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At least 73 records · Page 4Linked to original sources
PubMed2001

Topographic variations in normal skin, as viewed by in vivo reflectance confocal microscopy.

Near-infrared confocal microscopy is a new tool that provides skin images in vivo, with high resolution and contrast at a specific depth. Regional variations in live human skin viewed by confocal microscope have not been studied so far. In vivo reflectance confocal microscopy was performed in 10 adults (eight males, two females) of various skin phototypes. Six topographic sites were studied in each subject: forehead, cheek, inner and outer forearm surfaces, lower back and leg. Epidermal thickness at suprapapillary epidermal plates and rete pegs was measured during real-time imaging and the number and diameter of epidermal keratinocytes in each epidermal cell layer as well as the characteristics of dermal papillae were defined from the grabbed images. Stratum corneum appeared brighter in sun-exposed than in sun-protected areas and particularly pronounced in heavily pigmented individuals. The epidermal thickness at rete pegs, but not the suprapapillary epidermal plate, was greater in sun-exposed areas than in sun-protected sites except forearm flexor surface. The en face numerical density of granular keratinocytes is greater on the face as compared with all other sites, whereas the surface density of spinous keratinocytes is greater on sun-protected sites. Additionally, the number of basal keratinocytes per millimeter length of dermoepidermal junction is greater in sun exposed areas. Interestingly, the dermal papillae shape varies and their sizes increase in circumference from sun-exposed to sun-protected sites, as observed at a specific depth below the stratum corneum. In summary, our results demonstrate that near infra-red reflectance confocal microscopy is a feasible tool for microscopic analysis of skin morphometry in vivo.

Adult↗
PubMed2002

Contrast agents for confocal microscopy: how simple chemicals affect confocal images of normal and cancer cells in suspension.

Normal and malignant human cervical cancer cells were imaged in vivo with confocal, phase contrast, and brightfield microscopies. Results were compared between cells in growth medium before and after addition of acetic acid, hypertonic saline solution, toluidine blue, and Lugol's iodine. The exogenous agents changed the backscattering characteristics of the cells when measured with confocal microscopy at 808 nm. A tendency toward higher scattering was observed in treated cells. Acetic acid and toluidine blue increased the brightness of the nucleus with respect to the cytoplasm in normal and cancer cells. Hypertonic saline solution made the cytoplasm brighter than the nucleus in both types of cells. The results indicate that simple chemicals can be used to enhance confocal microscopy's ability to differentiate intracellular components, such as nuclear size and shape. This can further confocal microscopy's ability to assess disease in cells and tissues.

Acetic Acid↗
PubMed1992

Radial keratotomy. 1. The wound healing process and measurement of incisional gape in two animal models using in vivo confocal microscopy.

Using in vivo confocal microscopy, corneal wound healing was evaluated in both rabbit and cat eyes after radial keratotomy. A total of six rabbit and six cat eyes were evaluated sequentially over time for 1 mo after surgery by in vivo confocal microscopy, and quantitative measurements of changes in incisional wound gape were determined. In vivo histopathologic changes were correlated with conventional histopathologic findings in 18 rabbit and 4 cat eyes; the animals were killed at various intervals from 0-30 days after surgery. In the rabbit, in vivo corneal wound healing was characterized by the initial ingrowth of corneal epithelium followed by persistence within the wound without a marked fibrotic response. Measurement of incisional wound gape showed increasing gape from 144 +/- 32 microns on day 0 to 976 +/- 155 microns on day 26 at a distance of 2.4 mm from the optical zone. These in vivo measurements were not significantly different (P = 0.996) from those obtained using conventional histopathologic techniques which showed an incisional wound gape of 252 +/- 112 microns on day 0 and 917 +/- 216 microns on day 26 at 2.5 mm from the optical zone. In the cat eyes, healing of radial keratotomy wounds showed an initial increase in incisional wound gape from 135 +/- 56 microns on day 0 to 245 +/- 88 microns on day 7 at a distance of 2.4 mm from the optical zone. Starting at day 14 and continuing to day 30, there was a progressive decrease in incisional wound gape from 198 +/- 41 microns to 92 +/- 35 microns. Sequential, in vivo histopathologic analyses indicated that increasing incisional wound gape correlated with the retention of corneal epithelium in the wound. Initiation of decreasing incisional wound gape was associated with replacement of the incisional epithelial plug with fibroblastic tissue. These changes in the incisional wound gape observed in the cat suggest that healing of radial keratotomy wounds involves contraction of the wound in response to the ingrowth of fibroblastic cells. Furthermore, the contractile response appears to be biphasic involving a precontractile and contractile phase. Overall these data indicated that in vivo confocal microscopy provides quantitative histopathologic data on living tissue comparable with that obtained with conventional techniques on dead, fixed, and sectioned tissue. Additionally, the absence of wound fibrosis in the rabbit radial keratotomy model raises important questions as to the appropriateness of this experimental model for human radial keratotomy.

