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At least 847 records · Page 47Linked to original sources
PubMed2005

Fluorescence fibre-optic confocal microscopy of skin in vivo: microscope and fluorophores.

BACKGROUND/AIMS: Fibre-optic confocal imaging in vivo is a new approach in the assessment of human skin. The objective is to describe a novel instrument and its operation and use in combination with fluorophores. METHODS: The Stratum is a fibre-optic fluorescence confocal microscope especially developed for the study of skin and mucous membranes. The system is flexible and any body site can be studied with a hand-held scanner. The light source is a 488 nm argon ion laser. Horizontal (en face) images of the epidermis and outer dermis are produced with cellular resolution. Magnification is approximately 1000 x . Fluorescein sodium is routinely used as fluorophore (intradermal injection or application to the skin surface). This fluorophore is safe for human use in vivo, but other substances (rhodamine B, Acridine Orange, green fluorescent protein, curcumin) have also been studied. RESULTS: The instrument produces sharp images of epidermal cell layers from the epidermal surface to the sub-papillary dermis, with sub-cellular resolution. The scanner is flexible in use. The technique of intradermal fluorophore injection requires some skill. CONCLUSIONS: We consider this fibre-optic instrument a potentially important tool in skin research for non-invasive optical biopsy of primarily the epidermis. Present use is focussed on research applications, where the fluorophore distribution in the skin may illustrate morphological changes in the epidermis.

Dermoscopy↗
PubMed1992

Visualizing cells in three dimensions using confocal microscopy, image reconstruction and isosurface rendering: application to glial cells in mouse central nervous system.

This paper describes a general method for visualizing individual cells in intact tissue in three dimensions. The method involves immunostaining intact tissue to label specific cells, "optical sectioning" the stained tissue by laser scanning confocal microscopy, computationally reconstructing a three dimensional image data set from the digitized confocal optical sections, delineating isosurfaces of specific intensity within the reconstructed image by a "marching cubes" algorithm to generate polygon meshes defining boundaries of cells, and displaying individual cells, identified as three dimensional objects enclosed by contiguous polygon meshes, using computer graphics techniques. Each of the components of this method has been described previously in conjunction with other applications. However the combination of these techniques to visualize a variety of different individual cell types in three dimensions in intact tissue represents a new approach. To illustrate the application of this method, we have visualized three different glial cell types in mouse CNS tissue. Oligodendrocytes, specifically stained with antibody to myelin basic protein, were used as an example of cells labelled with an internal membrane antigen. Astrocytes, specifically stained with antibody to glial fibrillary acidic protein, were used as an example of cells labelled with a cytoplasmic antigen. Microglia, specifically stained with Mac.1 antibody, were used as an example of cells labelled with an external membrane antigen. The images that are generated contain remarkably detailed volumetric and textural information that is not obtainable by conventional imaging techniques.

Algorithms↗
PubMed1993

Clinical and diagnostic use of in vivo confocal microscopy in patients with corneal disease.

BACKGROUND: The purpose of this article is to introduce the practicing ophthalmologist to the optical principles and images produced by a tandem scanning confocal microscope (recently approved by the Food and Drug Administration for general clinical use). The tandem scanning confocal microscope allows real-time viewing of structures in the living cornea at the cellular level in four dimensions (x, y, z, and time). METHODS: Nine patients (2 males, 7 females), ranging in age from 7 to 52 years, were examined. Images were recorded on super VHS videotape, digitized and processed on a computer workstation, and photographed for presentation. RESULTS: Two-dimensional (x, y) 400 x 400-microns images (9-microns z-axis thickness) are presented for normal corneal structures and for the clinical conditions of herpetic keratitis, wound healing after myopic excimer ablation, Acanthamoeba infection, corneal dystrophies (granular, Reis-Buckler), contact lens abrasion, and the irido-corneal endothelial syndrome. CONCLUSION: Clinical confocal microscopy has the unique potential of providing noninvasive assessment of corneal injury and disease at the cellular level that is not available currently from other technologies.

