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PubMed1998

Structural organization of the perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter: a confocal microscopy study.

Despite the increasing evidence for a prominent role played by the perivascular endfeet of astrocytes in the functional metabolic coupling between astrocytes and neurons, a clear picture of their spatial organization is still lacking. To examine the three-dimensional structure of the astrocyte endfeet and their relationships with the endothelial cells, coronal rat brain sections immunolabeled for the two astroglial markers [glial fibrillary acidic protein (GFAP)/S-100beta] and the endothelial glucose transporter (GLUT1) were analyzed under the confocal microscope. Double immunolabeling of GFAP and S-100beta showed numerous well-defined astrocytes sending one or more endfeet to the vasculature. Examination of GFAP immunolabeling at higher magnification showed that these endfeet consist of well-defined rosette-like structures lying on the vessel wall. Double immunostaining of GFAP and GLUT1 showed that the endothelial cells were the main targets of these repeated geometrical units formed by the astrocyte endfeet. When three-dimensional images were reconstructed, obvious privileged anatomical relationships were observed between endfeet and individual endothelial cells. These anatomical data provide strong support for the involvement of astrocytes in cerebral metabolic coupling. The finger-like appearance of astrocyte endfeet could allow direct metabolic exchanges between intracerebral vessels and non-glial elements such as nerve terminals.

Animals↗
PubMed1992

In vivo assessment of corneal stromal toxicity by tandem scanning confocal microscopy.

Epithelial wound closure following superficial corneal abrasions precipitates a complex series of cellular changes in the stroma. Transmission electron microscopy and light microscopy have demonstrated that keratocytes in the anterior stroma show prominent cytoplasmic process and granularity within an hour after this injury. Cell degeneration follows rapidly, with almost complete depletion of these keratocytes by 6-12 hours. Regeneration then occurs by 24-48 hours. These findings were confirmed in-vivo by tandem-scanning confocal light microscopy in rabbits. The optical sectioning capability of this instrument allowed us to demonstrate the three-dimensional array of fibroblast process, their subsequent condensation, and loss by sequential examinations on the same animals. Cell shrinkage and other fixation artifacts common in corneal histological sections were avoided by this technique. Using this method, we investigated the corneal cytotoxicity of an antimitotic agent, mitomycin-C and a muscarinic antagonist, atropine sulphate. Mitomycin-C, an antimetabolite used in pterygium treatment, led to irreversible keratocyte depletion even after four days of observation. However, cellular reaction to the wound were attenuated by atropine sulphate suggesting that it may be useful as a novel inhibitor of fibroblast proliferation.

Animals↗
PubMed1997

Ultrasonic characterization of in vitro osteoarthritic articular cartilage with validation by confocal microscopy.

The majority of adults over the age of 65 y develop osteoarthritis (OA), a joint disease characterized by degeneration of articular cartilage and subchondral sclerosis. Early in the disease, the articular cartilage surface begins to change histologically from a smooth to a rough or fibrillated appearance. A prerequisite for any chondroprotective pharmacological intervention is detection of OA in its preclinical phase. Current diagnostic imaging modalities, such as radiographs or (nuclear) magnetic resonance imaging, either cannot directly image the cartilage surface or lack sufficient resolution to detect surface fibrillations. We have developed an ultrasonic technique that can be used to characterize these surface fibrillations directly. We present our in vitro results with validation by laser-based confocal microscopic imaging.

Adult↗
PubMed2005

A confocal microscopy study of the very early cellular response to oxidative stress induced by zinc phthalocyanine sensitization.

Oxidative stress has been involved in several biological and pathological processes. Reactive oxygen species have been shown to play both beneficial and deleterious roles. The present work contributes to the understanding of the very early events of cellular response to oxidative stress. Oxidative stress was produced intracellularly by light activation of zinc phthalocyanine (ZnPc) at a light dose that did not lead to apoptosis or necrosis. Phthalocyanine was photoactivated using the 647-nm laser line of a confocal microscope through the objective lens causing oxidative stress and allowing observation of the evoked phenomena at the single cell level and in real time. Mitochondria membrane potential (DeltaPsi(m)), intracellular pH, calcium concentration, and generation of reactive oxygen species (ROS) were recorded using specific vital fluorescent probes and quantified by image processing and analysis. Subcellular localization of ZnPc was also studied in order to determine the primary and intermediate ROS target.

Calcium↗
PubMed1995

A new form of primary, localized, corneal amyloidosis: a case report with confocal microscopy.

