Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Confocal microscopy”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37Linked to original sources
PubMed1996

Photolysis of caged compounds characterized by ratiometric confocal microscopy: a new approach to homogeneously control and measure the calcium concentration in cardiac myocytes.

Here we describe the subcellularly uniform control of the intracellular Ca2+ concentration ([Ca2+]i) by flash photolysis of caged Ca2+ or a caged Ca2+ buffer. A mixture of the two Ca2+ indicators Fluo-3 and Fura-red was used together with a laser-scanning confocal microscope to reveal spatial aspects of intracellular Ca2+ signals. The patch clamp technique in the whole-cell variant was applied to load the cells with the indicator mixture together with either DM-nitrophen or diazo-2 and to measure changes in the membrane current. An in vivo calibration was performed to convert the Fluo-3/Fura-red fluorescence ratios to [Ca2+] values. The resulting calibration curve suggested an apparent KD of 1.6 microM, Rmax of 2.15, Rmin of 0.08 and a Hill-coefficient of 0.75 for the indicator mixture. Controlled rupture of the cell membrane revealed a large fraction of immobile intracellular Fura-red fluorescence that may account for the reduced in vivo Rmax value when compared to the in vitro value of 3.1. In cardiac myocytes, flash photolytic release of Ca2+ from DM-nitrophen generated inwardly directed Na+/Ca2+ exchange currents and Ca2+ signals that were graded with the discharged flash-energy. Rapid line-scans revealed subcellularly homogeneous [Ca2+] jumps regardless of the discharged flash energy. Ca2+ signals evoked by L-type Ca2+ currents (ICa) could be terminated rapidly in a spatially homogeneous manner by UV flash photolysis of diazo-2. No side-effects of the photolytic products of DM-nitrophen or diazo-2 with the mixture of Fluo-3/Fura-red were detectable in our experiments. The combination of UV flash photolysis and laser scanning confocal microscopy enabled us to control [Ca2+]i homogeneously on the subcellular level. This approach may improve our understanding of the subcellular properties of cardiac Ca2+ signalling. The technique can also be applied in other cell types and with other signalling systems for which caged compounds are available.

Acetates↗
PubMed2000

Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy.

Abnormal fibrin architecture is thought to be a determinant factor of hypofibrinolysis. However, because of the lack of structural knowledge of the process of fibrin digestion, relationships between fibrin architecture and hypofibrinolysis remain controversial. To elucidate further structural and dynamic changes occurring during fibrinolysis, cross-linked plasma fibrin was labeled with colloidal gold particles, and fibrinolysis was followed by confocal microscopy. Morphological changes were characterized at fibrin network and fiber levels. The observation of a progressive disaggregation of the fibrin fibers emphasizes that fibrinolysis proceeds by transverse cutting rather than by progressive cleavage uniformly around the fiber. Plasma fibrin clots with a tight fibrin conformation made of thin fibers were dissolved at a slower rate than those with a loose fibrin conformation made of thicker (coarse) fibers, although the overall fibrin content remained constant. Unexpectedly, thin fibers were cleaved at a faster rate than thick ones. A dynamic study of FITC-recombinant tissue plasminogen activator distribution within the fibrin matrix during the course of fibrinolysis showed that the binding front was broader in coarse fibrin clots and moved more rapidly than that of fine plasma fibrin clots. These dynamic and structural approaches to fibrin digestion at the network and the fiber levels reveal aspects of the physical process of clot lysis. Furthermore, these results provide a clear explanation for the hypofibrinolysis related to a defective fibrin architecture as described in venous thromboembolism and in premature coronary artery disease.

Fibrin↗
PubMed2006

Confocal microscopy of the light organ crypts in juvenile Euprymna scolopes reveals their morphological complexity and dynamic function in symbiosis.