Acanthamoeba Keratitis↗
PubMed1993

Confocal microscopy and computer-assisted image reconstruction of astrocytes in the mammalian retina.

The distribution of astrocytes in the vascularized retina of pigs, rats and cats was investigated by confocal microscopy and computer-assisted image processing. In whole mounts, immunocytochemical identification was done by staining astrocytes for glial fibrillary acidic protein (GFAP), and blood vessels for alpha-smooth muscle actin or collagen IV. Double-staining was followed through consecutive optical sections and made it possible to precisely align the two markers in the inner retina. The resulting computer-assisted image reconstructions revealed asymmetric ensheathment of blood vessels by GFAP-positive fibres. The ultrastructural basis for this asymmetry, as studied by electron microscopy, was found to be different in pigs and cats. In the pig, astrocytes firmly ensheathed the vessel circumference, but glial filaments were much more abundant on the vitreal and lateral than on the scleral side. By contrast, in the cat astrocytes were generally confined to regions occupied by axonal bundles and constituted only part of the vascular glia limitans, else formed by Müller cells. Moreover, our observations unambiguously showed that individual astrocytes maintained simultaneous contact with axons and blood vessels and lined the vitreous body. The physical links provided by astrocytes suggest that they are able to function as central communicating elements between ganglion cells, the vasculature and the vitreous body.

Animals↗
PubMed2005

Co-localization of PARP-1 and lamin B in the nuclear architecture: a halo-fluorescence- and confocal-microscopy study.

A functional interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and lamin B has recently been proposed by nuclear fractionation, crosslinking, and immunoprecipitation experiments. Here we use fluorescence microscopy to verify and extend these findings. We analyze nuclear halo preparations by fluorescence in situ immuno staining (FISIS), which shares attributes with traditional nuclear fractionation techniques, and by confocal laser scanning microscopy (CLSM). The results agree in that a major part of the enzyme co-localizes with lamin B under physiological conditions, where PARP-1 only has basal activity. After DNA damage and the associated activation of PARP-1, and during the subsequent entry into apoptosis, dramatic changes occur: a gradual release of the enzyme from the lamina, accompanied by its accumulation in nucleoli. Our observations are in line with biochemical evidence for lamin B-PARP-1 interactions under physiological conditions and suggest ways by which these interactions are modified to support PARP-functions in damage and its fate in apoptosis.

Animals↗
PubMed1992

Effects of increasing Dk with rigid contact lens extended wear on rabbit corneal epithelium using confocal microscopy.

The effects of 24-h wear of various Dk-rigid gas-permeable (RGP) contact lenses on the rabbit corneal epithelium were studied by in vivo tandem scanning confocal microscopy (TSCM), and confirmed by scanning electron microscopy (SEM). Lenses used were polymethylmethacrylate (PMMA) (Dk/L = 0), RGP experimental A lens (siloxanylmethacrylate-fluoromethacrylate-methylmethacrylate , 33), experimental B (siloxanylmethacrylate-fluoromethacrylate, 56), and experimental C (siloxanylstyrene-fluoromethacrylate copolymer, 64 x 10(-9)) (cm/s) (ml O2/ml mm Hg) with 0.15-mm thickness (Dk/L measured by polarograph including boundary layer effect). After 24-h PMMA lens wear, TSCM showed no superficial epithelial cells but only exposed, underlying wing cells. The cornea with experimental A showed partial superficial epithelial desquamation. With experimental B wear, slight superficial epithelial cell swelling and desquamation were observed on the surface of the cornea. No changes were observed for the eye with experimental C and control. The observed severity of desquamation of superficial epithelial cells was dependent on the oxygen transmissibility (Dk/L) of RGP lenses worn. All in vivo findings were confirmed by SEM observations. Based on the results of this study, we conclude that (a) although Dk/L = 56 lens B shows no residual overnight corneal swelling, surface damage is still produced; (b) Dk/L = 64 lens C is best for epithelium showing the same corneal images as control; and (c) TSCM is a good way to evaluate the contact lens safety and efficacy in vivo at the cellular level noninvasively.

