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PubMed2001

Z-polarized confocal microscopy.

In light microscopy the transverse nature of the electromagnetic field precludes a strongly focused longitudinal field component, thus confining polarization spectroscopy and imaging to two dimensions (x,y). Here we describe a simple confocal microscopy arrangement that optimizes for signal from molecules with transition dipoles oriented parallel to the optic axis. In the proposed arrangement, we not only generate a predominant longitudinally (z) polarized focal field, but also engineer the detection scheme in such a way that in a bulk of randomly oriented molecules, the microscope's effective point-spread function is dominated by the contribution of those molecules that are oriented along the optic axis. Our arrangement not only implicitly allows for the determination of the orientation of transition dipoles of single molecules in three dimensions, but also highlights the contribution of z-oriented molecules in three-dimensional imaging.

Microscopy, Confocal↗
PubMed1999

The application of in vivo confocal microscopy and tear LDH measurement in assessing corneal response to contact lens and contact lens solutions.

PURPOSE: To evaluate differences in corneal response to daily wear (DW) of soft contact lens (CL) wear with different CL solutions and to assess the ability of in vivo confocal microscopy (CM) and tear lactate dehydrogenase (LDH) measurement to detect such differences in NZW rabbits. METHODS: Daily treatment of lenses consisted of a rub and rinse cleaning, then overnight soak in one of five solutions: Sauflon All in One (ALL), Compound A (CoA), OPTI-FREE((R) )Rinsing, Disinfecting, and Storage Solution (OPT), ReNu((R)) <?Pub Caret>Multipurpose Solution (REN), and UNISOL( (R)) Saline Solution (UNI). Rabbits (4/test group) wore 71% H( 2)O/type4 soft lenses approximately 7 hours daily. On days 0, 1, 3 and 7, slit lamp examination, tear LDH measurement, and in vivo CM were performed after removal of lenses. Using in vivo CM, epithelial thickness, epithelial cell area, and stromal thickness were measured, both centrally and peripherally. RESULTS: Epithelial thickness in ALL, CoA, and UNI-treated eyes showed a significant decrease of 15.6%, 13. 3%, and 10.6% (p < 0.05 in all groups), centrally, while CoA, OPT, and UNI showed a significant decrease of 9.3%, 7.1%, and 4.4% (p < 0. 05 in all groups), peripherally. ALL showed a significant 9.5% (p < 0.05) decrease of central cell area, while CoA showed a significant 21.5% (p < 0.01) decrease peripherally. UNI demonstrated a significant 3.2% (p < 0.05) decrease in central stromal thickness. ALL, CoA, and UNI showed a significant increase in LDH level of 152. 1%, 192.1%, and 308.2% (p < 0.05 in all groups) at day 3, respectively, but values declined at day 7. Significant changes in basal epithelial morphology were also observed with CoA on day 7 on in vivo CM. CONCLUSIONS: Overall, lens care solutions in combination with CL wear may interact to cause increased epithelial desquamation leading to decreased surface cell area and epithelial thickness. The clinical significance of these changes will require further investigation. In vivo CM combined with tear LDH assay is a quantitative, objective, non-invasive method of assessing CL wear and CL disinfecting solution effects on the cornea, and is able to detect differences in corneal response to different CL solutions.

Animals↗
PubMed2004

Surgical detachment of Descemet's membrane and endothelium imaged over time by in vivo confocal microscopy.

A 90-year-old woman developed a large circular capsulorhexis-like defect in Descemet's membrane as a complication of small incision cataract surgery. Nine months post-surgery, in vivo confocal microscopic examination of the temporal mid-peripheral cornea revealed an endothelial cell density of 934 +/- 69 cells/mm2 (normal range 1566-3088 cells/mm2). Endothelial pigmented deposits were visible as scattered hyper-reflective areas on the posterior endothelial surface. Descemet's folds were also noted. In vivo confocal microscopy performed 3 years later showed the temporal mid-peripheral corneal endothelial density (in the region of the break) was 948 +/- 66 cells/mm2. A reduction of endothelial polymegathism and pleomorphism was observed. Imaging in the region of the temporal portion of the original Descemet's defect showed well-defined linear structures with hyper-reflective edges. Compared to 3 years previously, the cornea at the level of Descemet's membrane appeared to have greater reflectivity. This case demonstrates how microstructural changes in the cornea can be described and analysed over time with the assistance of in vivo confocal microscopy.

