Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Microscopy, Confocal”

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 991 records · Page 55Linked to original sources
PubMed2002

Near real-time confocal microscopy of amelanotic tissue: detection of dysplasia in ex vivo cervical tissue.

RATIONALE AND OBJECTIVES: The authors performed this study to determine whether images of ex vivo tissue obtained with a near real-time confocal microscope can be used to differentiate between normal and dysplastic tissue. MATERIALS AND METHODS: Biopsy specimens of colposcopically normal and abnormal cervical tissue were obtained from 19 patients and imaged at various depths with a confocal microscope. Nuclear morphologic features were extracted from the confocal images; in addition, a group of reviewers examined the images and attempted to identify whether the specimen contained high-grade dysplasia. Results of both analyses were compared with the histopathologic findings of the same specimens provided by a board-certified pathologist with expertise in gynecologic pathology. RESULTS: The morphologic feature measurements compared well with the findings at pathologic examination. The use of the nuclear-cytoplasmic ratio to determine the presence of dysplasia resulted in a sensitivity of 100% and a specificity of 91%. The untrained reviewers had an average sensitivity of 95% and an average specificity of 69% in the determination of dysplasia. CONCLUSION: The results indicate the clinical potential of in vivo confocal imaging in the detection of dysplasia.

Carcinoma in Situ↗
PubMed2004

Measuring hearing organ vibration patterns with confocal microscopy and optical flow.

A new method for visualizing vibrating structures is described. The system provides a means to capture very fast repeating events by relatively minor modifications to a standard confocal microscope. An acousto-optic modulator was inserted in the beam path, generating brief pulses of laser light. Images were formed by summing consecutive frames until every pixel of the resulting image had been exposed to a laser pulse. Images were analyzed using a new method for optical flow computation; it was validated through introducing artificial displacements in confocal images. Displacements in the range of 0.8 to 4 pixels were measured with 5% error or better. The lower limit for reliable motion detection was 20% of the pixel size. These methods were used for investigating the motion pattern of the vibrating hearing organ. In contrast to standard theory, we show that the organ of Corti possesses several degrees of freedom during sound-evoked vibration. Outer hair cells showed motion indicative of deformation. After acoustic overstimulation, supporting cells contracted. This slowly developing structural change was visualized during simultaneous intense sound stimulation and its speed measured with the optical flow technique.

Acoustic Stimulation↗
PubMed1999

Efficient cell segmentation tool for confocal microscopy tissue images and quantitative evaluation of FISH signals.

In this paper we have presented a semi-automatic method for segmenting 3-D cell nuclei from tissue images obtained using Confocal Laser Scanning Microscope. This microscope can focus at different layers of the specimen and hence a stack of images giving a 3D representation can be obtained. The existing methods for segmenting the cells in 3-D confocal images are highly interactive and, hence, time consuming. We have developed an approach, where, given one segmented image-slice (optical section) of the set of confocal images, the remaining image-slices in the image stack can be automatically segmented in a layered approach. One of the image-slices in an image stack is considered as a representative image-slice. In this image-slice, overlapping boundary pixels are identified interactively while the remaining part of the cell boundary is marked using Laplacian of a Gaussian operator. This interactively traced portion of the boundary is considered as initial boundary for finding the overlapping boundary pixels in the neighboring image-slices. Simple basic search strategy is used for boundary search in the neighboring image-slices. The method minimizes the human interaction and is also found to be efficient and reasonably accurate. Some experimental results are presented to illustrate the usefulness of the technique. We have also given the application of our segmentation method to quantitative evaluation of fluorescence in situ hybridization (FISH) signals. A brief comparative study of visual FISH signal evaluation and the FISH signal counting by automatic image analysis is also given.

Automation↗
PubMed2006

In vivo laser confocal microscopy of Bowman's layer of the cornea.

