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PubMed1996

Confocal in vivo microscopy and confocal laser-scanning fluorescence microscopy in keratoconus.

The purpose of this study was the determination of morphological changes in the corneal epithelium and the keratocyte network in keratoconus. In all, 33 eyes of 19 patients were examined in vivo using the confocal slit-scanning microscope Microphthal. After penetrating keratoplasty, recipients' trephanates were stained with the Live/Dead kit and examined using the confocal laser-scanning fluorescence microscope Diaphot 300/Odyssey. The fluorescence images were reconstructed three-dimensionally. All findings were compared with data from healthy corneas. Morphological alterations were found only in the area of the corneal apex; obviously elongated superficial epithelial cells arranged in a whorl-like fashion were found. Near Bowman's membrane, highly reflective changes and fold-like structures were visible. The anterior stroma also showed an increased reflectivity. In the posterior stroma, typical findings were Vogt's striae and keratocytes with extremely long processes arranged nearly in parallel. In scarred stroma the keratocytes were spindle-shaped and arranged irregularly. The spatial organization of the living keratocyte network could be demonstrated through three-dimensional reconstructions.

Adult↗
PubMed2003

Imaging and non-contact profile analysis of Nd:YAG laser-irradiated teeth by scanning electron microscopy and confocal laser scanning microscopy.

Two types of non-contact optical profilometers, one performing scanning electron microscopy (SEM) installed with a 3D analyzer and another performing confocal laser scanning microscopy (CLSM) were applied to evaluate imaging and surface profiles simultaneously on enamel and dentin after Nd:YAG laser irradiation. The results were correlated with a stylus profilometer. Surface roughness (Ra) was also measured. Laser was applied perpendicularly to vertical sections of human extracted caries-free molars. Analysis was done on the same spot of the same specimen by both SEM and CLSM for comparison. After irradiation, enamel produced a flake-like surface and dentin resulted in a melted globule surface. SEM and CLSM gave similar surface profiles and different image contrast. The Ra obtained by CLSM was larger than that by SEM. Both SEM and CLSM provided a non-contact evaluation of tooth structural change by laser irradiation through surface analysis in selected micro areas, which was not possible using the stylus profilometer.

Dental Enamel↗
PubMed1997

Fluoride-induced precipitates on enamel surface and subsurface areas visualised by electron microscopy and confocal laser scanning microscopy.

The present study examined the enamel surface after in vitro topical treatments with a neutral 2% NaF solution. For minimising the risk of artefacts, samples were inspected without pre-treatment as fresh, naturally wet specimens by complementary techniques: variable pressure electron microscopy (VP-SEM) and confocal laser scanning microscopy (CLSM). VP-SEM provided information on the surface morphology, whereas CLSM allowed non-destructive visualisation of subsurface areas. Neutral NaF solutions induced globular precipitates on the enamel surfaces. If the globules formed may be described as "calcium fluoride like material", the additional information of this experiment is that, after interaction with neutral solutions, they also contain considerable amounts of NaF. When the NaF solutions were soaked on pellets and then were brought into contact with the enamel surfaces, NaF crystallites of cubic shape are formed. Confocal optical tomographies of subsurface enamel after treatments with neutral NaF solutions revealed partly coroded enamel structures, whereas VP-SEM showed intact surfaces. In between the coroded areas, a fine granulate precipitate could be observed. This is evidence that fluoride induces the formation of sub-surface precipitates only when applied during demineralisation. The precipitate could be readily removed by 24-h contact with a KOH solution.

Adolescent↗
PubMed1996

[Ca2+]i in rat afferent arteriole during constriction measured with confocal fluorescence microscopy.

Using confocal fluorescence microscopy, we followed the time course of intracellular calcium concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC) and endothelial cells (EC) of microperfused afferent arterioles isolated from rat juxtamedullary nephrons. Measurements were made while arterioles were exposed to pharmacological agonists or myogenic stimulation. Luminal addition of acetylcholine triggered an immediate increase of [Ca2+]i in EC and vasodilation. Addition of norepinephrine to the bath solution constricted the vessel and initiated periodic oscillations of [Ca2+]i in VSMC and synchronized vasomotion. Increase of perfusion pressure from 80 to 120 mmHg induced an immediate 9.6 +/- 2.9% (P < 0.05, n = 9) increase of [Ca2+]i in VSMC that was sustained. The arteriole dilated transiently when the perfusion pressure was increased, and persistent vasoconstriction to reduced diameter was observed after 35 s. When nitric oxide (NO) production in the perfused vessel was blocked with nitro-L-arginine methyl ester prior to the pressure step, similar profiles of change in VMSC [Ca2+]i were observed and persistent vasoconstriction began after 28 s. The same pressure step triggered an increase of [Ca2+]i in EC by 11.3 +/- 1.1% (P < 0.05, n = 5). These observations suggest that myogenic constriction of afferent arterioles was associated with an increase of [Ca2+]i in VSMC, and the constriction was delayed by endogenous NO production.