Animals↗
PubMed1994

Confocal microscopy of hair.

Confocal microscopy is an excellent method for studying the localization of fluorescent stains. Used in this way, superior 3D images can be obtained from multiple optical sections with very shallow depth of field. The main advantage of this technique is that the sample is not damaged. We have taken serial confocal sections of hair and via specific image enhancement routines have obtained high-quality 3D images enabling the visualization of cuticle scale and its pattern of distribution. This has been done on various types of hair: bleached, permed and in certain pathological conditions. This first step will allow us to characterize the hair surface in terms of its roughness, and the distribution and form of cuticular scale, parameters that have potential in the assessment of dermocosmetic efficacy.

Hair↗
PubMedDate not supplied

The attachment of mineralised tissues to coverslips for observing dynamic events by confocal microscopy.

In confocal scanning optical fluorescence microscopy, using high-aperture oil immersion lenses, the best images are obtained for focus planes immediately under the coverslip, or under a continuation of the coverslip with a medium with the same, high refractive index. Therefore placing the coverslip with a layer of oil underneath it is an advantage if the sample will allow it. If experimental manipulations that can displace the coverslip are involved,--for example, attempts to make dynamic observation of fluid flow--the coverslip is best cemented to the sample with a high-index material. This report describes the use of adhesive systems developed for restorative dentistry to achieve a durable attachment of dental tissues to microscope coverslips. The technique described in this paper has been used for monitoring real-time fluid movement in dentine. The samples were examined with a high-frame-rate confocal microscope (a tandem scanning microscope). The adhesive technology also could be utilised in the microscopic preparation of other porous translucent materials.

Dentin↗
PubMed1998

Characterization of the sperm-induced calcium wave in Xenopus eggs using confocal microscopy.

We have used confocal microscopy to examine the [Ca2+]i increase in the albino eggs of the frog Xenopus laevis after fertilization. Eggs were placed in agar wells with their animal poles downward so that fertilization occurred preferentially in the equatorial plane, and confocal microscopy was used to provide a two-dimensional optical section through the three-dimensional Ca2+ wave. These data indicate that the wave of increased [Ca2+]i traverses the entire egg and converges uniformly on the antipode. We show that ratioing two different fluorescent dyes to correct for variations in cell thickness is not a reliable technique for this very thick cell due to differential absorption with depth. Indo-1-dextran proves to be a more reliable Ca2+ indicator in this respect. Indo-1-dextran measurements indicate that the resting [Ca2+]i is not uniform throughout the egg but exhibits a 15% higher [Ca2+]i in the cortex than deep in the cytoplasm. This difference is accentuated during wave propagation and is not dependent on extracellular Ca2+. The average peak [Ca2+]i in the center of the egg as the wave propagates through it is 0.7 microM, approximately 60% of the peak cortical [Ca2+]i. The wave velocity through the center of the egg (5.7 micron/s) is slower than that in the cortex (8.9 micron/s), and both velocities vary slightly during transit. The cortical wave speed is particularly high at the beginning (15.7 micron/s) and end (17.2 micron/s) of the wave. Eggs injected with 30-80 microM of 3 kD heparin to compete with inositol-1,4,5,-trisphosphate for binding to its receptor exhibited multiple localized spots of elevated [Ca2+]i, and many of these did not initiate a wave. For those that did lead to a wave, it was usually slow moving and exhibited a reduced (60% reduction) amplitude compared with controls.

Animals↗
PubMed1994

Capillary perfusion of the rat brain cortex. An in vivo confocal microscopy study.