PURPOSE: To investigate in vivo microstructures of Bowman's layer in normal human subjects using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT2-RCM). DESIGN: Single-center, prospective, observational case series. PARTICIPANTS: Nineteen normal volunteers (10 male, 9 female; mean age, 46.2+/-21.7 years [range, 18-77]). METHODS: The central and peripheral cornea, specifically the epithelium, Bowman's layer, and its subjacent stroma, were examined using the HRT2-RCM. MAIN OUTCOME MEASURES: Selected images of the corneal layers were evaluated qualitatively for the shape and degree of light reflection of the microstructures. RESULTS: In all subjects, normal epithelial (superficial, wing, basal) cells, subbasal nerve plexus, Bowman's layer, and its subjacent stoma were observed clearly. However, in all subjects, polymorphic structures composed of fibrillar materials with less reflectivity than corneal nerves were observed beneath Bowman's layer. After application of pressure by a Tomo-cap, we observed numerous ridges that protruded into the epithelial basal and wing cell layers. Superficial stromal striae were also observed. These ridges and striae corresponded exactly to the orientation of the fibrous structures located beneath the epithelial cells. CONCLUSION: We report for the first time, the presence of polymorphic structures composed of fibrillar materials (K-structures) beneath Bowman's layer in normal human subjects, detected by HRT2-RCM. We surmise that these microstructures may correspond to the modified and condensed anterior stromal collagen fibers/lamellae that merge into Bowman's layer and that these fibrillar materials may be responsible for the formation of the anterior corneal mosaic. Further investigation of these microstructures in diseased eyes may provide insights into their pathophysiologic role in Bowman's layer.

Adolescent↗
PubMed1992

Confocal microscopy of the in-situ crystalline lens.

The fine structure of the in-situ rabbit crystalline ocular lens from the ex-vivo rabbit eye was observed with a confocal scanning laser microscope in the scattered light mode. The images were observed through the full thickness of the cornea and aqueous humour to a depth of 50 microns in the anterior ocular lens. The following structures were observed from optical sections of the ocular lens: two concentric regions of the lens capsule, epithelial cells, lens sutures, and surface and interior regions of individual lenticular fibres. The observed lateral resolution of the microscope objective was degraded by imaging across thick (millimetre) structures. This study shows the feasibility of obtaining high-contrast images of transparent objects across 1.7 mm of ocular tissue (cornea and aqueous humour) using confocal light microscopy.

Animals↗
PubMed1997

Two-dimensional millisecond analysis of intracellular Ca2+ sparks in cardiac myocytes by rapid scanning confocal microscopy: increase in amplitude by isoproterenol.

Two dimensional images of myocardial Ca2+ sparks, non-propagating local rises in cytoplasmic Ca2+ concentration, were obtained at 4 msec intervals with a rapid-scanning confocal laser microscope, Nikon RCM 8000, and fluo-3. Spontaneous Ca2+ sparks were observed at apparently random sites throughout the cytoplasm of rat ventricular cells. The duration of sparks was 30 to 40 msec and the time to peak intensity about 10 msec. Ryanodine (1 microM) completely inhibited Ca2+ sparks while nicardipine (3 microM) had no effect. Isoproterenol (1 microM) had no effect on the frequency and distribution of Ca2+ sparks but significantly increased their amplitude. These results suggest that myocardial Ca2+ sparks are the result of spontaneous release of Ca2+ from the sarcoplasmic reticulum and that beta-adrenergic stimulation may result in functional modification of the ryanodine receptor channel.

Adrenergic beta-Antagonists↗
PubMed1996

Intracellular free Ca2+ movements in cultured cardiac myocytes as shown by rapid scanning confocal microscopy.

Two-dimensional images of intracellular free Ca2+ movements in cultured cardiac myocytes were obtained at 33-ms intervals with a Ca(2+)-sensitive fluorescence probe, fluo-3, and a rapid scanning confocal laser microscope, a prototype of Nikon RCM8000. The cells used were isolated from the ventricular myocardium of neonatal mice, cultured for approximately 72 h and loaded with fluo-3. One type of cytoplasmic Ca2+ movement observed was a simultaneous increase in [Ca2+] throughout the cytoplasm, termed a "spike"; another type was a local increase in [Ca2+] propagating in the cytoplasm, termed a "wave." Cells with either spike or wave or both types of movements were observed. Tetrodotoxin (TTX) 10(-5) M, nicardipine 10(-6) M, and increased extracellular potassium concentration (40 mM) selectively inhibited spike, and ryanodine 10(-6) M and cyclopiazonic acid (CPA) 3 x 10(-6) M selectively inhibited wave. These results indicate that spike was triggered by depolarization-induced Ca2+ influx across the sarcolemma, whereas wave was a propagating local increase in Ca2+ due to Ca2+ release from the sarcoplasmic reticulum (SR). On spike, nuclear [Ca2+] was shown to increase and decrease synchronously with cytoplasmic [Ca2+], with a delay and slower time course.

