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PubMed1999

[The alterations in corneal structure at III/IV stage of keratoconus by means of confocal microscopy and ultrasound biomicroscopy before penetrating keratoplasty].

PURPOSE: The aim of the study was in-real time observation and morphological evaluation of the human corneas at III/IV stage of keratoconus, using the scanning slit confocal microscope Confoscan P4 and ultrasound biomicroscopy--UBM. MATERIAL AND METHODS: The patients with keratoconus were examined according to the Amsler scale. The material consisted of 12 corneas of 11 patients (8 men, 3 women), where assessment of the corneal structure was performed with the confocal microscope ConfoScan P4 (Tomey) and ultrasound biomicroscopy--UBM Model 840 (Humphrey Instruments). The comparison of different corneal regions (central and peripheral) was evaluated. RESULTS: The confocal microscopy and UBM revealed thinning of the layers of the corneal structure and pathological changes in the central area, especially at IV stage of keratoconus. The desquamating superficial cells were elongated, arranged around the apex of the cornea. Below the Bowman's membrane a considerable disarrangement of collagen fibers reflected by bright background illumination was observed. In the posterior part of the stroma the folds were detected. The examination of the cornea showed thickening in the peripheral part, central detachment of the Descemet's membrane and the endothelium from the posterior surface of the cornea. The thickness of the cornea varied from 0.201 to 0.384 mm in the central part and 0.675 to 0.740 mm in the peripheral area. CONCLUSION: Confocal scanning microscopy combined with ultrasound biomicroscopy enables the cornea to be examined in vivo. It can be used to localize pathological changes in individual corneal layers and to assess their extent.

Adolescent↗
PubMed2002

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).

Acetylcholine↗
PubMed1995

Immunocytochemical study of NA+ K(+)-ATPase alpha 1 and beta 1 subunits in human and rat normal hepatocytes using confocal microscopy.

Hepatic Na+ K(+)-ATPase was recently shown to be composed of two alpha 1 and beta 1 subunits similar to that of kidney. Its localization on hepatocyte plasma membranes was not clearly established. We have studied the localization of alpha 1 and beta 1 subunits using immunocytochemical method and confocal microscopy. In accordance with previous cytochemical findings, the catalytic alpha 1 subunit was distributed in basolateral and bile canalicular membranes of hepatocytes. The beta 1 subunit was not demonstrated, due to its very low amount in the liver. The controversy about the bile canalicular localization was discussed.

Animals↗
PubMed2003

Confocal microscopy of Thygeson's superficial punctate keratopathy.

PURPOSE: To describe the changes seen with the confocal microscope in Thygeson's superficial punctate keratopathy (TSPK). METHODS: Confocal microscopy was performed on six patients with TSPK presenting to Manchester Royal Eye Hospital from October 1999 to June 2001. Both eyes were examined including the normal fellow eyes of two patients with unilateral TSPK. Images were compared with age-matched controls. RESULTS: Irregular nerve fibers, masked by considerable haze, were seen in the subepithelial nerve plexus immediately above Bowman's membrane in TSPK. Generalized haze was also present in the anterior stroma along with areas of high reflectivity, microdots, and reflective bodies. Keratocytes had highly reflective nuclei and cell bodies of irregular size, orientation, and shape. The changes were related to the duration of TSPK, were not seen in normal fellow eyes, and were also present in areas without lesions. CONCLUSION: Confocal imaging of TSPK demonstrated considerable abnormalities in the subepithelial nerve plexus, Bowman's membrane, and the anterior stroma that were related to disease duration. Similar changes have been reported in a variety of corneal diseases and following laser corneal surgery. Recent evidence has supported interactions between the epithelium and Bowman's membrane and the anterior stroma in corneal disease and after laser corneal surgery. We speculate that such interactions may have a role in TSPK.

Adult↗
PubMed2005

Visualisation of the mechanosensitive channel of large conductance in bacteria using confocal microscopy.

The mechanosensitive channel of large conductance (MscL) plays an important role in the survival of bacterial cells to hypo-osmotic shock. This channel has been extensively studied and its sequence, structure and electrophysiological characteristics are well known. Here we present a method to visualise MscL in living bacteria using confocal microscopy. By creating a gene fusion between mscl and the gene encoding the green fluorescent protein (GFP) we were able to express the fusion protein MscL-GFP in bacteria. We show that MscL-GFP is present in the cytoplasmic membrane and forms functional channels. These channels have the same characteristics as wild-type MscL, except that they require more pressure to open. This method could prove an interesting, non-invasive, tool to study the localisation and the regulation of expression of MscL in bacteria.