Animals↗
PubMed2000

[Stereologic analysis of vacuolization of the T-system of frog muscle fibers, detected using confocal fluorescence microscopy].

The confocal fluorescence microscopy has been used for quantitative evaluation of the T-system reversible vacuoles produced by efflux of 80-120 mM glycerol from frog skeletal muscle fibers. The fibers were stained by membrane probe RH414 and by water-soluble dye fluorescein dextran that marks the vacuolar lumen. Using morphometrical and stereological methods the volume and surface densities of vacuoles were measured on single optical sections and Z-series during a 30 min glycerol efflux. Various methods of measurements (three-dimensional reconstruction of vacuoles, computer morphometry, point counting method) applied to the same Z-series provide similar results. The vacuolar membranes stained by RH414 look like bright rings 0.3-0.4 micron in width. It is concluded that the real position of vacuolar membrane corresponds to the middle of the vacuolar envelope. The measurements of the external dimensions of the envelope overestimate the stereological parameters up to 50%. The volume density of vacuoles reaches 10% within 20-30 min of glycerol efflux. It means that the volume of the T-system may increase by 25-30 times compared to the control value (0.3-0.4%). The surface density of vacuoles during reversible vacuolation is equal to 0.20-0.35 micron-1 and does not exceed the surface density of normal T-system. The sufficiency of membrane material for the T-system reversible vacuolation is discussed in addition to the role of geometrical factor in this phenomenon.

Animals↗
PubMed2005

Atomic force microscopy and laser confocal scanning microscopy analysis of callose fibers developed from protoplasts of embryogenic cells of a conifer.

Efficiency of novel fiber formation was much improved in protoplast culture of embryogenic cells (ECs) of a conifer, Larix leptolepis (Sieb. et Zucc.) Gord., by pre-culturing ECs in a medium containing a high concentration of glutamine (13.7 mM). The fibrillar substructures of large and elongated fibers of protoplasts isolated from Larix ECs were investigated by laser confocal scanning microscopy (LCSM) after Aniline Blue staining and atomic force microscopy (AFM) using a micromanipulator without any pre-treatment. Fibers were composed of bundles of fibrils and subfibrils, whose diameters were defined as 0.7 and 0.17 mum, respectively, by image analysis after LCSM and AFM. These fibers were proven to be composed of callose by using specific degrading enzymes for beta-1,4-glucan and beta-1,3-glucan.

Carbohydrate Conformation↗
PubMed2002

Imaging of fluorine and boron from fluorinated boronophenylalanine in the same cell at organelle resolution by correlative ion microscopy and confocal laser scanning microscopy.

PURPOSE: There is a clear need for a technique that provides subcellular locations of fluorine and boron atoms from fluorinated neutron capture agents because positron emission tomography is being tested as a tool for providing tumor boron concentrations in boron neutron capture therapy. EXPERIMENTAL DESIGN: Ion microscopy was used in combination with confocal laser scanning microscopy to investigate the subcellular locations of fluorine and boron from fluorinated p-boronophenylalanine (F-BPA) in human glioblastoma T98G cells. The fluorinated compound was also compared with p-boronophenylalanine (BPA) for delivery of boron after a clinically relevant 6-h exposure. Mitochondria were identified by rhodamine 123 labeling. A strict cryogenic sample preparation was used, and measurements were made in fractured freeze-dried cells. RESULTS: The nucleus, a perinuclear mitochondria-rich cytoplasmic region, and the remaining cytoplasm were the three subcellular regions identified in individual T98G cells. In cells treated with F-BPA, the mitochondria-rich perinuclear cytoplasmic region exhibited significantly lower fluorine and boron signals than the remaining cytoplasm and the nuclei. Ion microscopy observations revealed a nearly 1:1 distribution of fluorine and boron in subcellular compartments. Quantitative subcellular observations indicated that there was no significant difference in boron delivery to subcellular compartments between the F-BPA and nonfluorinated BPA. CONCLUSIONS: These observations provide the first direct evidence that fluorine and boron from fluorinated BPA are cocompartmentalized in cells and that the fluorinated compound is as efficient for boron delivery as the nonfluorinated BPA at a clinically relevant time point. These observations provide strong support for the use of F-BPA in positron emission tomography biodistribution studies for boron neutron capture therapy.