Aged↗
PubMed1995

Expression of fibronectin, laminin and ribosomes in normal and nocodazole-treated neonatal heart cells in culture: a study by laser scanning confocal microscopy and immunocytochemistry.

Polyclonal and monoclonal antibodies were used to examine the effects of the synthetic microtubule disruptive drug nocodazole on the subcellular expression of fibronectin, laminin, and ribosomes in primary cultures of neonatal cardiac ventricular cells. Non-invasive serial optical sectioning was carried out by immunolaser scanning confocal microscopy. In addition, fibronectin and laminin were immunolabelled with peroxidase or gold conjugates for electron-microscopic examination. Immunolabelling for the large 60S ribosome subunit in fibroblast-like non-myocytes showed that punctate ribosome structures with a multi-subunit composition were present in perinuclear region. Double immunostaining with antibodies directed against ribosomes and cellular fibronectin indicated that the punctate structures were cisternae of the rough endoplasmic reticulum. No clear effects of nocodazole treatment were detected on the distribution of cytoskeleton-bound ribosomes. Following immunolabelling for both glycoproteins and double immunolabelling for cellular fibronectin and the 60S ribosome subunit, fibronectin and laminin were found in the perinuclear cisternae of the rough endoplasmic reticulum and in pleomorphic secretory vesicles. The cisternal stacks of the Golgi complex appeared either unstained or were only weakly labelled. When these cells were exposed to nocodazole, fibronectin and laminin accumulated in peripheral parts of the cytoplasm, including cellular processes. These peripheral accumulation of immunostaining for fibronectin and laminin did not reflect Golgi staining, as shown by double labelling experiments versus wheat-germ-agglutinin staining, and, by exposing cultures to a high dose of brefeldin A.

Animals↗
PubMed2005

Direct observation of frits and dynamic air bubble formation in capillary electrochromatography using confocal fluorescence microscopy.

Confocal fluorescence microscopy has been used to study the capillary electrochromatography (CEC) frits and dynamic air bubble formation under real chromatographic conditions. Confocal fluorescence microscopy provides a nondestructive way to view the three-dimensional structure of the frits with high spatial resolution. Frits prepared with four different procedures were studied: (1) sintering bare silica beads with sodium silicate; (2) sintering bare silica beads wetted with water; (3) sintering C18 beads wetted with water; and (4) sintering C18 beads wetted with water and then surfaced-recovered with C18. Frits prepared with sintering silicate-wetted beads have a high degree of heterogeneity, while the other three types of frits have similar, more homogeneous packing structures. Confocal fluorescence microscopy also provides sufficient temporal resolution for in situ observation of the dynamic processes in air bubble formation. In this study, air bubble formation is imaged during the reorganization process of the packing bed and is shown to occur close to the border between the packing bed and the outlet frit. Confocal fluorescence microscopy opens a new avenue in studying dynamic processes in situ in CEC separations.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed1995

Innervation of cutaneous structures in the mouse hind paw: a confocal microscopy immunohistochemical study.

The normal innervation of structures in mouse foot pads was investigated with immunohistochemistry and confocal microscopy. Nerves were visualized by incubating Zamboni fixed, thick, frozen sections with antibodies to protein gene product 9.5 (PGP 9.5), vasoactive intestinal peptide, substance P, calcitonin gene-related peptide, and protein zero. The antibodies were localized using cyanine 3.18 labeled anti-rabbit gamma globulin. PGP 9.5 immunolocalization showed dense nerve bundles at the base of the foot pad with branches to larger blood vessels, sweat glands and epidermis. Sweat gland tubules were surrounded by numerous sudomotor axons; single fibers accompanied the sweat duct toward the skin's surface. Nerve bundles containing myelinated and unmyelinated axons ran through and around the centrally located sweat gland cluster to end in free nerve endings and Meissner's-like corpuscles at the apex of the foot pad. Other bundles running parallel to the epidermis gave arcuate branches that supplied epidermis on the sides of the pads with a rich nerve network, principally with free nerve endings that often reached the most superficial cell layers of epidermis. Calcitonin gene-related peptide-immunoreactive (-ir) nerves were distributed to dermis and epidermis in lower density than PGP 9.5-ir fibers. Substance P-ir fibers were less numerous; most terminated as free endings in deeper layers of epidermis. Vasoactive intestinal peptide-ir nerves almost exclusively innervated sweat glands, ducts and blood vessels, but not epidermis. The mouse hind paw has potential to serve as a model system for investigations of functional and morphological changes that affect peripheral and autonomic nerves under diverse experimental conditions.