Bacterial Physiological Phenomena↗
PubMed2000

Laser in situ keratomileusis flap margin: wound healing and complications imaged by in vivo confocal microscopy.

PURPOSE: To examine the healing response of laser in situ keratomileusis flap margin in vivo. METHODS: Forty-three eyes of 43 patients who had undergone myopic (n = 39) or hyperopic (n = 4) laser in situ keratomileusis were examined once after surgery. The flap margin was imaged by in vivo confocal microscopy at various depths, and the wound healing response, flap alignment, and complications were evaluated. Ten eyes were examined on day 3 postoperatively, 13 eyes at 1 to 2 weeks, 10 eyes at 1 to 2 months, five eyes at 3 months, and five eyes at 6 months or later. RESULTS: At 3 days after laser in situ keratomileusis, the surface epithelium and basal epithelium appeared normal. Keratocyte activation was strongest at 1 to 2 weeks and 1 to 2 months, and an increased amount of haze was observed correspondingly. Intrastromal epithelial cells forming a plug could occasionally be perceived in the wound gape. Wound constriction was completed in most cases by 3 to 6 months or later. Good alignment was observed in 12 of 43 flaps (27.9%) and moderate and poor alignment in 17 of 43 flaps (39.5%) and 13 of 43 flaps (30.2%), respectively. Poor alignment was not associated with lamellar epithelial ingrowth. Epithelial ingrowth was associated with dense haze at the interface. Diffuse lamellar keratitis was imaged in two corneas after hyperopic laser in situ keratomileusis. CONCLUSIONS: The laser in situ keratomileusis incision wound at the flap margin appears to heal after the sequence observed in incisional wounds in nonhuman primates. Complications, such as lamellar epithelial in growth and diffuse lamellar keratitis, were often observed, particularly after hyperopic laser in situ keratomileusis.

Adult↗
PubMed2002

Depth and age-dependent distribution of keratocytes in healthy human corneas: a study using scanning-slit confocal microscopy in vivo.

PURPOSE: To document keratocyte distribution and changes with age in the cellular network of the human cornea in vivo. SETTING: Department of Ophthalmology, University of Rostock, Rostock, Germany. METHODS: Forty-nine eyes of 31 healthy subjects of various ages were examined with a modified Microphthal scanning-slit confocal microscope (SSCM) (Hund) to document keratocyte distribution in the intact living cornea. Optical sections made by confocal microscopy were recorded on videotape, and the keratocyte density was determined for the total volume of the cornea and for the stromal sublayers. RESULTS: The highest cell density was in the anterior stroma of the cornea immediately posterior to Bowman's membrane (24 320 cells/mm(3) +/- 6740 [SD]), the lowest in the central area (11,610 +/- 4290 cells/mm(3)), and an intermediate density in the posterior stroma immediately adjacent to Descemet's membrane (18,850 +/- 4610 cells/mm(3)). The differences were statistically significant (P <.005). The keratocyte density was significantly lower in the anterior and posterior regions in the group older than 50 years: Cell density at 4% depth was 20,960 +/- 8200 cells/mm(3) and at 96%, 15 520 +/- 4290 cells/mm(3) (P <.05). CONCLUSIONS: In healthy living corneas, the keratocyte density was high in the areas adjacent to Bowman's and Descemet's membranes and was lower in patients older than 50 years than in those younger than 50 years. Further studies are needed to document the rate of change with age and to better understand the role and capacity of aging keratocytes in regenerative processes following corneal diseases or surgical procedures.

Adult↗
PubMed1996

The distribution of Ag-NORs proteins and ribosomal RNA genes in human spermatogonia: an original procedure associating silver-staining, fish and confocal microscopy.

It is generally accepted that Ag-NORs proteins are associated with nucleolar components engaged in rRNA synthesis. Since conflicting results have been reported concerning the intranucleolar sites of active ribosomal genes, the relationships between the silver-stained areas and the distribution of rDNA were analyzed in human spermatogonia using confocal microscopy of fluorescent in situ hybridized preparations previously stained with silver. The fluorescent signal strictly corresponded to the silver-stained area, identified at the ultrastructural level as the fibrillar center and the surrounding dense fibrillar component. A quantitative evaluation of nucleolar activity solely based on silver-staining may be misleading, taking into account that only Ag-positive proteins located in the dense fibrillar component can be associated with transcriptional activity, while those stored in the fibrillar center are associated with non-transcribed rRNA genes.