Boron↗
PubMed2004

Radial glia produce and align the ligand fibronectin during neuronal migration in the developing chick brain.

We demonstrated previously that alpha8beta1 integrin regulates the migration and survival of immature neurons during development of the chicken optic tectum; however, the potential extracellular ligand was unknown. We used immunohistochemistry to determine if several potential ligands (fibronectin, tenascin, vitronectin, and osteopontin) were expressed during neuronal migration along radial glia (RG). Fibronectin was localized in a pattern relevant to radial migration and survival of neurons; it was present before and during neuronal migration and appeared oriented along RG fibers by conventional fluorescence microscopy. Confocal microscopy confirmed that fibronectin was localized along RG cells during radial migration. It was more concentrated in some superficial laminae, which might support directional movement. Fibronectin was present after formation of definitive tectal laminae, but was diffuse and not aligned along RG, which persist. Flow cytometry analysis of dissociated optic tectum cells revealed that almost all RG were positive for fibronectin. Short-term cell culture experiments using an exocytosis inhibitor revealed that fibronectin accumulated in most RG cells. Thus, fibronectin is produced by RG and is aligned along their surfaces before and during migration. Fibronectin, therefore, is a potential ligand for general radial neuronal migration in the chick optic tectum. Its predominant source appears to be RG, in contrast with developing mammalian cortex, where fibronectin was not found in a pattern that could guide widespread radial migration and where neurons are the predominant producers of fibronectin during migration.

Animals↗
PubMed1998

Rubella virus replication complexes are virus-modified lysosomes.

Replication complexes are membrane-bound cytoplasmic vacuoles involved in rubella virus (RV) replication. These structures can be identified by their characteristic morphology at the electron microscopy (EM) level and by their association with double-stranded (ds) RNA in immunogold labeling EM studies. Although these virus-induced structures bear some resemblance to lysosomes, their exact nature and origin are unknown. In this study, the localization of two lysosomal markers, lysosomal-associated membrane protein (Lamp-1) and acid phosphatase, relative to the replication complexes was examined by light and electron microscopy. Confocal microscopy using antibodies to dsRNA and Lamp-1 showed colocalization of these two markers in the cytoplasm of RV-infected cells. Immunogold labeling EM studies using antibodies to Lamp-1 confirmed that Lamp-1 was associated with RV replication complexes. EM histochemical studies demonstrated the presence of acid phosphatase in the vacuoles of RV replication complexes. Taken together, these studies show that RV replication complexes are virus-modified lysosomes.

Acid Phosphatase↗
PubMed2005

Long-term histopathologic findings in human corneal wounds after refractive surgical procedures.

OBJECTIVE: To describe the human corneal histopathology after laser in situ keratomileusis, photorefractive keratectomy, astigmatic keratoplasty, clear corneal refractive lens surgery, conductive keratoplasty, and intrastromal corneal ring segment placement. DESIGN: Retrospective histopathologic study and selected literature review. METHODS: Ninety-four postmortem corneas from 49 patients and 10 corneal button specimens from 6 patients with previous refractive surgery were collected from eye banks and refractive surgeons in North America. This observational case series evaluated the pathologic findings in the wounds of these corneas in the context of previous studies. RESULTS: Three histopathologic types of corneal wound-healing responses were present in corneas after refractive surgery. The first was an epithelial cell modification that reestablished a smooth anterior corneal surface through basal epithelial cell hypertrophy, by epithelial hyperplasia, or both. The second was the production of a hypercellular fibrotic stromal scar, and the third was the production of a hypocellular primitive stromal scar. Although only the hypercellular fibrotic stromal scar could be identified on gross examination, all three wound responses were identified with light, electron, and immunofluorescence microscopy. Confocal microscopy also identified these three processes but with less detail and resolution. CONCLUSION: Understanding the locations, clinical appearance, and functional attributes of these wound-healing responses will help guide evolving techniques or adjunctive treatments.

