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 631 records · Page 35Linked to original sources
PubMed1993

Comparison of tear film thickness in three species determined by the glass fibre method and confocal microscopy.

Using optical methods to measure tear film thickness we previously found that values were substantially larger than earlier estimates by mechanical and chemical techniques. The predominant component was found to be mucus, not aqueous fluid. This article describes the results of using confocal microscopy to re-examine an earlier method of measuring thickness, the glass fibre technique. Confocal microscopy enabled visual identification of tear and epithelial surfaces and accurate measurement of the separation between them, giving the full film thickness. Consecutive measurements were made by each method in eyes of 22 animals of three species. Measurements using fibres in rabbits gave results of 7 microns, replicating earlier findings. However, the fibre technique gave the same result in all species. Values were not correlated with measurements by microscopy, which ranged from 10 to 14 microns. We propose that fibres were too flexible to be pressed through all mucus layers and contact epithelium, and as a result this method underestimated film thickness.

Animals↗
PubMed2005

Altered corneal nerves in aqueous tear deficiency viewed by in vivo confocal microscopy.

PURPOSE: To define the alterations of corneal nerves in aqueous tear deficiency dry eye patients with or without Sjögren syndrome and to identify the relationship between the morphologic changes of corneal nerves and the extent of dry eye. METHODS: Confocal microscopy was used to examine 38 consecutive aqueous tear deficiency patients (8 Sjögren syndrome and 30 non-Sjögren syndrome) and 30 age- and gender-matched normal controls. Images taken by Confocal2 slit-scanning microscope at subbasal epithelial cell layer of central cornea were analyzed. The number and density of corneal nerves and their size, beads, tortuosity, and branching pattern were compared. These data were correlated with age and the degree of dry eye. RESULTS: Sjögren syndrome patients showed a significant increase in average nerve number and tortuosity as compared with normal controls (P = 0.031 and 0.021, respectively). Severe nerve tortuosity (grade 4) and nerve branching appeared more frequently in aqueous tear deficiency than in normal subjects (P = 0.024 and 0.042, respectively). A decreased nerve number was observed with age in the normal controls (P = 0.002). However, such a correlation did not exist in aqueous tear deficiency. In aqueous tear deficiency, rose bengal staining score correlated positively with nerve density (P = 0.048) and nerve number (P = 0.001). Corneal fluorescein staining score was also positively correlated with nerve number (P = 0.027). CONCLUSIONS: Abnormal morphologic changes are observed in aqueous tear deficiency that are more severe in Sjögren syndrome. The distinct changes of corneal nerves include increased nerve number, tortuosity, and chances of branching, suggesting an attempted nerve regeneration. A strong correlation exists between the changes of nerve morphology and the degree of dry eye. These results provide some possible evidence for the abnormal corneal sensation in dry eye.

Adult↗
PubMed2001

Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo.

PURPOSE: To quantify keratocyte density according to stromal region and subject age and to measure the thickness of the normal human cornea and its layers in vivo. METHODS: Seventy normal corneas of 70 subjects were examined by confocal microscopy (contact lens wearers were excluded). Ages of subjects ranged from 12 to 80 years, with 10 subjects per decade. Images were recorded by continuously focusing the optical section through the full-thickness central cornea. Two independent human observers manually identified bright objects (keratocyte nuclei) against a dark background to quantify keratocyte density. This method was validated histologically in three human corneas. Thickness measurements were obtained by plotting mean reflected light intensity in images against corneal depth, and calculating distances between intensity peaks that corresponded to corneal layers. RESULTS: Full-thickness central keratocyte density was 20,522 +/- 2,981 cells/mm(3) (mean +/- SD, n = 69). The number of keratocytes in a full-thickness column of central stroma, which had a cross-sectional area of 1 mm(2), was 9624 +/- 1385 cells. Keratocyte density was highest in the anterior 10% of the stroma. Full-thickness keratocyte density was correlated with age (r = -0.62, P < 0.001), decreasing 0.45% per year. Central corneal thickness was 563.0 +/- 31.1 microm (mean +/- SD) and central epithelial thickness was 48.6 +/- 5.1 microm. CONCLUSIONS: This is the first study to quantify regional keratocyte density comprehensively in vivo across a broad age range of normal human subjects. The method was acceptable to both subject and observer, and may prove useful for quantifying keratocyte density in patients with corneal disorders or after corneal surgery.

Adolescent↗
PubMed1998

Spatial arrangement of fetal placental capillaries in terminal villi: a study using confocal microscopy.