Animals↗
PubMed1994

Confocal microscopy of F-actin distribution in Xenopus oocytes.

We have used rhodamine-conjugated phalloidin and confocal microscopy to examine the organisation of filamentous actin (F-actin) during oogenesis in Xenopus laevis. F-actin was restricted to a thin shell in the cortex of oogonia and post-mitotic oocytes less than 35 microns in diameter. In oocytes with diameters of 35-50 microns, F-actin was observed in three cellular domains: in the cortex, in the germinal vesicle (GV) and in a network of cytoplasmic cables. Initially, actin cables were sparsely distributed in the cytoplasm, with no evidence of discrete organising centres. In larger stage I oocytes, a dense network of actin cables extended throughout the cytoplasm, linking the GV and mitochondrial mass to the cortical actin shell. Apart from the F-actin associated with the mitochondrial mass, no evidence of a polarised distribution of F-actin was apparent in stage I oocytes. F-actin was observed also in the cortex and the GV of stage VI oocytes, and a network of cytoplasmic cables surrounded the GV. Cytoplasmic actin cables extended from the GV to the animal cortex, and formed a three-dimensional network surrounding clusters of yolk platelets in the vegetal cytoplasm. Finally, disruption of F-actin in stage VI oocytes with cytochalasin resulted in distortion and apparent rotation of the GV in the animal hemisphere, suggesting that actin plays a role in maintaining the polarised organisation of amphibian oocytes.

Actin Cytoskeleton↗
PubMed2002

Use of confocal microscopy for the study of spermatogenesis.

Spermatogenesis consists of spermatogonial proliferation, meiosis and spermatid differentiation. Laser scanning confocal microscopy (LSCM) may be used as an advanced analytical tool to follow spermatogenesis inside the seminiferous tubules without performing histological sections. For this purpose, separated seminiferous tubules are fixed in 0.5% paraformaldehyde, stained for DNA with propidium iodide and analyzed by LSCM. By producing longitudinal optical sections in the layer of spermatogonia, spermatocytes and spermatids, stage-specific changes in their structure may be followed within the tubules by LSCM. Longitudinal z-sections may be obtained to produce three-dimensional images of the seminiferous tubules. In addition, different proteins may be followed during spermatogenesis in a stage specific manner within the tubule by incubation of the fixed seminiferous tubules with appropriate antibodies. As an example of the spermatogenesis studies using described LSCM techniques, detailed examination of spermatogonia, spermatocytes and spermatids during golden hamster spermatogenesis is presented. LSCM analysis of c-kit and SC3 protein expression at different stages of hamster spermatogenesis is demonstrated.

Animals↗
PubMed2000

Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae.

Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were rapidly internalized within hyphae. FM4-64 was found best for imaging the dynamic changes in size, morphology and position of the apical vesicle cluster within growing hyphal tips because of its staining pattern, greater photostability and low cytotoxicity. FM4-64 was taken up into both the apical and subapical compartments of living hyphae in a time-dependent manner. The pattern of stain distribution was broadly similar in a range of fungal species tested (Aspergillus nidulans, Botrytis cinerea, Magnaporthe grisea, Neurospora crassa, Phycomyces blakesleeanus, Puccinia graminis, Rhizoctonia solani, Sclerotinia sclerotiorum and Trichoderma viride). With time, FM4-64 was internalized from the plasma membrane appearing in structures corresponding to putative endosomes, the apical vesicle cluster, the vacuolar membrane and mitochondria. These observations are consistent with dye internalization by endocytosis. A speculative model of the vesicle trafficking network within growing hyphae is presented.