Cornea↗
PubMed2001

Use of solid corrugated particles to enhance powder aerosol performance.

PURPOSE: To study the dispersion performance of non-porous corrugated particles, with a focus on the effect of particle surface morphology on aerosolization of bovine serum albumin (BSA) powders. METHODS: The solid-state characteristics of the spray-dried BSA powders, one consisting of smooth spherical particles and another corrugated particles, were characterized by laser diffraction, X-ray powder diffraction, scanning electron microscopy, confocal microscopy, thermogravimetric analysis, surface area analyzer, and buoyancy method. The powders were dispersed using the Rotahaler and the Dinkihaler coupled to a four-stage liquid impinger operating at 30 to 120 L/min. Fine particle fraction (FPF) was expressed as the wt. % of BSA particles of size < or =5 microm collected from the liquid impinger. RESULTS: Apart from the morphology and morphology-related properties (specific surface area, envelope density), the corrugated particles and spherical particles of BSA had very similar solid-state characteristics (particle size distribution, water content, true density, amorphous nature). Using the Dinkihaler, the FPFs of the corrugated particles were 10-20 wt. % higher than those of the smooth particles. Similar FPF differences were found for the powders dispersed by the Rotahaler, but the relative changes were larger. In addition, the differences were inversely proportional to the air flows (17.3% at 30 L/min, 25.2% at 60 L/min, 13.8% at 90, 8.5% at 120 L/min). Depending on the inhaler, capsule and device retention and impaction loss at the impinger throat were lower for the corrugated particles. CONCLUSIONS: Enhanced aerosol performance of powders can be obtained by surface modification of the particles. The surface asperities of the corrugated particles could lower the true area of contact between the particles, and thus reduce the powder cohesiveness. A distinct advantage of using corrugated particles is that the inhaler choice and air flow become less critical for these particles.

Administration, Inhalation↗
PubMed2001

Variant Alzheimer disease with spastic paraparesis: neuropathological phenotype.

Variant Alzheimer disease (varAD) is clinically characterized by the combination of presenile dementia with spastic paraparesis and is caused by certain mutations of the presenilin 1 (PS-1) gene. We now present the unusual neuropathological phenotype of varAD as seen in 5 affected members of the original Finnish family with a genomic deletion encompassing exon 9 of the PS-1 gene. Their primary and association cortices and hippocampus showed a profusion of eosinophilic, roundish structures with distinct borders termed "cotton wool" plaques (CWPs). The CWPs were immunoreactive for Abeta42/43 but weakly or not at all for Abeta40 isoforms of the amyloid beta peptide (Abeta). They were devoid of a congophilic core, and fibrillar amyloid could not be identified within them by electron microscopy. Confocal microscopy showed reduced density of axons within individual CWPs and only few CWP-related PHF-tau-positive dystrophic neurites. CWPs were particularly numerous in the medial motor cortex representing the lower extremities, and degeneration of the lateral corticospinal tracts was observed at the level of the medulla oblongata and the spinal cord. In addition to the predominant CWPs, variable numbers of diffuse and cored plaques were found in the cerebral cortex. Diffuse and non-neuritic cored amyloid plaques but no CWPs occurred in the cerebellum. In conclusion, varAD in this Finnish family is distinct from classic AD because of the degeneration of lateral corticospinal tracts, predominance of CWPs devoid of fibrillar amyloid cores in the cerebral cortex, and presence of non-neuritic amyloid plaques in the cerebellum.

Alzheimer Disease↗
PubMed2005

Quantitation of microparticles released from coated-platelets.

Dual agonist stimulation of platelets with thrombin and convulxin results in generation of coated-platelets, a sub-population of cells known formerly as COAT-platelets (collagen and thrombin). Coated-platelets retain several procoagulant proteins on their surface and express phosphatidylserine (PS). In this report, we utilize a new methodology to demonstrate that coated-platelets also release microparticles. Platelets were prelabeled with 2.5 microm Bodipy-maleimide and then stimulated with convulxin plus thrombin. Microparticles, 0.3-0.5 microm in diameter, were observed by fluorescence confocal microscopy. Confocal microscopy was also used to demonstrate that microparticles were positive for glycoprotein IIb/IIIa, glycoprotein Ib, CD9, and PS, but negative for fibrinogen and thrombospondin. Furthermore, microparticles released from Bodipy-labeled platelets were observed by flow cytometry, and activation with convulxin plus thrombin produced 15 +/- 5 microparticles per coated-platelet. In contrast, platelets stimulated with thrombin or convulxin alone produced few microparticles. Phenylarsine oxide and diamide, both of which potentiate the mitochondrial permeability transition pore and coated-platelet production, significantly increased the number of microparticles released per coated-platelet.