Confocal laser-scanning microscopy was used to visualize subsurface cerebral microvessels labeled with intravascular fluorescein in a closed cranial window model of the anesthetized rat. In noninvasive optical sections up to 250 microns beneath the brain surface, plasma perfusion and blood cell perfusion of individual capillaries were studied. Under resting conditions, in all cerebral capillaries the presence of plasma flow as demonstrated by the appearance of an intravenously injected fluorescent tracer within 20 seconds after injection. Plasma flow was verified even in capillaries that contained stationary erythrocytes or leukocytes; 91.1% of the capillaries contained flowing blood cells, 5.2% contained stationary blood cells, and no blood cells were seen in 3.6%. Mean blood cell velocity was 498.3 +/- 443.9 microns/s, and the mean blood cell supply rate was 35.75 +/- 28.01 cells per second. When capillaries were continuously observed for 1 minute, "on" and "off" periods of blood cell flow were noted. During hypercapnia (increase of PCO2 from 33.25 to 50.26 mm Hg), mean blood cell flux increased from 38.6 +/- 17.2 to 55.5 +/- 12.2 per second (P < .005, paired t test of mean values in six animals), and blood cell velocity increased from 519.5 +/- 254.8 to 828.5 +/- 460.8 microns/s (P = .074, paired t test of mean values in six animals). Homogeneity of blood cell flux increased as indicated by the coefficient of variation decreasing from 44.6% to 22.0%, and the portion of poorly perfused capillaries (blood cell flux, < 40 per second) decreased from 59.2% to 22.4%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Modern microscopy in biofilm research: confocal microscopy and other approaches.

Microscopy is the only technique whereby bacterial biofilms can be studied at the single-cell level in situ. Our understanding of biofilm structure, physiology and control hinges on the application of confocal scanning laser microscopy and other advanced microscopic techniques. Gene expression in four dimensions (x,y,z,t), interspecies interactions, and the role of exopolymer are being defined.

Biofilms↗
PubMed2005

[Evaluation of antifungal chemotherapeutic effects on fungal keratitis by confocal microscopy].

OBJECTIVE: To evaluate the validity of confocal microscopy in estimating curative effect and in directing the treatment for fungal keratitis in the process of antifungal chemotherapy. METHODS: Fifty-eight patients, who were confirmed fungal infection by confocal microscopy, were selected from 328 patients with fungal keratitis. All patients received routine topical and/or oral antifungal medication, and were examined by confocal microscopy once a week and one week after discontinuation of the treatment. The density of hyphae in the corneal lesion, the configuration of inflammatory cells and keratocyte were recorded. Antifungal chemotherapy was adjusted according to examination results and medicines were changed accordingly. If no hyphae were detected by confocal microscopy, antifungal medication was maintained for one week and then discontinued. All patients were followed up for two months to ensure no relapse of fungal infection. RESULTS: Fifty three patients were cured. The area of corneal lesions began to reduce 7 days after the beginning of antifungal chemotherapy. Confocal microscopy examination revealed that the hypha positive sites and the density of hypha were reduced gradually; inflammatory cells also decreased, the configuration of corneal lesion was transformed from asymmetry to symmetry; and normal keratocytes could be detected gradually. After 14 days of treatment, ulcers healed up in 37 cases and no hyphae and inflammatory cells were found in 23 cases. After 28 days of treatment, all corneal ulcers healed up; hyphae and inflammatory cells were completely disappeared in 31 patients, but a few hyphae still could be found in 22 patients. Antifungal chemotherapy was tapered gradually if no hyphae and inflammatory cells were detected by confocal microscopy. There was no relapse of fungus infection during 2-month follow-up. Infection deteriorated in the other five patients within 7 days, which showed increased density of hypha and inflammatory cells under confocal microscopy examination. All of them were treated with a penetrating keratoplasty to save the eyeball. CONCLUSIONS: Confocal microscopy is an ideal method for the evaluation of curative effects of fungal keratitis in the process of antifungal chemotherapy. This is also a valuable objective tool in directing antifungal medication.

Adolescent↗
PubMed1999

[Confocal microscopy in corneal dystrophies].

BACKGROUND: Confocal microscopy represents a methodology that allows in vivo examination of corneal morphology, particularly of the epithelium and stroma. MATERIAL AND METHODS: Using the confocal microscope "ConfoScan Modell P2" epithelial, stromal and endothelial changes were evaluated in 11 patients with corneal dystrophies. All findings were compared with data from healthy individuals. RESULTS: Confocal images could be correlated to conventional (slitlamp) biomicroscopic findings in all patients with corneal dystrophies. In addition, confocal microscopy provided more detailed images particularly of epithelial and stromal changes. CONCLUSION: Our data indicate that confocal microscopy provides information on living tissue that correlates with that obtained with conventional techniques on fixed and sectioned tissue.