In the hours to days following hatching, the Hawaiian bobtail squid, Euprymna scolopes, obtains its light-emitting symbiont, Vibrio fischeri, from the surrounding environment and propagates the bacteria in the epithelial crypts of a specialized light organ. Three-dimensional analyses using confocal microscopy revealed that each of the three crypts on either side of the juvenile light organ is composed of four morphological regions. Progressing from the lateral pore to the medial blind end of each crypt, the regions consist of 1) a duct, 2) an antechamber, 3) a bottleneck, and 4) a deep region. Only the deep region houses a persistent bacterial population, whereas the duct, antechamber, and bottleneck serve as conduits through which the bacteria enter during initial colonization and exit during diel venting, a behavior in which approximately 90% of the symbionts are expelled each dawn. Our data suggest that, like the duct, the antechamber and bottleneck may function to promote and maintain the specificity of the symbiosis. Pronounced structural and functional differences among the deep regions of the three crypts, along with previously reported characterizations of embryogenesis, suggest a continued developmental progression in the first few days after hatching. Taken together, the results of this study reveal a high degree of complexity in the morphology of the crypts, as well as in the extent to which the three crypts and their constituent regions differ in function during the early stages of the symbiosis.

Aliivibrio fischeri↗
PubMed2004

Immunolocalization of CB1 receptor in rat striatal neurons: a confocal microscopy study.

Several lines of evidence indicate that cannabinoids, among other functions, are involved in motor control. Although cannabinoid receptors (CB(1)) mRNA has been observed in medium-sized spiny neurons of the striatum, a description of the precise localization of CB(1) at a protein level among striatal cells is still lacking. Therefore, we performed immunohistochemical studies with light and confocal microscopy to identify neuronal subpopulations that express CB(1) and to assess the distribution of the receptor within these neurons. In our single label light microscopy study, CB(1) was observed in most medium-sized neurons of the caudate-putamen. However, CB(1) was also present in large-sized neurons scattered throughout the striatum. Our dual-label study showed that 89.3% of projection neurons in matrix contain CB(1), and that 56.4% of projection neurons in patch are labeled for CB(1). To investigate the presence of CB(1) among the different subclasses of striatal interneurons we performed a double-labeling study matching CB(1) and each of the striatal interneuron markers, namely, choline acetyl-transferase, parvalbumin, calretinin, and nitric oxide synthase. Our double-label study showed that most parvalbumin immunoreactive interneurons (86.5%), more than one-third (39.2%) of cholinergic interneurons, and about one-third (30.4%) of the NOS-positive neurons are labeled for CB(1). Calretinin-immunolabeled neurons were devoid of CB(1).

Animals↗
PubMed2002

Evaluation of hepatic histology by near-infrared confocal microscopy: a pilot study.

Liver biopsy is a necessary procedure in establishing the tissue diagnosis of many liver conditions and often guides therapeutic strategies. Current histopathologic techniques are either time-consuming or tissue-destroying; hence the potential need for a fast and nondestructive imaging technique of unfixed human liver. This pilot study evaluates the use of near-infrared reflectance confocal microscopy (CM) in the study of human liver histopathology. Without cutting or staining the tissue, CM provides images of bulk parenchyma showing cellular and subcellular detail and depicting morphologic features of hepatic parenchyma in both diseased and nondiseased states. This article presents a series of 12 human liver biopsy samples, providing an overview on the potential of this technique in assessing common findings from light microscopy.

Adult↗
PubMed1997

Confocal microscopy of cataracts from animal model systems: relevance to human nuclear cataract.

A recent study demonstrated that cytosolic lipid membrane structures, independent of the plasma membrane, preferentially occurred in human cataractous lenses. Animal model systems of cataractogenesis (selenite treated rats: galactose fed rats; buthionine-sulfoxime treated mice; Emory mice) were screened for possible relevant structures using the lipid membrane probe DiI and confocal microscopy. Well delineated plasma membranes of lens fiber cells with independent cytosolic staining structures were only observed in the selenite model system. These cytosolic structures were not observed in aged matched control lenses or within the transparent cortical regions of selenite treated animals with intense nuclear opacification. These results suggested that the morphological changes in DiI staining structures seen in the nucleus of the human cataractous lens were best approximated by those seen in the selenite model system.