Animals↗
PubMed1993

Confocal microscopy reveals coordinated calcium fluctuations and oscillations in synaptic boutons.

Calcium ions are one of the main factors regulating quantal transmitter release and thus synaptic transmission in the nervous system. Using confocal microscopy, fluorescent imaging with the calcium indicator Rhod-2, and time series analysis, we show that the levels of calcium ions inside single synaptic boutons of the lizard neuromuscular junction are not constant at rest, but undergo coordinated fluctuations in the space domain, which cover a large fraction of the synaptic bouton. Furthermore, oscillations in intracellular calcium were frequently observed in the time domain. Control experiments showed no coordinated fluctuations or oscillations at locations outside the synaptic boutons. Edge detection analysis showed that the coordinated fluctuations and oscillations were not due to movement artifacts. No coordinated fluctuations and oscillations were seen when similar measurements and analyses were performed on artificial fluorescent beads. A variance analysis was performed on artificial fluorescent beads and on synaptic boutons. The variance of the fluorescent signal at the synaptic boutons was larger than the variance in artificial beads with the same mean fluorescence. This extra variance was greatly reduced when the extracellular calcium concentration was decreased from 2.0 mM to 0.4 mM. We conclude that the coordinated fluctuations and oscillations in the calcium-induced fluorescence at the synaptic boutons are genuine biological phenomena and may be of significance in the regulation of transmitter release.

Animals↗
PubMed2003

Fluorescence confocal microscopy and image analysis of bladder cancer using 5-aminolevulinic acid.

5-aminolevulinic acid mediated changes in tissue specific fluorescence were studied in bladder cancer. Bladders of normal patients and also patients diagnosed with cancer were instilled with 5-aminolevulinic acid and the resultant protoporphyrin IX mediated fluorescence intensity was imaged and quantified with confocal laser microscopy and fluorescence image analysis. Urothelial tumour cells were observed to fluoresce more intensely than normal urothelial cells. Submucosa and muscle tissues exhibited minimal fluorescence compared to urothelial cells of malignant origin and also normal urothelial cells. Degree of fluorescence intensity was in the order of malignant urothelium > normal urothelium > normal submucosa > normal muscles. Fluorescence intensity was also found to increase with duration of ALA instillation. Grade 3 malignant cells produced more fluorescence compared to grade 2 and grade 1. Similarly, T1 transitional cell carcinoma (TCC) showed increased fluorescence intensity than that of Ta TCC. Also, tumour blood vessels fluoresced more intensely compared to blood vessels found in normal bladder tissue. Tissue specific ALA mediated PpIX micro fluorescence can be used as a diagnostic technique for early detection of neoplasms and confocal laser microscopy and fluorescence image analysis are advantageous diagnostic tools for the photodynamic diagnosis of bladder neoplasms in vivo.

Administration, Intravesical↗
PubMed2004

Low-cost, frequency-domain, fluorescence lifetime confocal microscopy.

We describe the theory and implementation of a frequency-domain fluorescence lifetime confocal microscope using switched diode laser illumination. Standard, communications-type, radio-frequency electronics are used to provide inexpensive modulation references and to perform phase-sensitive detection. This allows the rapid acquisition of fluorescence intensity and lifetime images and their display in real time. We show fluorescence lifetime images of bead objects and fluorescence lifetime images of biological specimens from a single confocal scan.

Electronics↗
PubMed2005

Distribution of CdSe quantum dots within swollen polystyrene microgel particles using confocal microscopy.