The spatial arrangement of capillaries was studied in terminal villi of placentas at term by using confocal microscopy and methods for different types of 3-D reconstructions. Fixed specimens embedded in paraffin or glycol methacrylate resin were cut and stained with eosin. Digitized images of serial optical sections (approximately 0.5 microm) of individual terminal villi lying completely inside physical sections (100 microm) were captured by confocal laser scanning microscopy and analyzed. Topological reconstruction of the capillary bed and measurements of its Euler number, surface and volume rendering and wire-frame visualization were performed. Our findings showed that villous capillaries are arranged either in a single loop or in a more or less complicated anastomosing system. The results suggest that the combination of confocal microscope capture, methods for 3-D rendering and characterization of topological features reveals valuable information about spatial relationships of tissues in placental villi and the arrangement of the villous microcirculation, e.g. about the branching pattern of capillaries.

Adult↗
PubMed1993

Measurements of motoneuron somal volumes using laser confocal microscopy: comparisons with shape-based stereological estimations.

Previous studies on motoneuronal morphometry have assumed the geometry and orientation of neuronal soma in estimating somal volumes based on two-dimensional measurements. In this study, the optical sectioning property of the confocal microscope was used to make direct measurements of phrenic moto-neuron somal volume. These measured volumes were compared to shape-based stereological estimates of volume. Phrenic motoneuron pools in adult rats were retrogradely labeled with fluorescent dye. Labeled motoneurons, in 150-micron-thick tissue sections, were imaged using a Bio-Rad MRC 500 confocal microscope. Somal volumes were directly measured using ANALYZE, a comprehensive image processing software package. These volumes were compared to volume estimates based on five geometrical shapes previously used to study spinal motoneurons. In the adult phrenic motoneuron pool, overestimations of somal volume up to 300% were observed in cases where the Z-axis dimensions of the neurons were not simply related to the X and Y dimensions. None of the five geometrical shapes were found to be suitable for estimating either mean or individual somal volumes of the phrenic motoneuron pool. Confocal microscopy allowed accurate reconstruction along X, Y, and Z axes, and therefore provided a more direct method of measuring motoneuron somal volumes. We conclude that significant and inconsistent errors can be introduced by using shape-based stereological methods for estimating neuronal somal volumes.

Animals↗
PubMed1997

Cellular aspects of vascular remodeling in hypertension revealed by confocal microscopy.

Cellular aspects of remodeling in intact arteries have not been fully investigated, mainly due to the lack of an appropriate methodology that allows for simple measurements. The aim of this study was to develop a method based on laser scanning confocal microscopy (LSCM), compare it with previous methodology, and apply it to the study of remodeling in hypertension. The morphology of mesenteric resistance arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) was determined with wire myography on one segment with a standardized diameter setting (0.9[d100]) and with perfusion myography on a second segment from the same artery at the calculated equivalent pressure. The second segments were stained with the nuclear dye Hoechst 33342 (live tissue) or propidium iodide (fixed tissue) and measured with LSCM and MetaMorph software. Compared with wire myography, perfusion myography showed similar differences from those previously reported. Compared with LSCM, perfusion myography showed a similar lumen but significantly smaller wall thickness in both live and fixed tissue, probably due to measurement underestimation. In the study with LSCM, arteries from SHRSP compared with those from WKY showed (1) reduced lumen, (2) altered cell density that was significantly increased in the adventitia, decreased in the media, and unchanged in the intima, (3) significantly increased medial volume, (4) significantly smaller endothelial cell nuclei, and (5) adventitial-like cells in the media. We conclude that (1) LSCM is a reliable and straightforward method to study morphology in intact vessels, (2) it provides new information on the cellular changes in remodeling, (3) adventitia might play an active role in the process of remodeling in hypertension, and (4) endothelium "remodels" in hypertension.

Animals↗
PubMed2001

In vivo confocal microscopy of corneal epithelial ingrowth through a laser in situ keratomileusis flap buttonhole.

We report a case of epithelial ingrowth through a buttonhole 6 months after laser in situ keratomileusis. Elevation topography showed irregular astigmatism and an unreliable pachymetric map. In vivo confocal microscopy showed an epithelial basal cell mosaic with prominent borders and distinct nuclei. No corneal nerves were identified in any image. Cells in the anterior stroma possessed bright, reflective nuclei and appeared to form clusters. The interface between the stromal bed and the flap had formation of nests of fibrotic tissue and epithelial cells.