Animals↗
PubMed2000

Colocalization of dihydropyridine and ryanodine receptors in neonate rabbit heart using confocal microscopy.

Because of undeveloped T tubules and sparse sarcoplasmic reticulum, Ca(2+)-induced Ca(2+) release (CICR) may not be the major mechanism providing contractile Ca(2+) in the neonatal heart. Spatial association of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), a key factor for CICR, was examined in isolated neonatal rabbit ventricular myocytes aged 3-20 days by double-labeling immunofluorescence and confocal microscopy. We found a significant increase (P < 0.0005) in the degree of colocalization of DHPR and RyR during development. The number of voxels containing DHPR that also contained RyR in the 3-day-old group (62 +/- 1.8%) was significantly lower than in the other age groups (76 +/- 1.3 in 6-day old, 75 +/- 1.2 in 10-day old, and 79 +/- 0.9% in 20-day old). The number of voxels containing RyR that also contained DHPR was significantly higher in the 20-day-old group (17 +/- 0.5%) compared with the other age groups (10 +/- 0.7 in 3-day old, 11 +/- 0.6 in 6-day old, and 11 +/- 0.5% in 10-day old). During this period, the pattern of colocalization changed from mostly peripheral to mostly internal couplings. Our results provide a structural basis for the diminished prominence of CICR in neonatal heart.

Animals↗
PubMedDate not supplied

Quantification of dental microwear by tandem scanning confocal microscopy and scale-sensitive fractal analyses.

Dental microwear analysis is among the most commonly used approaches to reconstructing the diets of extinct animal species and past peoples. The usual procedure involves imaging tooth wear surfaces by scanning electron microscopy (SEM). Surfaces are characterized quantitatively by measurement of individual wear features (pits and scratches) on photomicrographs. Recent studies of living animals have shown associations between diets on one hand and patterns of dental microwear on the other. Furthermore, patterns on fossil teeth have been used to reconstruct diets in extinct forms. However, conventional methods for microwear analysis are limited. Scanning electron microscopy does not provide a true representation of these surfaces in three dimensions, and identification and measurement of individual features is time consuming, subjective, and subject to high interobserver error. This paper describes a new approach to the analysis of dental microwear using tandem scanning confocal microscopy and scale-sensitive fractal analyses. The instrument used in this study provides three-dimensional coordinates representing surfaces at a resolution equivalent to that employed by most SEM microwear studies. Fractal analyses offer objective, repeatable, quantitative characterization of surfaces. This approach eliminates major sources of error and increases power to resolve differences between species. Moreover, rapid surface characterization will allow examination of large samples to assess within species variation and to make finer distinctions between species.

Animals↗
PubMed2003

Development of virtual histology and virtual biopsy using laser-scanning confocal microscopy.

The aim of this project is to acquire a direct image of histology from in vivo gastrointestinal mucosa. In other words, the task of 'endo-microscope' is to observe the cellular architecture of tissue in vivo during routine endoscopic examination. As the first step to completing this study, resected fresh specimens from the oesophagus. stomach and colon were examined by laser-scanning confocal microscopy (LCM) (Fluoview, Olympus, Tokyo). Fresh untreated mucosal specimens obtained by endoscopic pinch biopsy, polypectomy or endoscopic mucosal resection were collected and placed in normal saline and examined by LCM, collecting the reflective light of a 488-nm wavelength argon laser beam. As the second step, a probe-type LCM 'endo-microscope' was designed and applied to observe the human oral-cavity mucosa. The probe has 4.5-mm outer diameter and 20-cm length, which enables easy access to oral cavity mucosa. The estimated special resolution of the probe is 1-5 microm. A real-time microscopic image directly from ex vivo fresh specimens was acquired. The acquired LCM images corresponded well with the conventional H-E light microscopic images. Cell wall, nucleus and cytoplasm were simultaneously visualized by LCM scanning. This novel method enables serial imaginary microscopic sections on fresh specimens. In addition, a probe-type LCM 'endo-microscope' was designed and was applied to observe human oral cavity mucosa. Virtual histological images from the living oral squamous cell were successfully obtained. LCM images from ex vivo fresh specimens demonstrated the features of the H-E staining histological image. In the next step to accomplish our project, we developed a LCM probe with 4.5-mm outer diameter to obtain a virtual image of human oral cavity mucosa.