CdSe quantum dots (QDs) are semiconducting nanoparticles that fluoresce when stimulated by visible light. This property has been exploited in their use as tracer particles in biomedical applications. In this study, confocal microscopy has been used to determine the distribution of QDs within polystyrene microgel particles, dispersed in an organic solvent. It was found that the extent of microgel swelling affected the penetration of the QDs into the particles. Only when the microgel particles were swollen to their maximum extent were the QDs able to penetrate into the central core region of the particles.

Journal Article↗
PubMed2004

Application of red laser video-rate scanning confocal microscopy to in vivo assessment of tubular function in the rat: selective action of diuretics on tubular diameter.

This study examined the use of a red laser illuminated, video-rate scanning confocal reflection microscopy (VRSCM) system, with improved structural and functional imaging at high temporal resolution, to visualize physiological changes in the kidney in response to pharmacological stimuli. We applied VRSCM to superficial nephrons in vivo and measured temporal changes in the diameter of proximal and/or distal tubular segments in response to the administration of three major classes of diuretics with known selective actions at specific nephron sites. Mannitol caused measurable increases in both proximal and distal tubular diameter, whereas frusemide and hydrochlorothiazide caused dilation of the distal tubules only. The findings indicate that VRSCM is capable of detecting and quantifying predicted dynamic changes in renal tubular diameter.

Animals↗
PubMedDate not supplied

Confocal microscopy of corneas with an intracorneal lens for hyperopia.

PURPOSE: We evaluated short-term results and confocal microscopic corneal changes following intracorneal lens implantation. METHODS: In six eyes of three patients with hyperopia between +3.00 and +6.00 diopters (D), an intrastromal hydrogel lens (Permavision, Anamed, Anaheim, Calif) was implanted. Mean baseline hyperopia was +3.90 D. Manifest refraction, uncorrected visual acuity, and spectacle-corrected visual acuity were evaluated. We also performed confocal real-time microscopy with a water immersion objective. Corneal optical sections were recorded and reviewed frame by frame. Examinations were done at months 3, 6, and 12 after intracorneal lens implantation. RESULTS: After surgery, the spherical equivalent refraction was within +/- 0.50 D in 83% (five of six eyes) at 3 months and 100% (six eyes) at 6 and 12 months. Uncorrected visual acuity (UCVA) at 3 months was within 20/40 or better in 67% (four eyes) and in 100% (six eyes) at 6 and 12 months; no eyes had 20/20 or better UCVA at 3 and 6 months. One eye (17%) had 20/20 or better UCVA at 12 months. On confocal microscopy, one eye had an amorphous deposit adjacent to the lens and presumed fibroblastic activity in the same stromal area at 6 months, which was non-progressive up to 12 months. CONCLUSION: Intracorneal lenses may be a treatment option for correction of spherical hyperopia. Predictability must be improved but results in these six eyes were stable up to 1 year. Confocal miscroscopy confirmed biocompatibility and showed no abnormal changes, except two spots of hypercellularity in one eye.

Adult↗
PubMed1993

The spatial arrangement of Allium triquetrum chain quadrivalents at metaphase I as reviewed by confocal microscopy.

We examined the three-dimensional arrangement of bivalents and, in particular, a chain of four chromosomes (chain quadrivalent) in the metaphase I spindle of pollen mother cells of Allium triquetrum by confocal microscopy. Firstly, we show by optical sectioning and three-dimensional image reconstruction that the cooriented pairs of centromeres of all seven bivalents lie virtually parallel to each other in the metaphase I spindle, parallel to the long axis of the spindle. Secondly, we likewise show that the four centromeres of the chain quadrivalent are aligned in the metaphase I spindle in, essentially, a two-dimensional array, not in a three-dimensional array, as proposed by some other authors. This two-dimensionality has its basis, we argue, in the principle that poleward directed spindle forces minimise centromere-to-pole distances and therefore align pairs of centromeres connected to opposite poles most axially (vertically) in the spindle. These distances are minimised for the quadrivalent as a whole only when it lies in two dimensions, i.e. in a plane parallel to the spindle axis.