Aniline Compounds↗
PubMed2005

Thickness of the gerbil tympanic membrane measured with confocal microscopy.

Thickness data for the gerbil tympanic membrane, an extremely thin biological membrane, are presented. Thickness measurements were performed on fresh material using fluorescence images taken perpendicular through the membrane with a commercial confocal microscope. Thickness varies strongly across the membrane. Similar thickness distributions in all samples (pars tensa n = 11; pars flaccida n = 3) were observed. The pars tensa has a rather constant thickness of about 7 microm in the central region curving as a horse shoe upwards around the manubrium. In the most superior parts of the pars tensa thickness becomes gradually twice as large. Thickness increases also steeply from the central region towards the edges (about 35 microm near the annulus and 20 microm near the manubrium). A pronounced, local thickening of about 30 microm is present close to the edge and extends as a ring along the entire annular periphery of the pars tensa. Overall, the pars flaccida is thicker than the pars tensa and has a rugged surface. Its central region has a mean thickness of about 24 microm with a mean variation of about 4 microm. The average thickness in the inferior region is slightly larger than in the superior region. The pars flaccida thickens steeply, up to about 80 microm, near the edges.

Animals↗
PubMed1995

[ACh and ATP induced calcium mobilization in outer hair cells of the guinea pig cochlea: confocal microscopy].

Effects of acetylcholine (ACh) and ATP as the candidates of efferent neurotransmitter on intracellular Ca2+ concentrations ( [Ca2+]i) of the isolated outer hair cells (OHCs) from guinea pig cochlea were studied with laser scanning confocal microscope. The OHCs were loaded with Ca2+ sensitive dye Fluo-3, whose fluorescent intensity was strongest at the basal end in resting OHC. The presence of ACh gradually increased [Ca2+]i to a higher level at the basal end of OHCs. Continuous application of ATP caused a rapid [Ca2+]i increase followed by a gradual exhaustion throughout the whole OHC. Magnitude of the increase at the apex was greater than that at the base. In OHCs whose calcium had been exhausted by ATP, ACh induced a temporary increase in [Ca2+]i. It seems likely that the ACh-induced [Ca2+]i rise is partly due to an influx of extracellular Ca2+. The bursting nature of ATP-induced [Ca2+]i rise may be a consequence of Ca2+ influx through the ATP-gated cation channels and of the mobilization of intracellular store mediated by ATP.

Acetylcholine↗
PubMed1999

Characterization of epithelial downgrowth by confocal microscopy.

A 43-year-old white woman with a history of multiple ocular surgeries, including 4 penetrating keratoplasties, developed a concentric retrocorneal membrane at the graft periphery in the right eye. A white-light, tandem, scanning confocal microscope using a 24x/0.60 contact objective was used to examine the right eye in vivo. At the endothelial layer, confocal microscopic images similar to corneal epithelial cells were detected at the graft periphery. Unlike normal endothelial cells, the imaged cells demonstrated easily recognizable nuclei.

Adult↗
PubMed1996

Advances in automated 3-D image analyses of cell populations imaged by confocal microscopy.