Biopsy↗
PubMed2006

The use of confocal microscopy to assess surface roughness of two milled CAD-CAM ceramics following two polishing techniques.

OBJECTIVE: To assess the effects of two polishing techniques on the surface roughness of a commercial and experimental dental ceramic for use in a chairside CAD-CAM system. METHODS: A standard onlay was reproduced fifteen times in each of the two materials. Each stub was retained and examined using confocal microscopy in reflection mode. Five random samples of each were polished with Sof-Lex aluminium oxide discs, five polished with diamond impregnated polishing wheels and five left in the as-machined state. Surface roughness parameters were measured for each material group in each condition and statistical analyses carried out to determine the efficacy of the polishing techniques for each material. RESULTS: The as-machined surfaces of both materials had a statistically similar surface finish. Polishing with Sof-Lex discs was effective at reducing surface roughness of both materials. Whilst the polishing wheels were effective in reducing surface roughness for the commercial material, there was no statistically significant difference in the surface roughness of the experimental material in the as-machined or wheel polished states. SIGNIFICANCE: Although Sof-Lex discs were effective in reducing surface roughness of the two materials examined in this study, wheel polishing should not be used to reduce surface roughness of the experimental material. This will need to be taken into consideration in any future clinical studies of this material.

Aluminum Oxide↗
PubMed2003

Constitutive nuclear localization and initial cytoplasmic apoptotic activation of endogenous caspase-3 evidenced by confocal microscopy.

The localization of caspases and their substrates in different cellular compartments may be one way to regulate apoptosis. Caspase-3-dependent proteolysis of inhibitor caspase-activated deoxyribonuclease (ICAD) activates caspase-activated deoxyribonuclease (CAD), which induces apoptotic internucleosomal DNA degradation. The nuclear localization of ICAD, pro- and active-caspase-3 molecules remains a controversial issue. Using a combination of immunodetection of endogenous molecules and confocal microscopy, we analysed the kinetics of the procaspase-3 and CAD activation induced by FAS triggering in Jurkat cells. Through a semi-quantitative image analysis, we showed a constitutive nuclear localization of pro-caspase 3 and ICAD in non-apoptotic cells. FAS stimulation induced 7A6 apoptotic antigen expression, which could be related to three different sequential patterns of nuclear chromatin organization. Active-caspase-3 first appeared in the cytoplasm and was next observed in the nucleus. Simultaneously, the amount of ICAD located in the nucleus decreased, whereas the amount of ICAD located in the cytoplasm remained unchanged. Thus, our experiments using in situ immunodetection of endogenous molecules show that the ICAD cleavage by the active-caspase-3 probably takes place in the nucleus. These results provide new perspectives about the subcellular compartmentation and traffic of caspases during the apoptotic process.

Apoptosis↗
PubMed1994

Quantitative three-dimensional confocal microscopy of synaptic structures in living brain tissue.

In order to study changes in synaptic structure that accompany learning and memory, we have developed optical methods to visualize dendritic spines and presynaptic terminals in living, electrically monitored brain slices maintained in vitro. Focal microapplication of the fluorescent lipophilic dye DiI provides Golgi-like staining of small numbers of cells and processes that can be resolved clearly using confocal microscopy; viability of stained cells is established by exclusion of the fluorescent DNA-binding dye ethidium bromide. Serial optical sections are enhanced by deconvolution and other image processing methods. The resulting high-resolution images are combined in an automated procedure to generate three-dimensional reconstructions, in which submicron synaptic structures can be viewed and measured. These unbiased methods allow volume changes in individual, living synaptic structures to be assessed quantitatively over periods of hours or days in development or in response to stimulation, drug application, or other perturbations.