Diphenylhexatriene↗
PubMed2005

Evaluation of hydrogen ion concentrations in prostates from rats and dogs using fluorescent confocal microscopy.

The knowledge of intracellular spatial distribution of pH in prostates in animal models reflective of human prostate may have implications for drug development upon pH dependent drug delivery and activity. Freshly dissected prostate tissues (in vitro) or the entire prostate gland (in vivo) were loaded with fluorescent dyes and viewed using confocal microscopy. Images were initially taken in tissues perfused with RPMI-1640 medium. Calibration in situ was performed with high potassium buffers of known pH containing nigericin. Acetoxymethyl ester carboxy-SNARF-1 was visible in epithelial cells (but not stroma) in rat and dog prostates. The pH of lysosomes in prostate epithelial cells was 5.2 as determined by fluorescence of Lyso Sensor Green DND-189. A method of in situ confirmation of tissue viability was developed by a secondary loading and visualization of the BCECF fluorescent dye. Besides the direct measurement of the pH in rat and dog tissues (pH approximately 7.0), a method of pH measurement in prostate tissue (rather than in cell culture) was developed.

Animals↗
PubMed1995

Uptake kinetics and intracellular localization of hypocrellin photosensitizers for photodynamic therapy: a confocal microscopy study.

Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The intracellular localization and uptake kinetics of hypocrellin B and several selected hypocrellin congeners were determined semiquantitatively by fluorescence confocal microscopy in monolayer cultures of EMT6/Ed murine tumor cells. Each compound had unique uptake kinetics. Although no compound tested to date has demonstrated nuclear labeling, most could be detected in lysosomes, Golgi, endoplasmic reticulum and, to a minor extent, in cellular membranes. No two compounds gave identical labeling distributions. The differences are assumed to originate in physicochemical properties characteristic of each compound, which may ultimately impact upon the primary modality of phototoxicity.

Animals↗
PubMed1998

Laser scanning confocal microscopy of the hearing organ: fluorochrome-dependent cellular damage is seen after overexposure.

In order to combine laser confocal microscopy with physiological measurements, a number of conditions have to be met: the dye must not be toxic to the cells the laser light itself must not damage the cells; and the excitation of the fluorochrome during imaging must not generate products with toxic effects. We have investigated these conditions the hearing organ of the guinea pig. Two dyes were used, namely, calcein-AM, which is metabolized in vital cells to a fluorescent product in the cytoplasm, and a lipophilic membrane dye. The effect of the dyes on cell function was tested in the intact hearing organ, maintained in the isolated temporal bone, by measuring the electrophysiological potentials generated by the sensory cells in response to tone pulses. The loading of the cells with the dyes had no adverse effects. The effect of the laser beam was explored on isolated coils from the cochlea. In two preparations, the specimens viewed in the confocal system were fixed and processed for electron microscopy. Identified cells were followed before, during, and after laser exposure and could ultimately be examined at the ultrastructural level. Exposure to the laser beam did not cause damage in unstained cells, even at high intensities. In stained tissue, confocal microscopy could safely be performed at normal beam intensity without causing ultrastructural changes. At high intensities, about 100 times normal for 60 times as long, irradiation damage was seen that was selective in that the cells stained with the different dyes exhibited damage at the different sites corresponding to the subcellular location of the dyes. Cells stained with calcein showed lysis of mitochondria and loss of cytoplasmic matrix, whereas cells stained with the styryl membrane dye showed swelling of subsurface cisternae, contortion of the cell wall, and shrinkage. The styryl dyes, in particular, which selectively stain the sensory and neuronal cells in the organ of Corti, could be exploited for phototoxic use.

Animals↗
PubMed2005

[Advantages of confocal microscopy in the analysis of epiretinal membranes and internal limiting membranes after macular surgery].