Automated three-dimensional (3-D) image analysis methods are presented for rapid and effective analysis of populations of fluorescently labeled cells or nuclei in thick tissue sections that have been imaged three dimensionally using a confocal microscope. The methods presented here greatly improve upon our earlier work (Roysam et al.:J Microsc 173: 115-126, 1994). The principal advances reported are: algorithms for efficient data pre-processing and adaptive segmentation, effective handling of image anisotrophy, and fast 3-D morphological algorithms for separating overlapping or connected clusters utilizing image gradient information whenever available. A particular feature of this method is its ability to separate densely packed and connected clusters of cell nuclei. Some of the challenges overcome in this work include the efficient and effective handling of imaging noise, anisotrophy, and large variations in image parameters such as intensity, object size, and shape. The method is able to handle significant inter-cell, intra-cell, inter-image, and intra-image variations. Studies indicate that this method is rapid, robust, and adaptable. Examples were presented to illustrate the applicability of this approach to analyzing images of nuclei from densely packed regions in thick sections of rat liver, and brain that were labeled with a fluorescent Schiff reagent.

Algorithms↗
PubMed1996

Restricted propagation of cytoplasmic Ca2+ oscillation into the nucleus in guinea pig cardiac myocytes as revealed by rapid scanning confocal microscopy and indo-1.

Two-dimensional images of cytoplasmic and nuclear free Ca2+ movements in cardiac myocytes were obtained at 67-msec intervals using a Ca(2+)-sensitive fluorescence probe, indo-1, and a rapid scanning confocal laser microscope, Nikon RCM8000. Isolated guinea pig ventricular cells were loaded with indo-1 and stimulated at 0.5 Hz through patch pipettes. On stimulation, nuclear Ca2+ concentration ([Ca2+]) was observed to rise and fall following cytoplasmic [Ca2+] with an obvious delay. Application of isoproterenol significantly increased the peak [Ca2+] on stimulation in both the cytoplasm and nucleus with no substantial change in the basal [Ca2+]; the increase in peak [Ca2+] produced by application of isoproterenol was larger in the cytoplasm than in the nucleus. Under a low [Na+] condition, the basal [Ca2+] was increased from the control values in both the cytoplasm and nucleus; no difference in basal [Ca2+] was observed between the two regions. The increase in peak [Ca2+] by low [Na+] in the cytoplasm was significantly larger than that in the nucleus. When the cells were voltage clamped at 0 mV for 3 sec, no difference in the steady state [Ca2+] was observed between the cytoplasm and nucleus. Nuclear [Ca2+] was also observed to increase following a Ca2+ wave, a local increase in [Ca2+] propagating within the cytoplasm, with a delay. Thus, we demonstrated in isolated myocardial cells that cytoplasmic Ca2+ movements, although hampered by the nuclear envelope, are propagated into the nucleus, a mechanism through which factors affecting cytoplasmic Ca2+ may influence intranuclear events.

Animals↗
PubMed1996

Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement.

The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with Cl-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units.

Calibration↗
PubMed1994

In-vivo analysis of angiogenesis and revascularization of transplanted pancreatic islets using confocal microscopy.

A technique to measure angiogenesis and revascularization in pancreatic islets transplanted at the renal subcapsular site in the rat has been developed. In-vivo imaging of the microcirculation of transplanted pancreatic islets was conducted using a confocal scanning laser microscope (CSLM) to achieve optical sectioning through the graft in order to perform a computer reconstruction of the three-dimensional neovascular morphology. Individual islets were harvested by enzymatic digestion of excised pancreas from Fischer 344 rats. Isolated islets were cultured for 24 h, and approximately 300-350 islets were transplanted at the renal subcapsular site of the left kidney in an anaesthetized rat. Six to 14 days post-transplantation, the animal was anaesthetized and prepared for in-vivo imaging of the microvasculature on a Zeiss LSM-10. Optical contrast of the microvasculature was enhanced by the administration of fluorescein-labelled dextran into the circulating blood. The transplant site was identified and serial sections were obtained through the vascular bed at varying z-intervals. Complementary fluorescence video images were also obtained via a silicon intensifier tube camera mounted on the CSLM. At completion of the imaging procedure, the kidney was returned into the body cavity, the area was sutured and the animal was allowed to recuperate for subsequent examinations. Image processing algorithms, such as grey-level thresholding, median filtering, skeletonization and template matching, were applied to compute the vessel density and diameters and extrapolated to measure 3-D vessel lengths and the tortousity index of the neovasculature.