We present a case of bilateral, primary, localized, corneal amyloid deposition with a unique appearance not, to our knowledge, previously described. The patient was a 73-year-old white man who complained of glare. Best corrected visual acuity was 20/25 in both eyes. Slit lamp examination revealed subepithelial/anterior stromal white-gray deposits in the peripheral cornea in both eyes. The corneal surface was smooth with no elevated areas. Superficial corneal scrapings showed focal deposits of an amorphous, glass-like material below the epithelial surface and within Bowman's membrane, which stained with Congo red. We examined the amyloid deposits using the confocal microscope to obtain high magnification images in vivo. The confocal microscopic views showed intercellular, cotton candy-like, fibrillar amyloid material scattered throughout the anterior stroma. The use of this instrument may permit clinical diagnosis of this rare disorder without the need for biopsy.

Aged↗
PubMed2003

Effect of composition and complexity of dentin-bonding agents on operator variability--analysis of gap formation using confocal microscopy.

The variability of gap formation due to different operators and bonding agents around dentin-bonded composites in cylindrical cavities (internal diameter, 3 mm; depth, 1 mm; n = 210) was evaluated in vitro. Seven dentists evaluated two experimental bonding systems (A & B) and one commercial product (C) by each treating 10 teeth with each system. One of the investigators restored all cavities with composite (Z100, 3M ESPE) and removed excess material to expose the cavity margins. After storage in water for 1 wk, the maximum gap-width was measured using a confocal microscope and statistically analysed. The relative gap-widths (%) for the three bonding systems were not statistically different. Means +/- SD were 0.18 +/- 0.06, 0.19 +/- 0.08, and 0.20 +/- 0.06 for groups A, B, and C, respectively. The variation caused by the operator-material interaction was significant. When the operator's variability (SD) within the different materials was examined, group C displayed a significantly lower variability than the other two. It was concluded that the operator variable is a more important consideration than the material variable. The self-etching system performed better than the more complex A and B systems when variability rather than gap size was considered.

Acid Etching, Dental↗
PubMed2000

Protein profiled features patterned via confocal microscopy.

Protein patterns were printed using conventional microlithographic materials in a bilayer arrangement and unconventional exposure tools. The bilayer resist stack consisted of a lower poly(tert-butyl methacrylate) layer and an upper diazonaphtoquinone/novolak layer. The protein features were printed in either 'contact printing', or 'step and repeat' mode. The latter printing mode can be managed in a flow-cell consisting of a standard microscope slide and cover slip, spaced apart by about 20 microm, as follows: (i) the exposure step is carried out in the cell using focused 488 nm beam of a confocal laser scanning microscope; (ii) the development step is performed by flowing the photoresist developer through the cell; (iii) the selective deposition of the protein (FITC-labelled avidin) is achieved via the flow of the protein solution through the cell until a desired contrast has been reached; (iv) the control of the process is assured using on-line monitoring of the photo-activated red fluorescence of the developing resist layer, and of the green fluorescence of the FITC-protein patterns, respectively. The protein printing technique uses equipment routinely available in biological laboratory. The 'step and repeat' patterning yields high and controllable resolution. The process can be applied in the fabrication of medical microanalysis devices.

Biosensing Techniques↗
PubMed1995

Distribution of caldesmon and of the acidic isoform of calponin in cultured cerebellar neurons and in different regions of the rat brain: an immunofluorescence and confocal microscopy study.

Caldesmon and calponin are two F-actin-binding and calcium-calmodulin-dependent proteins. In smooth muscle and nonmuscle cells both proteins are localized on actin filaments. Using one- or two-dimensional gel electrophoresis followed by the Western blot technique, and by immunofluorescence studies, we have given evidence that calponin is also present in rat and pig brain. In the present study, for the first time, we demonstrate caldesmon- and calponin-specific immunoreactivities in cerebellar cultured neurons. In the rat central nervous system these antibodies mainly stain neuronal cell bodies and dendrites. By confocal analysis we observed that calponin and caldesmon are located in the actomyosin domain although the total actin and myosin were not saturated. In many cases it is clear that these two proteins are adjacent rather than superimposed in the same domain of the cell. These results are compatible with the functional role of caldesmon and calponin in the regulation of the actomyosin activity as described by others and suggest that they are part of the contractile apparatus of neural cells.

Actins↗
PubMed2003

Physiological and morphological characterization of honeybee olfactory neurons combining electrophysiology, calcium imaging and confocal microscopy.