Animals↗
PubMed2000

Three distinct types of Ca(2+) waves in Langendorff-perfused rat heart revealed by real-time confocal microscopy.

Although Ca(2+) waves in cardiac myocytes are regarded as arrhythmogenic substrates, their properties in the heart in situ are poorly understood. On the hypothesis that Ca(2+) waves in the heart behave diversely and some of them influence the cardiac function, we analyzed their incidence, propagation velocity, and intercellular propagation at the subepicardial myocardium of fluo 3-loaded rat whole hearts using real-time laser scanning confocal microscopy. We classified Ca(2+) waves into 3 types. In intact regions showing homogeneous Ca(2+) transients under sinus rhythm (2 mmol/L [Ca(2+)](o)), Ca(2+) waves did not occur. Under quiescence, the waves occurred sporadically (3.8 waves. min(-1) x cell(-1)), with a velocity of 84 microm/s, a decline half-time (t(1/2)) of 0.16 seconds, and rare intercellular propagation (propagation ratio <0.06) (sporadic wave). In contrast, in presumably Ca(2+)-overloaded regions showing higher fluorescent intensity (113% versus the intact regions), Ca(2+) waves occurred at 28 waves x min(-1) x cell(-1) under quiescence with a higher velocity (116 microm/s), longer decline time (t(1/2) = 0.41 second), and occasional intercellular propagation (propagation ratio = 0.23) (Ca(2+)-overloaded wave). In regions with much higher fluorescent intensity (124% versus the intact region), Ca(2+) waves occurred with a high incidence (133 waves x min(-1) x cell(-1)) and little intercellular propagation (agonal wave). We conclude that the spatiotemporal properties of Ca(2+) waves in the heart are diverse and modulated by the Ca(2+)-loading state. The sporadic waves would not affect cardiac function, but prevalent Ca(2+)-overloaded and agonal waves may induce contractile failure and arrhythmias.

Aniline Compounds↗
PubMed2003

Microanatomical and biochemical origins of normal and precancerous cervical autofluorescence using laser-scanning fluorescence confocal microscopy.

Clinical studies have shown that in vivo fluorescence spectroscopy can improve the diagnosis of cervical precancer. Recent work suggests that epithelial fluorescence increases, whereas stromal fluorescence decreases, with precancer. However, the microanatomic and biochemical sources of fluorescence in living cervical tissue have not yet been established. This study aims to characterize the origins of living normal and precancerous cervical fluorescence at microscopic levels using laser-scanning fluorescence confocal microscopy. Ten pairs of colposcopically normal and abnormal biopsies were obtained; transverse, 200 microm thick, short-term tissue cultures were prepared and imaged when viable with UV (351-364 nm) and 488 nm excitation before and after addition of the vital dye, Mitotracker Orange. In normal epithelium basal epithelial cells showed cytoplasmic fluorescence; parabasal, intermediate and superficial cells showed fluorescence only at the periphery of the cell. In low-grade precancers cytoplasmic fluorescence was visible in the bottom one-third of the epithelium; in high-grade precancers cytoplasmic fluorescence was visible throughout the lower two-thirds of the epithelium. Cytoplasmic fluorescence was colocalized with the MitoTracker probe and is attributed to mitochondrial reduced form of nicotinamide adenine dinucleotide at UV excitation and mitochondrial flavin adenine dinucleotide at 488 nm excitation. Stromal fluorescence originated from matrix fibers; with the development of precancer the density and fluorescence intensity of matrix fibers decrease. Autofluorescence properties of precancerous cervix reflect an increased number of metabolically active mitochondria in epithelial cells and a reduced stromal fluorescence, which can be an indicator for altered communication between precancerous epithelium and stroma. These changes can explain differences in in vivo fluorescence spectra of normal and precancerous cervical tissue.

Cervix Uteri↗
PubMed1997

Confocal microscopy using oblique sections for measurement of corneal epithelial thickness in conscious humans.