Corneal Dystrophies, Hereditary↗
PubMed1995

Confocal microscopy of corneal graft rejection.

Corneal allografts were transplanted into inflamed and vascularized graft beds in rabbit eyes. The grafts were examined every 4 days by slit-lamp biomicroscopy and scanning confocal microscopy. Confocal images were recorded with a video camera and computer enhanced in real-time. Layers of the cornea were visualized in serial optical sections parallel to the epithelium. In the third postoperative week, signs of graft rejection were observed; slit-lamp examination revealed a circumferential line of epithelial rejection, along with cloudiness and edema. Vessels were observed growing into the graft. By confocal microscopy, infiltrating cells were seen in the graft stroma. Foci of cells were especially pronounced around the sutures. Scattered leukocyte infiltrates were prominent at capillary terminals. There was an accompanying reduction in the stromal keratocyte density in the region of the infiltrate. Additionally, various degrees of fibrosis were noted around each suture and at the host-graft interface. Confocal microscopy may provide a valuable clinical tool for determining the earliest indicators of an antigraft immune response, and as an aid in the differential diagnosis of other inflammatory conditions of the cornea.

Animals↗
PubMed1994

Calcium signalling in individual T-cells measured by confocal microscopy.

Laser-scanning confocal microscopy was used in conjunction with a highly fluorescent Ca2+ indicator fluo-3 to visualize real-time alterations in the intracellular Ca2+ concentration ([Ca2+]i) in individual living Jurkat T-cells during the first minutes of activation by phytohaemagglutinin (PHA) at the physiological temperature (37 degrees C). With a delay of 30-120 s, PHA induced a strong [Ca2+]i peak in the micromolar range (1-3 microM). The rise in [Ca2+]i lasted for 1-2 minutes, and was followed by a sustained plateau of elevated [Ca2+]i in the 0.2-0.5 microM range. Some cells (10-20%) responded with additional asynchronous 0.5-1.5 microM peaks during the plateau phase. These oscillations continued for 10-20 minutes. The spans of the peaks ranged from 30 to 100 s, intervals between peaks varied from 60 to 300 s. It was shown that the initial [Ca2+]i peak was associated with Ca2+ mobilisation from internal sources, whereas the plateau was maintained by an influx of Ca2+ from external medium. In K(+)-rich medium or in the presence of quinine, a K+ channel blocker, no secondary response to PHA-activation characterised by an elevated plateau was observed. The data suggest that the Ca2+ influx was dependent on the membrane potential and/or the extracellular K(+)-concentration. Optical sectioning showed that the intracellular Ca2+ distributed almost homogeneously throughout the cell volume both in control and in PHA-stimulated cells including those exhibiting Ca2+ oscillations. This suggests that Ca2+ signals are localized not only in cytoplasm at the cell plasma membrane but can be also transferred directly into the nucleus.

Aniline Compounds↗
PubMed1999

High resolution quantitation of microvascular plasma perfusion in non-ischemic and ischemic rat brain by laser-scanning confocal microscopy.

Laser-scanning confocal microscopy (LSCM) was used to measure at high resolution cerebral plasma volumes (perfusion) using two fluorescent plasma markers in a rat model of embolic stroke. This application of LSCM to study the microvascular circulation in embolic stroke was developed as an alternative to autoradiography to measure cerebral perfusion. An additional benefit of LSCM is that it quantitates with great accuracy the structural relationships of the microcirculation to cells and the pathological alterations of the ischemic brain. Autoradiography allows only a quantitative analysis of cerebral perfusion. For example, in order to study the microcirculation and its relationship to blood brain barrier damage, the volume of perfused cerebral capillaries was measured by administering two fluorescent plasma markers (FITC-dextran and Evans blue) intravenously to a rat. Evans blue was administered before cerebral ischemia and FITC-dextran administered post-ischemia 1 min before sacrifice. Volumes of plasma perfusion were analyzed by means of a system developed for 3D analysis of fixed and stained serial brain histologies. Plasma volumes for the non-ischemic cerebral cortex were 1.00%+/-0.38% while plasma volumes in the caudate/putamen were 0.69%+/-0.17% in good agreement with the previously published values using the autoradiography method. The architecture of the capillaries in the ischemic core showed perfusion of Evans blue but there was no flow of FITC dextran. Our work represents a novel application of this technology to investigation of cerebral vascular disease and identifies its potential to become an important tool for investigation of cerebral pathology.