The insect antennal lobe is the first brain structure to process olfactory information. Like the vertebrate olfactory bulb the antennal lobe is substructured in olfactory glomeruli. In insects, glomeruli can be morphologically identified, and have characteristic olfactory response profiles. Local neurons interconnect glomeruli, and output (projection) neurons project to higher-order brain centres. The relationship between their elaborate morphology and their physiology is not understood. We recorded electrophysiologically from antennal lobe neurons, and iontophoretically injected a calcium-sensitive dye. We then measured their spatio-temporal calcium responses to a variety of odours. Finally, we confocally reconstructed the neurons, and identified the innervated glomeruli. An increase or decrease in spiking frequency corresponded to an intracellular calcium increase or decrease in the cell. While intracellular recordings generally lasted between 10 and 30 min, calcium imaging was stable for up to 2 h, allowing a more detailed physiological analysis. The responses indicate that heterogeneous local neurons get input in the glomerulus in which they branch most strongly. In many cases, the physiological response properties of the cells corresponded to the known response profile of the innervated glomerulus. In other words, the large variety of response profiles generally found when comparing antennal lobe neurons is reduced to a more predictable response profile when the innervated glomerulus is known.

Action Potentials↗
PubMed2002

Live-cell nucleocytoplasmic protein shuttle assay utilizing laser confocal microscopy and FRAP.

In eukaryotes, protein trafficking to and from the nucleus, or shuttling, has been demonstrated to be an important function for proteins that have vital roles in one or both subcellular compartments. Current techniques of detecting protein nuclear shuttling are extremely labor intensive and only statically visualize evidence of shuttling. Fluorescence recovery after photobleaching (FRAP), or fluorescence microphotolysis, has proven to be an effective method of analyzing protein dynamics in live cells, especially when coupled to GFP technology. Here, we describe a relatively simple in vivo protein nuclear shuttling assay that utilizes red fluorescent and green fluorescent protein fusions as substrates for FRAP using a laser confocal microscope. This technique is less time consuming than established shuttle assays, is internally controlled, and visualizes nucleocytoplasmic shuttling in living cells of the same species and cell type. This technique can be potentially used to detect the ability of any nuclear protein to shuttle from the nucleus to any other subcellular compartment for any eukaryotic species in which GFP or dsRed1 fusion protein can be expressed.

Cell Fusion↗
PubMed1996

Confocal microscopy analysis of living Xenopus eggs and the mechanism of cortical rotation.

The dorsoventral body axis in amphibian embryos is established by a rotation of the outer cortex relative to the inner cytoplasmic core. This cortical rotation depends on microtubules and is correlated with a parallel array of microtubules just inside the vegetal cortex. Since the parallel array moves with the inner cytoplasm and most of its microtubules are oriented with their plus ends facing the direction of cortical movement, it has been suggested that plus end-directed motor molecules attached to the cortex drive the rotation by moving along microtubules of the parallel array. Using an inverted confocal microscope to examine living eggs, however, we found that rotation movements precede the formation of a detectable parallel array at the vegetal pole, that the parallel array consists of multiple layers of microtubules at depths ranging from 4 to 8 microns inside the plasma membrane and that the velocity of rotation is immobilized eggs increases with depth in this region. These findings suggest that (1) early cytoplasmic movements are due to something other than the fully formed parallel array and (2) the motor molecules responsible for the bulk of the rotation movement are not restricted to a monolayer at the subcortical interface but may be distributed throughout the parallel array, perhaps causing microtubules to slide along other microtubules by a mechanism similar to that seen in cilia and eukaryotic flagella.

Animals↗
PubMedDate not supplied

The use of confocal microscopy in evaluating corneal wound healing after excimer laser keratectomy.

Corneal wound healing following excimer laser keratectomy is the major cause of regression of treatment results. The amount of anterior stromal haze that develops may be influenced by topical medications. Over a period of 6 months, we followed 15 New Zealand white rabbit eyes that underwent excimer laser keratectomy with the VISX 193-nm ArF laser at a fluence of 150 mJ/cm2 for a depth of 130 microns. Eyes were randomized to treatment with prednisolone acetate, diclofenac sodium (Voltaren), a combination of both, and a control group. Drops were administered four times a day for 1 week, two times a day for 3 weeks, and the drops were then tapered. All eyes were reepithelialized by 5 to 7 days. The tandem scanning confocal microscope (TSCM) was used to evaluate the corneal wound in vivo weekly for a month and monthly for 6 months. During the early postoperative period, the TSCM revealed significant anterior stromal keratocyte activation with cell elongation and the spindle-shaped appearance of fibroblasts in all groups. Collagenous stromal scarring was evident initially, then slowly decreased in all treatment groups. This study shows that TSCM is clinically useful for successive in vivo examinations of corneal wounds after excimer laser keratectomy and for comparing the effects of various topical medications.