Adult↗
PubMed1999

Electron microscopic diagnosis of human flavivirus encephalitis: use of confocal microscopy as an aid.

The distinction between intracranial viral infections and inflammatory conditions requiring immunosuppression is important. Although specific laboratory reagents are readily available for some viruses, diagnosis of arbovirus infection is more difficult. Transmission electron microscopy (TEM) theoretically allows identification of viral particles independent of reagent availability, but it has limited sensitivity. We report two cases of human flavivirus encephalitis diagnosed by TEM. Laser scanning confocal microscopy (LSCM) was used in one case to survey unembedded tissue slices for focal abnormalities, from which fragments smaller than 1 mm2 were excised for epoxy embedding. This facilitated TEM identification of intracytoplasmic, budding, 35-40 nm spherical virus particles, confirmed by serology as St. Louis encephalitis. In contrast to mosquitoes and newborn mice, in which high viral loads are associated with minimal tissue responses, these biopsies showed florid angiodestructive inflammation and microgliosis, with rare virions in necrotic perivascular cells and astrocytes. To our knowledge, this represents the first ultrastructural study of St. Louis encephalitis in humans, indicating the potential value of LSCM-aided TEM.

Adult↗
PubMed2005

Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy.

The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59+/-2 pN, which correlated nicely to the 51+/-1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.

Antibodies, Monoclonal↗
PubMed2006

In vivo confocal microscopy and ex vivo flow cytometry: new tools for assessing ocular inflammation applied to rabbit lipopolysaccharide-induced conjunctivitis.

PURPOSE: Lipopolysaccharide (LPS) may act as a key stimulatory agent in ocular surface diseases (OSDs) through TNF-alpha release. We used in vivo confocal microscopy (CM) and ex vivo flow cytometry, two new tools for assessing ocular inflammation induced by LPS. METHODS: We investigated a model of acute inflammation in rabbits by subconjunctival injection of LPS and developed new evaluation techniques for animal models: CM, to observe inflammatory infiltrates, and conjunctival impression cytology (IC) specimens processed with in vitro CM and flow cytometry for assessing TNF-alpha and TNF receptor-1 (TNFR-1) expression. A neutralizing anti-TNF-alpha antibody was used to assess the role of TNF-alpha. RESULTS: In vivo CM provided high-resolution images of inflammatory infiltrates and leukocyte rolling in blood vessels. It showed that the LPS group presented strong conjunctival inflammation, reaching its maximum level 4 h after injection. Flow cytometry and immunostaining in IC specimens showed an increased expression of TNF-alpha and TNFR-1 in the epithelium. Immunohistology confirmed these results and showed infiltration of vimentin+, CD4(+), and CD8(+) cells in the conjunctiva. TUNEL-positive cells were found 4 h after injection. Neutralizing anti-TNF-alpha significantly inhibited LPS-induced inflammation and apoptosis evaluated by in vivo CM; and inhibited LPS-induced TNF-alpha and TNFR-1 expression by ex vivo conjunctival IC specimens evaluated by flow cytometry. CONCLUSIONS: IC specimens and new-generation in vivo CM were thus in good agreement with immunohistology and appeared to be reliable, effective, and nonharmful methods to investigate experimental models of OSDs. The two new tools applied here evaluate the animal models in vivo on the cellular lever. This study is consistent with the experimental research's strategy by reducing the number of experimental animals used.

Animals↗
PubMed1998

Analysis of 3-D microstructure of porous poly(lactide-glycolide) matrices using confocal microscopy.

Porous matrices of biodegradable polymers are extensively used as scaffolds in tissue engineering and as drug delivery devices. A critical component of the design, processing, and utility of such polymeric systems concerns the local void microarchitecture. In this study, a novel approach based on confocal fluorescence imaging was employed to visualize and quantify in 3 dimensions (3-D) the individual and population-level void morphology within porous polymeric matrices. Poly(lactic acid-glycolic acid) copolymer matrices were cast to yield void configurations of variable void sizes but constant cumulative voidage. Using confocal microscopy, fluorescently saturated polymer matrices were optically sectioned into serial 2-D images, and 3-D void contours were reconstructed via object discrimination and connectivity analysis. The resultant data was used to quantitate the matrix microstructure and map its evolution following polymer degradation. Under conditions of accelerated degradation, matrix erosion was found to cause a significant change in the disposition of voids; this involves two processes (void formation and void enlargement), the extent of which was influenced by the initial void size and the duration of erosion. By virtue of providing both static and dynamic descriptions of the void morphology in poly(lactic acid-glycolic acid) matrices, this is the first spatiotemporal study of the 3-D microarchitecture of porous, bioerodible tissue analog matrices.