Allium↗
PubMed1990

The application of real-time confocal microscopy to the study of high-speed dental-bur-tooth-cutting interactions.

A tandem scanning reflected light microscope (TSM) has been used to study the cutting interactions of dental burs with enamel. Confocal microscopy produces high-resolution images of subsurface structures in semi-transparent specimens such as teeth. The TSM has a real-time imaging ability which allows the visualization of the high-speed failure of a substrate which is being machined. This paper describes the criteria for successful imaging and their implementation in the design of a stage for controlling these cutting interactions. Examples of the results achieved are given and further applications for this technique in the field of biomaterials suggested.

Dental Enamel↗
PubMed1993

Quantitative comparison of intracellular concentration and volume of Clara cell 10 KD protein in rat bronchi and bronchioles based on laser scanning confocal microscopy.

We used an antibody to rat Clara cell 10 KD secretory protein (CC10) to compare the abundance of CC10 in non-ciliated epithelium of bronchioles and bronchi by immunohistochemistry and laser scanning confocal microscopy (LSCM) in the reflectance mode. Three zones of reflectance intensity (high, medium, low), directly related to CC10 content, were used to distinguish subcellular compartments of differing densities. Two major compartments contained CC10: granules and endoplasmic reticulum. Bronchiolar cell granules had relatively even reflectance, a high CC10 concentration (0.92 mg/ml), and occupied 7.5% of the cell volume. Bronchial cell granules were bi-zonal, lower in CC10 concentration (0.83 mg/ml), and occupied less cell volume (2.3%). Low-reflecting endoplasmic reticulum occupied a greater cell volume in bronchioles than in bronchi (32.8% and 22.1%, respectively). An intermediate-density zone consisted of endoplasmic reticulum close to granules. CC10 abundance, expressed as ng stored per unit surface area of epithelial basal lamina, was more than three times greater in bronchioles than in proximal bronchi (1.50 ng/mm2 vs 0.42 ng/mm2). These data demonstrate that the process of CC10 secretion differs markedly in non-ciliated epithelium of bronchi and bronchioles. Identifiable mucous-goblet cells did not contain CC10. The LSCM provides a method for quantifying intracellular CC10 pools in intact lung cells and has the potential for quantifying other proteins in subcellular compartments.

Animals↗
PubMed1993

Confocal microscopy and cellular bioinformatics.

An exhaustive description of most biological data requires, besides a set of analytic information, an eidetic representation of the data itself. In this paper, two topics are presented. The first one is confocal microscopy, an advanced technique to produce multidimensional cellular and subcellular structures images. The second one is a software application, named BIOCELL, based on a relational database management system, developed to provide the biologist with an integrated tool to handle simultaneously cell biology linguistic and eidetic information.

Biotechnology↗
PubMed1989

Three-dimensional imaging of intact isolated islets of Langerhans with confocal microscopy.

We present a new technique for analyzing the three-dimensional structure of intact isolated islets of Langerhans. Adult rat and human islets were stained with whole-mount immunofluorescence techniques and optically sectioned with a confocal microscope. This has several advantages over traditional methods: 1) the technical difficulties in serial sectioning and handling the large numbers of sections are avoided, 2) optical sectioning by confocal microscopy gives improved resolution and strongly suppresses light from out-of-focus structures, and 3) entire islets can be rapidly imaged for the presence of positive staining. This new technique should facilitate the study of the three-dimensional structure of islets of Langerhans.

Animals↗
PubMedDate not supplied

In vivo spatio-temporal visualization of the human skin by real-time confocal microscopy.

A modified tandem scanning confocal microscope is used to obtain in vivo images of the human skin in real time. Three-dimensional and temporal visualizations are demonstrated with volume reconstruction and blood flow images. Two image processing methods based on Fourier transform and logarithmic processing are presented. Their applications in noise removal of the scanning disk lines and of the heterogeneity of light are illustrated.

Adult↗