated bone cell differentiation, supported new bone formation on resorbed and unresorbed surfaces, and underwent bone turnover. The type and organization of new bone reflected the growth rate and maturation of the graft rather than the age of the patient.

Adult↗
PubMed1996

4D confocal microscopy for visualisation of bone remodelling.

Until recently it was very time consuming and difficult to make three-dimensional (3D) images of newly formed bone. With the advent of confocal technologies and increased computer power, 3D imaging is greatly facilitated. In this paper we demonstrate that enhanced confocal visualisation of newly formed bone is possible when bone is labelled in vivo sequentially with two osteotropic markers (xylenol orange and tetracycline). Computer-assisted reconstruction of the confocal optical sections was achieved through the use of the CONVEX Application Visualisation System (AVS). The computerised image data provides the researcher with ample flexibility in displaying the results. It was found that CSLM combined with AVS is excellent for visualising the remodelling process in three and four dimensions, in which the fourth dimension is time. With this approach visualised bone remodelling has become possible in a manner not easily achieved by other techniques.

Animals↗
PubMed2005

Low-cost, scalable laser scanning module for real-time reflectance and fluorescence confocal microscopy.

We present a low-cost, high-speed, retrofitted laser scanning module for microscopy. The cage-mounted system, with various available fiber-coupled sources, offers a real-time imaging alternative to costly commercial systems with capabilities for conventional or confocal reflectance and fluorescence applications as well as advanced laser scanning microscopy implementations. Reflectance images of a resolution target and confocal images of fluorescent polystyrene beads are presented for system characterization. Confocal fluorescence image stacks of T84 epithelial cancer cells are presented to demonstrate application to biological studies. This laser scanning module is a flexible, scalable, high-speed alternative to commercial laser scanning systems suitable for applications requiring a simple imaging tool and for teaching laboratories.

Algorithms↗
PubMed2000

Confocal microscopy of electro-optic materials: effect of aberrations on the axial response in ac mode

Confocal scanning optical microscopy can be used to investigate the structure of electro-optic materials. Application of an ac electric field allows one to measure sensitively small changes in the reflection of light from a sample surface, and those changes can be related to the electro-optic properties. We observe the axial dependence of the ac light intensity to be a linear combination of the dc component and its axial derivative. Our analysis shows that astigmatic aberrations and the azimuthal dependence of the optical index in anisotropic materials can explain this behavior.

Journal Article↗