Biocompatible Materials↗
PubMed2000

Subcellular localization of glutamate-stimulated intracellular magnesium concentration changes in cultured rat forebrain neurons using confocal microscopy.

Glutamate can stimulate increases in intracellular magnesium concentration ([Mg2+]i) and induce neurotoxicity, both independent of Ca2+ changes. Although Mg2+ is essential within the cell, very little is known about how it is regulated, especially in neurons. Therefore we used the fluorescent indicator, magindo-1 and confocal microscopy to examine possible intracellular pools of Mg2+ in cultured neurons that can be dynamically regulated by glutamate. The magindo-1 fluorescence signal was present throughout the cell body and extends into the neuronal processes. The magindo-1 405 nm/490 nm ratio signal was similar in the cytoplasm and nucleus, suggesting that resting [Mg2+]i is uniform across the neuron. The addition of 100 microM glutamate/10 microM glycine in an extracellular Ca2+- and Na+-free buffer stimulated an increase in [Mg2+]i in both the nuclear and cytoplasmic regions of similar magnitude and duration. This glutamate exposure also stimulated a [Mg2+]i increase in neuronal processes which was inhibited by the N-methyl-D-aspartate receptor antagonist, MK-801 (10 microM). The glutamate-stimulated [Mg2+]i increase in both the cell body and neuronal processes was dependent on the extracellular Mg2+ concentration. These findings suggest glutamate-stimulated [Mg2+]i changes may not only impact cytoplasmic processes, but also directly trigger nuclear events involved, for example, in neuronal injury.

Acetates↗
PubMed2007

In vivo confocal microscopy of polymorphic amyloid degeneration and posterior crocodile shagreen.

PURPOSE: To report the in vivo confocal microscopic findings of combined polymorphic amyloid degeneration and posterior crocodile shagreen and correlate these findings with clinical presentations and previous histopathologic analysis. METHODS: Case reports with confocal microscopic analysis and literature review. RESULTS: Three patients with bilateral corneal opacities presented to the Emory Eye Center Cornea Clinic for evaluation. The first patient, a 65-year-old woman, was noted to have bilateral, discrete, punctate, and filamentous refractile stromal opacifications that appeared to be concentrated in the deep stroma. The second patient, a 50-year-old man, had bilateral deep central stromal haze in a mosaic pattern with intervening cracklike clear zones. The third patient, an 80-year-old woman, had bilateral deep stromal deposits similar to patient 1 in addition to bilateral deep central stromal haze in a mosaic pattern with intervening cracklike clear zones similar to patient 2. Confocal microscopy showed bright, enhancing punctate lesions in the deep stroma and anterior stroma, with unaffected areas between these regions in patients 1 and 3, and central acellular opacification of irregular intensity with intermittent linear clear zones within the normal posterior stromal bed in cases 2 and 3. The first patient was diagnosed with isolated polymorphic amyloid degeneration, the second patient was diagnosed with isolated posterior crocodile shagreen, and the third patient was diagnosed with combined polymorphic amyloid degeneration and posterior crocodile shagreen. CONCLUSION: These cases are, to our knowledge, the first to report confocal microscopic findings in isolated polymorphic amyloid degeneration and combined polymorphic amyloid degeneration with posterior crocodile shagreen.

Aged↗
PubMed1996

A simple and rapid technique for the immunofluorescence confocal microscopy of intact Arabidopsis root tips.

Visualisation of immunofluorescence labelling of Arabidopsis roots has previously been limited to single cell layers. A simple, rapid method has been devised in which the whole root can be processed to allow antibody penetration into several cell layers. When optically sectioned using confocal microscopy, cellular arrangements of microtubules, callose, calmodulin and a phosphoprotein epitope have been visualised using this technique. As the root is not physically sectioned, information regarding the three-dimensional position of individual cells in relation to each other and the tissue as a whole is retained. Using this technique, we have assessed the effect of brefeldin A on the frequency of mitotic arrays in root tip cortical and epidermal cells, and found that the occurrence of phragmoplasts increases significantly with brefeldin A treatment. This study demonstrates the possible future use of the whole root technique to assess rapidly the developmental, mutational and inhibitor-induced changes in the organisation of cellular components in Arabidopsis.