DNA, Ribosomal↗
PubMed1997

Murine thymic nurse cells and rosettes: analysis of adhesion molecule expression using confocal microscopy and a simplified enrichment method.

Thymic nurse cells (TNC) and T-cell stromal rosettes (ROS) are two in vivo models for stromal cell-thymocyte interactions. We describe a simplified enrichment method for TNC and ROS that overcomes the necessity for large amounts of tissue. The complexes were further analyzed with confocal microscopy, and three subunits of ROS were defined on the basis of their central cell phenotype, i.e., macrophage, dendritic, or epithelial cell rosettes. Because adhesion molecules are proposed to play a crucial role in T-cell development, we investigated CD44, LFA-1, and ICAM-1 expression in such complexes. The epithelial component of TNC expresses CD44 and ICAM-1, whereas intra-TNC thymocytes are LFA-negative. With regard to ROS, all subsets expressed CD44, and macrophage and dendritic cell ROS were also ICAM-1-positive and LFA-1-positive. The current protocol opens the possibility for further in vivo analysis of stromal cell-thymocyte interactions, e.g., for studies of scarce gene mutant mice.

Animals↗
PubMed2005

Confocal microscopy studies of a model oligoribonucleotide HIV inhibitor.

Previous work has shown that novel amphipalhic oligo and polyribonucleotides are potent inhibitors of HIV. It was hypothesized that the mechanism(s) of action for these compounds might be inhibition of retroviral reverse transcriptase (RT) and/or viral uptake by cells. A fluorescent oligonucleotide analog was prepared, and confocal microscopy studies were undertaken in order to examine oligonucleotide-cell interactions.

Anti-HIV Agents↗
PubMed2004

Simultaneous 1H PFG-NMR and confocal microscopy of monolayer cell cultures: effects of apoptosis and necrosis on water diffusion and compartmentalization.

We induced apoptosis and necrosis in monolayer cultures of Chinese hamster ovary cells using okadaic acid and hydrogen peroxide (H2O2), respectively, and examined the effect on water diffusion and compartmentalization using pulsed-field-gradient (PFG) 1H-NMR and simultaneous confocal microscopy. In PFG experiments characterized by a fixed diffusion time (<4.7 ms) and variable b-values (0-27000 s/mm2), 1H-NMR data collected with untreated cells exhibited multiexponential behavior. Analysis with a slow-exchange model revealed two distinct cellular water compartments with different apparent diffusion coefficients (ADCs; 0.56, 0.06 x 10(-3) mm2/s) and volume fractions (0.96 and 0.04). During the first 12 hr of necrosis or apoptosis, the amount of water in the smallest compartment increased twofold before significant changes in cell density or plasma membrane integrity occurred. Over the same period, water content in the largest compartment decreased by a factor of >2 in apoptotic cells, in accordance with observed cell shrinkage, and changed little in necrotic counterparts, where only slight swelling was evident. These results indicate that PFG 1H-NMR serves as a sensitive indicator of early cell death in monolayer cultures, and can be used to distinguish apoptosis from necrosis. Measurements of restricted diffusion and water exchange are presented to elucidate the compartment origins and justify the model assumptions.

Animals↗
PubMed2000

Distinct subcellular localization of transiently expressed types 1 and 2 iodothyronine deiodinases as determined by immunofluorescence confocal microscopy.

We compared the subcellular localization of FLAG-epitope tagged Types 1 and 2 deiodinases (D1 and D2) transiently expressed in human embryonic kidney (HEK-293) and mouse neuroblastoma (NB2A) cells. D2 is an integral membrane protein based on resistance to extraction at pH 11 with the NH2 terminus in the endoplasmic reticulum (ER). Immunofluorescence confocal microscopy using anti-FLAG and anti-GRP78/BiP antibodies showed the FLAG-D1 signal was found in the periphery of the cells and not co-localized with the ER specific marker GRP78/BiP. On the other hand, FLAG-D2 protein was found in the ER co-localized with the GRP78/BiP protein. These differential distribution patterns indicate subcellular sorting of D1 and D2 is determined by intrinsic protein sequence and can explain the ready access of D2-generated T3 to the nucleus.

Animals↗
PubMed2000

A ruthenium probe for cell viability measurement using flow cytometry, confocal microscopy and time-resolved luminescence.