Animals↗
PubMedDate not supplied

Confocal microscopy of disposable and nondisposable heads for the Moria M2 microkeratome.

PURPOSE: To study the quality of the flap obtained with two different heads, disposable and nondisposable, and two different speeds, low and high, using the same Moria M2 microkeratome (Moria, Antony, France). METHODS: In a prospective randomized study, 51 consecutive eyes (28 patients) underwent myopic LASIK (-4.39 +/- 2.15 diopters). The eyes were randomly assigned to four groups related to cutting head model (disposable and nondisposable) and cut speed (low and high). In all cases, the intended cut was 160 microm and the same M2 unit was used. During surgery, superficial measurements of the flap and the surgeon's opinion about the quality of the cut were recorded. One month after LASIK, the cut depth, interface particle density, and transparency of the cornea (confocal wound healing opacity index) were measured with a confocal microscope. RESULTS: Flaps performed with a disposable cutting head and high speed were statistically significantly larger than the flaps created in the other groups (Kruskal-Wallis test; P = .041). No statistical differences were noted in cut depth, number of particles, and wound healing opacity index. The surgeon's opinion parameters were similar in all groups. CONCLUSIONS: The Moria M2 microkeratome is safe and provided reproducible results, regardless of cutting head and speed. Disposable and nondisposable head models were similar in their outcome in the quality control model used in this study.

Cornea↗
PubMed2003

Gross anatomy of the muscle systems and associated innervation of Apatemon cobitidis proterorhini metacercaria (Trematoda: Strigeidea), as visualized by confocal microscopy.

The major muscle systems of the metacercaria of the strigeid trematode, Apatemon cobitidis proterorhini have been examined using phalloidin as a site-specific probe for filamentous actin. Regional differences were evident in the organization of the body wall musculature of the forebody and hindbody, the former comprising outer circular, intermediate longitudinal and inner diagonal fibres, the latter having the inner diagonal fibres replaced with an extra layer of more widely spaced circular muscle. Three orientations of muscle fibres (equatorial, meridional, radial) were discernible in the oral sucker, acetabulum and paired lappets. Large longitudinal extensor and flexor muscles project into the hindbody where they connect to the body wall or end blindly. Innervation to the muscle systems of Apatemon was examined by immunocytochemistry, using antibodies to known myoactive substances: the flatworm FMRFamide-related neuropeptide (FaRP), GYIRFamide, and the biogenic amine, 5-hydroxytryptamine (5-HT). Strong immunostaining for both peptidergic and serotoninergic components was found in the central nervous system and confocal microscopic mapping of the distribution of these neuroactive substances revealed they occupied separate neuronal pathways. In the peripheral nervous system, GYIRFamide-immunoreactivity was extensive and, in particular, associated with the innervation of all attachment structures; serotoninergic fibres, on the other hand, were localized to the oral sucker and pharynx and to regions along the anterior margins of the forebody.

Animals↗
PubMed2006

[Distribution and morphological changes of corneal nerves in type 2 diabetic patients detected by confocal microscopy].

OBJECTIVE: To evaluate corneal nerve distribution and morphological changes in subjects with type 2 diabetes. METHODS: Sixty-five eyes of 59 patients with type 2 diabetes (26 males, 39 females) and 26 eyes (10 males, 16 females) of control subjects were included in the present study. Based on the indirect ophthalmoscopic examination and fluorescein angiography, the diabetic group was divided into three sub-groups according to the stage of retinopathy: without diabetic retinopathy (NDR), non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). The central cornea was scanned with Confoscan 3.0. The images of subbasal nerve plexus and stromal nerves were analyzed. RESULTS: Corneal branch nerve fiber density of subbasal nerve plexus decreased in diabetic group as compared with control group, the difference was statistically significant. The decrease of nerve fiber density appeared only in the PDR sub-group, while the decrease was not significant in NDR and NPDR sub-groups. No obviously difference was found among control group, NDR and NPDR sub-groups. The proportion of patients with an abnormal morphologic nerve fibers in mid-stroma in diabetes group was greater than that in the control group and the difference was statistically significant (chi(2) = 46.613, P = 0.000). CONCLUSIONS: Corneal confocal microscope allows rapid and noninvasive in vivo evaluation of corneal nerves. The subbasal and stromal nerves of subjects with type 2 diabetes were abnormal in morphology.

Aged↗
PubMed2000

Genome organization in the human sperm nucleus studied by FISH and confocal microscopy.