Antigens, CD↗
PubMed2006

Visualization of single and aggregated hulless oat (Avena nuda L.) (1-->3),(1-->4)-beta-D-glucan molecules by atomic force microscopy and confocal scanning laser microscopy.

Surfactants were used to disperse oat beta-glucan. Atomic force microscopy (AFM) images of the resulting samples revealed a distribution of extended chainlike molecules and allowed, for the first time, direct visualization of single oat beta-glucan molecules with cross-sectional heights of about 0.44 nm. The number-average contour length (L(n)) and root-mean-square end-to-end distance ((R(ee)2)(1/2)) measured from the AFM images were 938 and 912 nm, respectively. The calculated persistence length (L(p)) was 526 nm. The weight-average molecular weight (M(w)) calculated from single beta-glucan molecules was 4.43 x 10(5). Samples without surfactant showed a strong tendency to form aggregates. The sample concentration, reserving time, and calcofluor as well as freezing could affect the formation of aggregates. These aggregates were visualized by both AFM and confocal scanning laser microscopy. The shape of the aggregates changed from small dots with diameters of approximately 20-50 nm to microfibrils over 3 microm long with the increasing of the concentration of oat beta-glucan from 10 to 100 microg/mL. The particle size distribution obtained by a laser particle size analyzer was 926 nm, which confirmed the size of oat beta-glucan molecules obtained from AFM images.

Avena↗
PubMed1995

A comparative study of the application of scanning acoustic microscopy and confocal laser scanning microscopy to the structural assessment of human bones.

The Scanning Acoustic Microscope (SAM) is used to image mechanical structure and to measure the sound velocity in micrometers of in vitro samples from human bone. A similar principle is used in confocal laser scanning microscopy. The same sample is imaged by both methods and the results compared. We concentrated on the SAM, the function of which is described in greater detail. We demonstrate that the confocal scanning principle is applied in both microscopes, although information about structure differs, depending on the method used.

Acoustics↗
PubMed2006

Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy: differentiation of agonists and antagonists.

Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit beta-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon beta-arrestin-mediated clathrin-coated pits, whereas, beta-arrestin-2 conjugated green fluorescence protein (beta-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.

Arrestins↗
PubMed1994

Quantitative and 3-dimensional analysis of Langerhans cells in basal cell carcinoma. A comparative study using light microscopy and confocal laser scanning microscopy.

We have analysed Langerhans cells (LCs) in basal cell carcinoma (BCC) and in healthy skin in 15 patients, using three different techniques: light microscopic examination of horizontal sheets, and of 6-micron-thick vertical skin sections, and confocal laser scanning microscopy (CLSM) of 25-micron-thick vertical sections. The use of CLSM enables both a quantitative and a three-dimensional (3-D) analysis of the cells in the same tissue volume. A statistically significant reduction in the relative volume of epidermal CD1a reactivity confined to tumour areas was found with CLSM. This difference was confirmed when the number of LCs in horizontal sheets were counted. In contrast, no significant reduction in epidermal CD1a+ cells was found in thin vertical sections. This is probably due to the smaller tissue sample examined, and to variations in the number of CD1a+ cells, with less cells directly overlying the tumour nests. The ratio of CD1a-expressing cells in the epidermis/dermis was significantly reduced in BCCs, compared with healthy looking skin. Few LCs were observed in tumour nests, but they were numerous in the surrounding stroma of the dermis. Three-dimensional reconstructions of CD1a+ cells in BCC revealed striking morphological changes; they had a reduced number of dendrites, and these were often short and had few branches. The results demonstrate that CLSM is a suitable technique for quantitative and morphological analysis of CD1a-expressing cells in the skin. We suggest that the alterations in LC numbers, distribution and morphology in BCC most probably are secondary to changes in the local environment.

Adult↗