Arabidopsis↗
PubMed1998

Confocal microscopy. Applications in research and practice of pathology.

OBJECTIVE: To discuss recent advances in the use of confocal microscopy to perform quantitative analysis of physiologic and pathologic intracellular events in the living organ. STUDY DESIGN: We developed a new confocal microscope system equipped with a microlens-arrayed multipinhole scanning disc and water-immersion objective lenses. The confocal microscope enables us to quantitate nonhomogeneous changes in the three-dimensional intracellular calcium ion concentration in a living, whole heart. RESULTS: The new system allows in situ quantitative imaging of the calcium wave in the living heart at a full video rate. Calcium waves propagating from cell to cell were frequently interrupted by calcium transients from spontaneous sinus rhythm, but low transparency of the living tissues and cell injury with laser light still remained unsolved. CONCLUSION: The evolution of the confocal microscope with high spatial and temporal resolution renders the investigation of biologic phenomena even in the living organ.

Calcium↗
PubMed1999

In vivo confocal microscopy of a family with Schnyder crystalline corneal dystrophy.

OBJECTIVE: To analyze corneal morphology in Schnyder crystalline corneal dystrophy (SCCD) in vivo. DESIGN: Observational case series. PARTICIPANTS: Five eyes of four patients of various belonging to the same family were examined. METHODS: The eyes were examined using in vivo confocal microscopy (CM). MAIN OUTCOME MEASURES: The corneal morphology including keratocytes and stromal extracellular matrix, as well as basal epithelial/subepithelial nerves is, described. RESULTS: The right eye of a 48-year-old male patient had been treated with anterior keratectomy and the left eye with phototherapeutic keratectomy (PTK). The right eye presented with increased stromal reflectivity owing to accumulation of extracellular matrix and large subepithelial crystalline deposits. Far fewer crystals could be observed in the left eye. The haze, however, was increased, either because of the dystrophy or the excimer laser treatment. The anterior keratocytes appeared irregular, and the subepithelial nerves were undetectable in both eyes. His 78-year-old mother showed more advanced changes with dense crystals, highly fibrotic stroma, and severely damaged corneal innervation. The partly irregular anterior keratocytes of the 9- and 7-year-old children contained intracellular deposits, although the corneas were clinically clear with only subtle subepithelial crystalline formation. Accumulation of similar reflective material was also observed in association with the prominent subepithelial nerves. CONCLUSIONS: In the early stages of SCCD, highly reflective deposits accumulate intracellularly and around anterior keratocytes and along subepithelial nerves. With time, the normal corneal architecture becomes disturbed by large extracellular crystalline deposits and accumulation of highly reflective extracellular matrix resulting in central opacity and disruption of the subepithelial nerve plexus. Furthermore, neural regeneration after keratectomy appears delayed in SCCD.

Aged↗
PubMed1997

A rapid method for the assessment of bone architecture by confocal microscopy.

Conventional ways of demonstrating and analysing the components of osseous tissue have always been hampered by the difficulty of physically sectioning bone. In this study, we have used Acridine Orange staining of 100-micron-thick unembedded bone slices and then assessed the cellular and tissue architecture by confocal microscopy. The result showed the Acridine Orange, by differential staining of the cellular nucleic acids, permits ready assessment of cell shape and cell organization as well as variations in growth patterns. Our studies have provided a new and relatively easy way of assessing the morphology of bone specimens by rendering unnecessary the need for embedding, decalcification and thin sectioning of the osseous tissue.

Acridine Orange↗
PubMed1995

Vagal afferent innervation of rat abdominal paraganglia as revealed by anterograde DiI-tracing and confocal microscopy.

Abdominal vagal afferent fibers were selectively labeled by injecting the fluorescent carbocyanine dye DiI into the left nodose ganglion of rats. Almost all paraganglia that were distributed along the five major abdominal vagal branches and their subbranches were found to be innervated by labeled vagal afferents. Laser scanning confocal microscopy with its single optical sectioning and three-dimensional reconstruction capabilities were used to analyze this innervation in more detail for paraganglia near the vagal hepatic branch and liver hilus. Furthermore, in double-labeling studies, it was demonstrated that a large percentage of the vagally innervated glomus cells were capable of catecholamine synthesis on the basis of their positive staining for tyrosine hydroxylase antibody. These findings support the concept of a chemoreceptive function for the abdominal paraganglia.

Animals↗