Algorithms↗
PubMed2006

Thickness distribution of fresh and preserved human eardrums measured with confocal microscopy.

HYPOTHESIS: In this study, the thickness distribution of the fresh human eardrum was measured and possible thickness changes in successive stages of preservation and preparation were studied. METHODS: The thickness measurement was performed on axial fluorescence images taken perpendicularly through the membrane with a confocal microscope. The influence of fixation and preservation (in Cialit solution) on the thickness was also investigated. The same eardrum was prepared (decalcified, dehydrated, and stained) for histologic sectioning and the thickness was measured on the sections using conventional light microscopy. RESULTS: Similar thickness distributions in the measured samples (n = 3) were observed. The pars tensa has a rather constant thickness in a central region curving as a horseshoe upward around the manubrium. The membrane thickens slightly from the inferior to the superior side. The anterior region is thicker than the posterior region. In narrow bands along the manubrium, peripheral rim, and in the region inferior to the umbo, a much larger thickness in comparison with that in the central region was found. Mean thicknesses of approximately 40, 50, and 120 microm were observed in the central region of the studied eardrums, respectively. CONCLUSION: Whereas the thickness distribution of the human eardrums shows similar features, the absolute thickness seems to vary a lot from one specimen to another. There is no significant difference in thickness of the same membrane in fresh, fixed, or preserved condition. Thus, human eardrums may be safely preserved in fixative for later thickness measurements. The histologic preparation process, however, causes a significant location-dependent shrinkage.

Age Factors↗
PubMed1993

Nitric oxide synthase immunoreactive neurons anatomically define a longitudinal dorsolateral column within the midbrain periaqueductal gray of the rat: analysis using laser confocal microscopy.

Nitric oxide has recently been proposed as a neuronal messenger in both the central and peripheral nervous system. Antibodies against nitric oxide synthase (NOS), the synthesizing enzyme for nitric oxide, were used in combination with immunocytochemistry and confocal laser microscopy to analyze the distribution of this enzyme in the midbrain periaqueductal gray (PAG) of the rat. NOS immunoreactive neurons were localized predominantly in a longitudinally oriented column in the dorsolateral PAG. NOS immunoreactive fibers and processes were scattered throughout the PAG but were most prevalent in the dorsolateral column and in the juxta-aqueductal column. This study provides neurochemical support for the existence of longitudinal columns in the PAG which are postulated to underlie the functional organization of this complex brainstem region.

Amino Acid Oxidoreductases↗
PubMed2001

Three-dimensional study of the capillary supply of skeletal muscle fibres using confocal microscopy.

Three-dimensional (3D) study of capillary network of individual muscle fibres in rat extensor digitorum longus (EDL) and soleus (SOL) muscles is presented. Stereology and 3D reconstruction techniques were applied to stacks of serial optical sections recorded by a confocal microscope from thick muscle slices. The results suggest that SOL muscle fibres have a larger surface area and volume as well as a larger length of capillaries per fibre length than EDL. On the other hand, these two muscles have a similar ratio of capillary length to fibre surface area. The 3D approach to evaluation of muscle fibre capillarization brings many advantages over traditional measurements made on single muscle sections and could also be applied to the study of angiogenesis in other tissues.

Animals↗
PubMed2006

Experimental study on meiotic spindles and chromosomes of mouse mature (M II) stage oocytes under laser scanning confocal microscopy.

Taking the mouse as a model, the experimental method of observing the morphology of meiotic spindles and chromosomes in mature oocytes were investigated in order to evaluate the effects of various interventions on the quality of oocytes accurately and rapidly. Laser scanning confocal microscope (LSCM) was used to examine the meiotic spindles and chromosomes by the technologies of optical section and three-dimensional (3D) image reconstruction. The results showed that the configurations of meiotic spindles and chromosomes could be observed clearly by LSCM. The normal rate of meiotic spindles and chromosomes was 82% and 86% respectively. It was concluded that the LSCM was a valid instrument to observe the meiotic spindles and chromosomes of mature oocytes and could be used as a valid method to evaluate the quality of M II oocytes.

Animals↗