The capability of the new luminescent probe (dibenzo[h,j] dipyrido[3,2-a:2',3'-c]phenazine)bis(2,2'-bipyridine)ruthenium(II) dication, (RB2Z), to discriminate live and dead cells has been tested on rat hepatocytes and mouse lymphocytes. RB2Z-stained cells were analyzed using flow cytometry, fluorescence (confocal) microscopy and time-resolved luminescence measurements. The established viability probes propidium iodide (PI) and SYTOX green (SG) were used as controls. The intense luminescence of RB2Z at 606 nm is localized in the nucleus of nonviable cells. Viability measurements by flow cytometry and fluorescence microscopy using RB2Z as dead-cell marker yield the same results as PI and SG. The luminescence lifetime of RB2Z also displays sensitivity to cell viability (0.45 and 0.82 microsecond in presence of fully viable and dead cells, respectively). This ruthenium complex is photostable under laser sources and its 200 nm Stokes shift facilitates multicolor labeling experiments in flow cytometry and fluorescence microscopy. Unlike the currently available probes, the long-lived excited state of RB2Z also allows assays based on luminescence decay measurements.

Animals↗
PubMed1996

Section thickness and mitotic counts in ovarian mucinous carcinoma. Methodological study with scanning confocal microscopy.

To study the effect of section thickness on mitotic counts, paraffin sections of 28 cases of mucinous ovarian carcinomas were analyzed. Mitotic counts, estimated with the number of mitoses per mm2 of neoplastic epithelium (M/Vv index) and per mm3 from optical sections and through the whole section thickness were done. Section thickness was measured with scanning confocal microscopy from each specimen of 3 series of sections with different nominal thicknesses (5, 8, and 10 microns). Section thickness varied considerably within each series of sections. The sections of the 5-microns group tended to be thicker and the sections of the 10-microns group thinner than expected on the basis of their nominal values. As expected, mitotic counts through the section gave higher M/Vv index values than counts done using optical sections. M/Vv values obtained using optical sections increased with increasing nominal section thickness. The study suggests that, when the section thickness cannot be measured, it is advisable to do mitosis counting by using optical sections in lightly stained tissue sections in the thickness range of 8-15 microns.

Adenocarcinoma, Mucinous↗
PubMed1997

Confocal microscopy of human lens membranes in aged normal and nuclear cataracts.

PURPOSE: To visualize the structure and determine the continuity of lipid membranes in lens fiber cells (LFCs) from human aged normal and cataractous lenses. METHODS: Thick sections from human nuclear cataracts and aged normal lenses were stained with the lipophilic probe DiI, and then analyzed by confocal microscopy. Staining patterns of membranes were observed in individual optical sections or three-dimensional projections of z-series taken in longitudinal section and cross-section of LFCs from different regions within the lens nucleus. RESULTS: DiI bound to and delineated the plasma membrane of LFCs from all regions of the lens nucleus. Three-dimensional projections of z-series from aged normal and cataractous lenses suggested that some of the stained lipid membranes were not continuous with LFC plasma membrane of cataractous lenses. CONCLUSIONS: The results obtained using these methods demonstrated that lipid membranes, discontinuous with the plasma membrane of LFCs, were indicative of a novel process occurring predominately in cataractous human lenses.

Aged↗
PubMed1998

Quantification of the nucleocytoplasmic distribution of wild type and modified proteins using confocal microscopy: interaction between 90-kDa heat shock protein (Hsp90 alpha) and glucocorticosteroid receptor (GR).

The investigation of molecular interactions in whole cells by immunofluorescence was developed recently, based on the targeting of the protein partners to different cellular compartments and analysis of the modifications in their subcellular distribution resulting from their interaction. This paper describes the adaptation of the confocal microscopy to the quantification of the partitioning of transiently coexpressed proteins between nucleus and cytoplasm. We defined a nucleocytoplasmic ratio R, corresponding to the difference between nuclear and cytoplasmic fluorescence intensities divided by their sum (N - C/N + C), which does not refer to absolute fluorescence intensities. Interaction was detected by statistically comparing the distribution of R value frequencies in cell populations expressing one or both proteins. The convenience of this whole cell method was demonstrated by detecting and analyzing interaction between the human glucocorticosteroid receptor (GR) and the chick 90-kDa heat shock protein (Hsp90), using various combinations of wild-type and nuclear- or cytoplasmic-targeted GR and Hsp90. In addition, three Hsp90 deletion/ truncation mutants were tested: the C-terminal truncated mutant NC4 interacted slightly, indicating the contribution of this part of the molecule to the interaction with GR, while the shorter truncated mutant NC6 did not interact with GR, likely resulting from an incorrect folding of the molecule. No role for the first charged region (delta A') was found as shown by the strong interaction detected for the delta A'Hsp90. This method can fruitfully be applied to the delimitation of the amino-acid sequences involved in protein-protein interaction by mutational analysis, especially to seek confirmation of other methods or when other approaches have failed.