Animals↗
PubMed2000

Principles and practices of laser scanning confocal microscopy.

The laser scanning confocal microscope (LSCM) is an essential tool for many biomedical imaging applications at the level of the light microscope. The basic principles of confocal microscopy and the evolution of the LSCM into today's sophisticated instruments are outlined. The major imaging modes of the LSCM are introduced including single optical sections, multiple wavelength images, three-dimensional reconstructions, and living cell and tissue sequences. Practical aspects of specimen preparation, image collection, and image presentation are included along with a primer on troubleshooting the LSCM for the novice.

Animals↗
PubMed2004

Detection and spectroscopy of gold nanoparticles using supercontinuum white light confocal microscopy.

We combine confocal microscopy using supercontinuum laser illumination and an interferometric detection technique to identify single nanoparticles of diameter below 10 nm. Spectral analysis of the signal allows us to record the plasmon resonance of a single nanoparticle. Our results hold great promise for fundamental studies of the optical properties of single metal clusters and for their use in biophysical applications.

Journal Article↗
PubMed1992

Spontaneous and propagated calcium release in isolated cardiac myocytes viewed by confocal microscopy.

Laser scanning confocal microscopy of the Ca(2+)-sensitive fluorophore fluo-3 has been used to investigate spontaneous and propagated calcium release at high temporal and spatial resolution in enzymatically dispersed rat cardiomyocytes. Waves of fluorescence which propagated throughout the cytosol were evident in spontaneously contracting cardiac cells containing fluo-3, but not in cells containing Ca(2+)-insensitive fluorophores [2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein, SNARF-1, rhodamine-123, or tetramethylrhodamine-labeled dextran]. These waves represent localized areas of elevated [Ca2+] [975 +/- 13 (SE) nM, range 800-1,500 nM; n = 16 cells]. Ca2+ waves were initiated by the spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR) and propagated through cells at rates of 50-150 microns/s. Ca2+ waves were usually initiated at the cell ends, but multiple and variable initiation foci were observed in some cells. Where waves intersected within a single cell there was extinction of wave propagation, confirming the SR as the direct source of Ca2+ and revealing a refractory period in SR Ca2+ release. In some cells high-frequency Ca2+ waves lead to synchronized elevation of [Ca2+] throughout the entire cytosol and within the time period associated with cell depolarization. These observations support the hypothesis that some cardiac arrhythmias are initiated by spontaneous and propagated Ca2+ release and involve subsequent depolarization, global elevation of intracellular [Ca2+], and cell contraction.

Aniline Compounds↗
PubMed1990

Localization of cerium-based reaction products by scanning laser reflectance confocal microscopy.

Scanning laser confocal microscopy was utilized to visualize sites of hydrogen peroxide release from stimulated neutrophils and lysosomal acid phosphatase in these and other cells using cerium in the detection systems. Imaging of the cerium-containing reactions was achieved by employing the reflectance mode of this instrument. Localization of these products at the light microscope level was direct and did not require other reactions to generate a visible product. This new approach to cerium cytochemistry should prove useful for many applications.

Acid Phosphatase↗
PubMed1991

Improved detection of in situ hybridization by laser scanning confocal microscopy.

Laser scanning confocal microscopy (LSCM) offers a significant improvement over conventional bright-field and dark-field light microscopy for producing images of silver grains in autoradiograms of specimens prepared by in situ hybridization. The out-of-focus image of the background silver grains present in the emulsion is eliminated from the in-focus image of the radioactive probe associated with the cells by optical sectioning with the LSCM operated in a reflected light mode. The improved images produced by the LSCM provide a significant increase in the sensitivity of detecting positively labeled cells and tissues prepared by in situ hybridization. The power of this detection method is demonstrated using samples of HIV-infected human peripheral blood cells, tissue sections of human placenta and human skin. It is anticipated that the method can be universally applied to samples prepared by in situ hybridization techniques.

Cell Line↗