PURPOSE: Thickness measurements by confocal microscopy in conscious human subjects may be liable to error as a result of instability of the eye or instrument. Our aim was to evaluate a technique which was expected to be less sensitive to such problems. METHOD: Thickness of corneal epithelium was determined from oblique confocal sections through cornea. A contact lens of known thickness worn by subjects was used to calibrate images. RESULTS: There were two layers in images which could have corresponded to the stromal/epithelial interface. The mean result in each subject ranged from 38 to 53 microm using the more superficial layer and 46 to 60 microm using the deeper one. The smaller values gave the distance between the epithelial surface and the sub-epithelial nerve plexus and thus seemed to correspond to epithelial thickness. CONCLUSIONS: Measurements of epithelial thickness by our new method are comparable with results of earlier studies.

Adult↗
PubMed2002

Endothelial cell injury induced by preservation solutions: a confocal microscopy study.

BACKGROUND: We evaluated the effects of standard preservation solutions on cultured human greater saphenous vein endothelial cells. METHODS: Endothelial cells (eight strains) were preincubated for 6 or 24 hours at 4 degrees C in Celsior, Euro-Collins, St. Thomas Hospital II, and University of Wisconsin solutions, reincubated in warm oxygenated culture medium 199, and observed up to 48 hours. Culture viability was assessed through cell counting and confocal microscopy of calcein loaded cells. RESULTS: Incubation in both Euro-Collins and St. Thomas, but not in Celsior or University of Wisconsin solutions, caused significant cells losses and diffuse morphological damages characterized by solution-specific distinctive alterations. Injury caused by 6-hour, but not by 24-hour treatment, was reversible. CONCLUSIONS: The incubation with Celsior and University of Wisconsin solutions substantially preserved endothelial viability and proliferative capability. Conversely, a prolonged incubation in either Euro-Collins or St. Thomas solutions caused severe and potentially irreversible damage referable to the induction of, respectively, apoptotic or necrotic changes.

Aged↗
PubMed1999

Characterization of psoriasis in vivo by reflectance confocal microscopy.

Among available non-invasive imaging tools, confocal reflectance microscopy (CM) provides the highest-resolution optical sectioning of skin in vivo, to a controlled depth of 200-350 microns, the level of the upper reticular dermis. In this study, CM was used to view the histological features of psoriasis in vivo in lesional and non-lesional skin of five patients with stable psoriasis vulgaris. Stereohistological analysis of non-invasive confocal sections, and the correlation with transverse (en face) hematoxylin- and eosin-stained sections from biopsies, was also performed. In psoriatic lesions, nucleated corneocytes and collections of infiltrating inflammatory cells were clearly seen. Morphometric parameters such as epidermal height, length of papillary dermis, and the count of dermal papillae were also easily quantified. In the upper dermis, dilated capillary loops were always present. Since CM sections are en face, the presence or absence of the granular layer could not be visualized in single frames, but could be monitored in a sequence of real-time videotaped images. In summary, CM provides a new technique for histologically evaluating psoriasis in vivo.

Adult↗
PubMed2005

Confocal microscopy and biochemical analysis reveal spatial and functional separation between anandamide uptake and hydrolysis in human keratinocytes.

The signaling activity of anandamide (AEA) is terminated by its uptake across the cellular membrane and subsequent intracellular hydrolysis by the fatty acid amide hydrolase (FAAH). To date, the existence of an AEA membrane transporter (AMT) independent of FAAH activity remains questionable, although it has been recently corroborated by pharmacological and genetic data. We performed confocal microscopy and biochemical analysis in human HaCaT keratinocytes, in order to study the cellular distribution of AMT and FAAH. We found that FAAH is intracellularly localized as a punctate staining partially overlapping with the endoplasmic reticulum. Consistently, subcellular fractionation and reconstitution of vesicles from membranes of different compartments demonstrated that FAAH activity was localized mainly in microsomal fractions, whereas AMT activity was almost exclusively in plasma membranes. These results provide the first morphological and biochemical evidence to support the view that transport and hydrolysis are two spatially and functionally distinct processes in AEA degradation.

Amidohydrolases↗