Animals↗
PubMed2004

In vivo confocal microscopy of keratic precipitates.

OBJECTIVE: To evaluate the heterogeneity of keratic precipitates (KP) in varying subtypes of uveitis by in vivo confocal microscopy (IVCM). METHODS: The KP were viewed with a scanning confocal microscope in patients (n = 33) who sought care at a tertiary referral uveitis service for immune-mediated and infectious forms of uveitis, including HLA-B27-associated uveitis, sarcoidosis, Vogt-Koyanagi-Harada syndrome, juvenile chronic arthritis, Fuchs heterochromic iridocyclitis, cytomegalovirus retinitis, herpes zoster ophthalmicus, ocular toxoplasmosis, and idiopathic uveitis. Images were captured and digitalized in real time. RESULTS: Forty-two eyes of 33 patients were examined in this study. Patient age ranged from 22 to 84 years, with a mean age of 49.4 years. Seventeen (52%) of the patients were women, and 16 patients (48%) were men. The KP ranged in diameter from 10 to 350 mum. We observed the following absolute and speculative outcomes: KP are markedly heterogeneous and variable as documented by IVCM; KP in individual patients are consistent throughout the cornea; the morphologic features of KP change across time; infectious vs noninfectious causes of uveitis seem to be readily distinguishable by using IVCM; and KP may have consistency for specific disease states and therefore may have diagnostic importance. CONCLUSIONS: To our knowledge, this is the first time that IVCM has been used to describe the architecture and heterogeneity of KP in uveitis. Such observations reveal a heterogeneity that could not be appreciated by conventional slitlamp microscopy and may have diagnostic relevance.

Adult↗
PubMed1997

Confocal microscopy of cementocytes and their lacunae and canaliculi in rat molars.

The present study was designed to analyze the morphological characteristics of cementocytes and osteocytes. The maxillae of 10-week-old Wistar rats were used for observations. Non-decalcified ground sections stained vitally with fluorescence dyes and decalcified frozen sections stained with FITC-phalloidin were examined by confocal microscopy. Calcein and alizarin red stained the calcification front of bone, cementum, and dentin intensely. In addition, lacunae and canaliculi of cementocytes and osteocytes as well as dentinal canals were stained with the fluorescent dyes. The staining of lacunae and canaliculi was less intense than that of the calcification front of bone, cementum and dentin. The canaliculi of cementocytes and osteocytes were connected with the canaliculi extending from the calcification front of cementum and bone, respectively. The canalicular density was less in the cellular cementum than in the bone. Areas devoid of canaliculi were numerous in the cellular cementum, whereas areas devoid of canaliculi were scarce in the alveolar bone. Further, the lacunae of cementocytes showed various shapes, from oval to tubular, while the lacunae of osteocytes were invariably oval. The cell body and the cytoplasmic processes of cementocytes were positive for FITC-phalloidin within the extracellular matrix of cellular cementum, which was negative. The distribution of actin filaments in the osteocytes and the cementocytes was predominantly cortical and appeared to be closely associated with the cell membrane of the cell bodies and the cytoplasmic processes. Intense staining was seen at the proximal part of the cytoplasmic processes in both osteocytes and cementocytes, showing a punctuated structure of the cells that was more frequent in osteocytes than in cementocytes. The stress fiber known to be present in most of the cultured cells was not evident in the these cells in situ. The cells incorporated in the cementodentinal junction were strongly stained with FITC-phalloidin. The distribution pattern of the cytoplasmic processes stained with FITC-phalloidin was similar to that of the canaliculli stained vitally. The cytoplasmic processes of osteocytes and cementocytes were connected with those of cells lining the surface of bone and cementum. The present result-that lacunae and canaliculi of cementocytes were stained vitally with the fluorescence dyes-suggests that cementocytes may have a role in secondary calcification of cellular cementum. Further, the lower density of cytoplasmic processes in cementocytes than in osteocytes suggests a lack of complexity in the intercellular network within the cellular cementum.

Actins↗