PURPOSE: To study specimens obtained from epiretinal membrane (ERM) and macular hole surgery with confocal microscopy and to evaluate its advantages in understanding their pathophysiology. MATERIAL AND METHODS: This retrospective study included 50 patients undergoing surgery for epiretinal membrane (40 patients) or idiopathic macular hole (10 patients). Infracyanine green was used in 45 cases. Immunohistological examination of the excised tissues was undertaken using confocal microscopy after labeling with antibodies to glial fibrillary acidic protein (GFAP) and vimentin. RESULTS: Confocal microscopic analysis of epiretinal membranes identified three types of tissues: 13 ERMs, which appeared thick and rich in cells, 20 internal limiting membranes (ILMs), which appeared thin, wrinkled and poor in cells, and seven composite tissues, where ERM and ILM were removed at the same time. The analysis of ten specimens obtained from macular hole surgery revealed nine ILMs and one composite tissue that were quite similar to those removed in epiretinal membrane surgery. In all, 27 specimens (10 ERMs, 12 ILMs and five composite tissues) contained GFAP-positive glial cells. CONCLUSION: The confocal microscope provided a fast, three-dimensional analysis of ERMs and ILMs. An immunohistological study identified cells that take part in the genesis of the membranes. Glial cells were found both in ERM and ILM specimens. Our findings suggest that Müller's cells may contribute to the pathogenesis of ERM and macular hole development.

Adult↗
PubMed2001

Assessment of internalization and viability of Porphyromonas gingivalis in KB epithelial cells by confocal microscopy.

Porphyromonas gingivalis (P. gingivalis) is considered to be one of the main periodontal pathogens. The goal of this work was to confirm the ability of P. gingivalis to invade host cells. We detected P. gingivalis inside KB cells by confocal microscopy and analyzed the various aspects of the adherence and internalization process. Lysates of P. gingivalis-infected KB cells were also examined using anaerobic growth techniques. The results showed the viability and ability to replicate, inside the host cells, of the internalized pathogen. The production of vesicles was also tracked for the first time. Confocal microscopy revealed P. gingivalis in a perinuclear position.

Endocytosis↗
PubMed2003

Virtual histology of colorectal lesions using laser-scanning confocal microscopy.

BACKGROUND AND STUDY AIMS: Histological examination of gastrointestinal lesions is currently based on light-microscopic examination of thin-slice specimens, with hematoxylin and eosin staining. A study of the use of laser-scanning confocal microscopy (LCM) to obtain immediate microscopic images of untreated specimens for examining colorectal lesions was carried out. A probe-type LCM prototype endomicroscope that can be passed through the working channel of an endoscope has also been developed. MATERIALS AND METHODS: The study materials consisted of colorectal lesions resected either endoscopically or surgically at Showa University Northern Yokohama Hospital. One hundred untreated specimens were examined using LCM. The histopathological findings in the lesions were seven cases of normal colonic mucosa, five hyperplastic polyps, 68 adenomas with low-grade dysplasia, 10 adenomas with high-grade dysplasia, and 10 adenocarcinomas. An argon laser beam with a wavelength of 488 nm was used for the LCM study. Observation of the resected normal colonic mucosa (in vitro) and the rectal mucosa of a healthy volunteer (in vivo) was possible using the endomicroscope. The LCM images for each specimen were compared with the hematoxylin-eosin-stained histopathological cross-sections. RESULTS: The LCM images corresponded well with the conventional hematoxylin-eosin light-microscopic images. The nuclei were not visualized in normal mucosa or hyperplastic polyps. In adenomas with high-grade dysplasia and carcinomas, nuclei were more often visible than in adenomas with low-grade dysplasia. The rate of visualization of nuclei was significantly different ( P < 0.01) between these two groups (60.0 % vs. 10.3 %). In LCM images using endomicroscope, it was possible to recognize the orifices of the colonic glands and goblet cells both in vitro and in vivo. CONCLUSIONS: Laser-scanning confocal microscopy provides immediate images that correspond well with those of hematoxylin-eosin staining. An improved probe-type LCM endomicroscope is being developed which should provide better histological images of colorectal lesions in vivo.

Adenocarcinoma↗
PubMed1998

In vivo confocal microscopy after photorefractive keratectomy in humans. A prospective, long-term study.