The sperm nucleus has a unique chromatin structure where the DNA is highly condensed and associated with specific proteins, the protamines. It is a nondividing cell which is also transcriptionally inactive. After fusion with an oocyte, the sperm nucleus undergoes decondensation and, in the same time, starts replication and transcription. It has been suggested that somatic chromosomes during interphase are organized in territories which display a cell type and cell cycle specific distribution. The purpose of this work was to investigate whether chromosomes would also have a specific distribution in the sperm nucleus, which could be related to its inactive state, and have implications on the early stages of fertilization. In the present study, centromeric and telomeric sequences were detected by fluorescent techniques performed on human decondensed spermatozoa. Chromosome painting probes were used to detect the chromosome X and chromosome 13 on interphase sperm nuclei. The fluorescent signals were captured in 3D with a confocal microscope. For each of these chromatin structures, the volume, position, and distribution of the signals were analyzed in samples of 30 nuclei with the help of image analysis software. The centromeres appeared grouped in several foci that were randomly distributed within the sperm nucleus. The telomeres gave an approximately haploid number of small signals, evenly distributed throughout the nucleus. The chromosomes X and 13 occupied 4.7% and 3. 7% of the total nuclear volume, respectively. Interestingly, the X chromosome territory showed a preferential position in the anterior half of the volume of the nucleus, whereas chromosome 13 had a random position. This work shows a particular distribution of chromosome territories in the human sperm nucleus that could be related to mechanisms implicated in its specific functions. The analysis of more chromosomes and chromosomal structures, including the Y chromosome, would help to understand the structure of the human sperm chromatin, and its fundamental and clinical implications.

Cell Nucleus↗
PubMed2001

3-D confocal microscopy of etched nuclear tracks in CR-39.

Solid State Nuclear Track Detectors (SSNTD's) are used in a wide range of applications such as Geological Dating, Environmental Sciences (radon), life Sciences (Radiobiology, Dosimetry...), as well as Nuclear and Astro-Physics. In order to be observable under a microscope, the nanometric latent damaged trails due to the slowing down of charged particles into the SSNTD have to be specifically etched. In our laboratory, we are studying this chemical action and propose models that enable simulations to be performed. In the literature, the basic model uses two distinct etch-rates that are considered constant, VB; the Bulk and VT the Track etch-rate. A little bit more sophisticated and realistic, a model with a variable track etch-rate was established, taking into account the variation of energy deposition along the particle's trajectory. Up to now, the known methods used for determining the response function of CR-39 are very time consuming and strenuous. The method we present here is based on the use of the confocal microscope, which provides three-dimensional track images. The obtained set of 3-D co-ordinates can be treated mathematically, giving, in the framework of the two etch-velocity model, the response function. With this new approach, tracks are analysed one by one; response functions are obtained for each track and can be compared to fundamental characteristics of the charged Particle-Matter interactions. Moreover, the method we propose is applied semi-automatically and could easily be automated in the near future.

Image Processing, Computer-Assisted↗
PubMed1996

Anatomical pathways connecting lip sensory structures and central nervous system in hirudinid leeches visualized by carbocyanine dyes and laser scanning confocal microscopy.

Chemoreception in Hirudo medicinalis is thought to be mediated by ciliated cells grouped in sensory structures, the sensilla, arranged in bands on the animal's dorsal lip (Elliott, 1986; Zipser et al., 1994). Furthermore, chemical and/or thermal stimulation of the dorsal lip in reduced preparations evokes changes in the electrical activity of the cephalic nerves that connect the head with the central nervous system. However, the complete trajectory by which the sensory afferents reach the cerebral ganglia has not been demonstrated anatomically. In this study, we traced these pathways following retrograde and/or anterograde transport of carbocyanine dyes (DiI, DiA and DiD) in the cephalic nerves of Hirudo medicinalis and a closely related species, Macrobdella decora. While information regarding Macrobdella's chemoreception is scarce, the two species show some differences with regard to their chemical preferences. Dyes were applied to the sensillar structures along the dorsal lip, or to the cut ends of individual cephalic nerves in fixed preparations that included the lip and attached nerves with or without the head ganglia. After a two week incubation, specimens were mounted and imaged using a confocal microscope. The results show that the axons of the sensory neurons in the sensilla project through the four pairs of cephalic nerves. The sensillar projections are however more numerous in the dorsal nerves than they are in the ventral ones. In addition, the organization of the sensillar bands, the morphology of the pathways and the sensory structures themselves appear to be identical for Hirudo and Macrobdella and therefore the behavioral differences in response to appetitive stimuli cannot be readily explained by differences in morphology.

Animals↗