OBJECTIVE: To assess corneal morphological characteristics in vivo after photorefractive keratectomy (PRK) in humans. METHODS: Eighteen eyes were examined before and after PRK by means of in vivo confocal microscopy. Epithelial, stromal, and endothelial morphological characteristics were recorded. Minimum follow-up was 12 months. RESULTS: Immediately after PRK, the anterior stroma showed marked intercellular edema. At 1 month, fine linear structures were noted in the anterior stroma and midstroma, and a thin hyperreflective scar was present. The linear structures and the scar tissue were more marked at 4 months but were still present up to 26 months. Anterior stromal keratocyte density increased significantly 1 and 4 months after PRK, whereas midstromal and posterior keratocytes and endothelial cell densities did not change. Basal epithelial nerves were recognizable as early as 1 month after PRK. Contact lens-related microdots in the stroma remained unaffected. CONCLUSIONS: The stromal linear structures represent a finding that is detectable only by confocal microscopy at high magnification, is not related to previous contact lens wear, and is still visible 26 months after PRK. The extension of these structures as far as the midstroma indicates that the permanent corneal changes caused by PRK affect deeper stromal layers than the immediate subepithelial region.

Adult↗
PubMed2003

Corneal confocal microscopy: a non-invasive surrogate of nerve fibre damage and repair in diabetic patients.

AIMS/HYPOTHESIS: The accurate detection, characterization and quantification of human diabetic neuropathy are important to define at risk patients, anticipate deterioration, and assess new therapies. Corneal confocal microscopy is a reiterative, rapid, non-invasive in vivo clinical examination technique capable of imaging corneal nerve fibres. The aim of this study was to define the ability of this technique to quantify the extent of degeneration and regeneration of corneal nerve fibres in diabetic patients with increasing neuropathic severity. METHODS: We scanned the cornea and collected images of Bowman's layer (containing a rich nerve plexus) from 18 diabetic patients and 18 age-matched control subjects. RESULTS: Corneal nerve fibre density (F(3)=9.6, p<0.0001), length (F(3)=23.8, p<0.0001), and branch density (F(3)=13.9, p<0.0001) were reduced in diabetic patients compared with control subjects, with a tendency for greater reduction in these measures with increasing severity of neuropathy. CONCLUSION/INTERPRETATION: Corneal confocal microscopy is a rapid, non-invasive in vivo clinical examination technique which accurately defines the extent of corneal nerve damage and repair and acts as a surrogate measure of somatic neuropathy in diabetic patients. It could represent an advance to define the severity of neuropathy and expedite assessment of therapeutic efficacy in clinical trials of human diabetic neuropathy.

Cornea↗
PubMed1994

Regulation of Clara cell 10 kD protein secretion by pilocarpine: quantitative comparison of nonciliated cells in rat bronchi and bronchioles based on laser scanning confocal microscopy.

Standard immunohistochemical techniques and laser scanning confocal microscopy were used to assess changes in the abundance of Clara cell 10 kD protein (CC10) within granules and endoplasmic reticulum of rat nonciliated cells after the administration of the secretagogue pilocarpine. Intracellular pools of CC10 were compared over time with time-matched controls at two airway levels, proximal bronchi and terminal bronchioles. Three zones of reflectance intensity (high, medium, and low), corresponding to different densities of CC10 were used to quantify changes in CC10 levels. We observed a shift in the abundance of CC10 from endoplasmic reticulum to granules 30 min after injection of pilocarpine. In addition, the depletion of granule-based CC10 occurred earlier in bronchial cells (30 min) than in bronchiolar cells (60 min). We also noted that the density of CC10 within the two cellular compartments remained steady even though the levels of CC10 dropped due to degranulation. Following degranulation, CC10 levels returned to near-control levels. In the absence of pilocarpine, the percentage of cell volume occupied by the granule-based CC10 indicated that two varieties of nonciliated cells exist in bronchial airways. However, administration of pilocarpine abolishes this difference in CC10 abundance. Our results demonstrate that bronchial cells respond sooner to the secretagogue than do their bronchiolar counterparts and/or possess biosynthetic capabilities that are more easily overwhelmed than those of the terminal bronchiolar cells. These differences suggest that proximal nonciliated cells are distinct from distal nonciliated cells.

Animals↗