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PubMed2003

Sub-diffraction-limit imaging based on the topographic contrast of differential confocal microscopy.

Using the nanometer depth sensitivity of differential confocal microscopy, we detect surface features of lateral dimensions smaller than the diffraction limit without fluorescence labeling. The lateral resolution of the topographic images is further enhanced by a maximum-likelihood estimation algorithm. Based on the comparison of signal and noise at high spatial frequency, we estimate the best lateral resolution of the enhanced images to be 0.15 lambda. In addition, on composite samples this technique can simultaneously display sub-diffraction-limit topographic features and reflectivity heterogeneity.

Journal Article↗
PubMed2003

Adaptive approximation of the boundary surface of a neuron in confocal microscopy volumetric images.

In biomedical visualisation, the isosurface is usually used to represent (approximate) the boundary surface of the structure within biomedical volumetric images. However, in many confocal microscopy volumetric images of neurons, the grey values of the object and/or background are usually uneven. Therefore a fixed isosurface is not suitable for use in approximating the boundary surface of the neuron. A method is proposed to construct the adaptively approximating surface of the boundary surface of the neuron. In this method, the boundary surface of the neuron could be locally and adaptively approximated with different surface patches in different local regions. Consequently, the approximation accuracy has been considerably improved.

Algorithms↗
PubMed2006

Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections.

We performed second harmonic generation (SHG) imaging of collagen in rat-tendon cryosections, using femtosecond laser scanning confocal microscopy, both in backscattering and transmission geometries. SHG transmission images of collagen fibers were spatially resolved due to a coherent, directional SHG component. This effect was enhanced with the use of an index-matching fluid (n(i) = 1.52). The average SHG intensity oscillated with wavelength in the backscattered geometry (isotropic SHG component), whereas the spectral profile was consistent with quasi-phase-matching conditions in transmission geometry (forward propagating, coherent SHG component) around 440 nm (lambda(p) = 880 nm). Collagen type I from bovine Achilles tendon was imaged for SHG in the backscattered geometry and its first-order effective nonlinear coefficient was determined (|d(eff)| approximately 0.085(+/-0.025)x10(-12)mV(-1)) by comparison to samples of inorganic materials with known effective nonlinear coefficients (LiNbO3 and LiIO3). The SHG spectral response of collagen type I from bovine Achilles tendon matched that of the rat-tendon cryosections in backscattered geometry. Collagen types I, II, and VI powders (nonfibrous) did not show any detectable SHG, indicating a lack of noncentrosymmetric crystalline structure at the molecular level. The various stages of collagen thermal denaturation were investigated in rat-tendon cryosections using SHG and bright-field imaging. Thermal denaturation resulted in the gradual destruction of the SHG signal.

Animals↗
PubMed1996

An improved mounting method for observation of thick specimens using confocal microscopy.

When applying aqueous media to mount thick specimens, air bubbles occur frequently between coverslips and slide glasses. This results in an increase of background signal by confocal microscopy. Using Spurr's resin as a mounting medium, we could observe thick specimens with oil immersion objective lens without the use of coverslips, then avoid air bubbles near the specimen. Fading of the fluorescence appeared to be less in specimens mounted by the resin than in specimens mounted by aqueous media. Further, the new method increased depth of field so that more planes of specimens could be analyzed.

Animals↗
PubMed2004

Structural survey of airway sensory receptors in the rabbit using confocal microscopy.

Information on the morphology of airway receptors is limited. The present study surveys rabbit airway receptors using immunohistochemical and fluorescent labeling to identify their structure with confocal microscopy. Various receptor types were observed to have multiple branches where a parent axon fed several structures. Receptors were located in different layers of the airway, i.e., smooth muscle, lamina propria (submucosa) and the epithelium. Smooth muscle and submucosal receptors were innervated by thick myelinated fibers, while epithelial receptors were supplied by thin-diameter axons. Structures of smooth muscle receptors and some submucosal receptors covered a relatively large area, while epithelial receptors were less extended. In addition, intrapulmonary ganglia were also labeled. Some were closely associated with the axons of smooth muscle receptors.

Animals↗
PubMed2004

[Experimental study on the microcosmic changes of compressed premaxillary suture with laser scanning confocal microscopy].

OBJECTIVE: The aim of this experiment is to know whether active orthopedic appliance is rationale to do presurgical treatment and seek some regularities. METHODS: Laser scanning confocal microscopy and triple fluorochrome labeling in bone were used to observe and study detailed changes occurred in compressed suture systematically. RESULTS: Widened suture and broken fibres between the adjoining bones was observed at the 3rd week postoperatively. At the 3rd week hyperplasia of organic tissue components was also seen. At the 6 and 9th week there were no apparent differences among all groups no matter whether in control group or experimental groups. CONCLUSION: Retarding injury of compressed suture and surrounding tissue hyperplasia exists. With growth and development wounded suture will gradually assimilate to control group structurally and functionally.

Animals↗
PubMed1996

Effects of denervation on acetylcholine receptor clusters on frog cardiac ganglion neurons as revealed by quantitative laser scanning confocal microscopy.

We examined the effects of denervation on clusters of nicotinic acetylcholine receptors (AChRs) on autonomic neurons in the frog heart using immunofluorescence techniques and laser scanning confocal microscopy. We showed previously that normally innervated neurons have both large, brightly stained AChR clusters and small, dim AChR clusters. A majority (80%) of the large/bright AChR clusters are located at synaptic sites, whereas the small/dim clusters are distributed widely over the cell surface. Here, we use image analysis to identify these two classes of clusters on images generated from stacks of optical sections through neuronal cell bodies and to examine the effects of denervation on their number, size, and brightness (pixel intensity). Denervation reduces the number of large/ bright AChR clusters per cell to < 10% of sham-operated values and increases the number of small/dim clusters per cell by two- to threefold. These changes occur at 4 d of denervation, the earliest time examined, and are sustained for 6 weeks. The size of large/bright AChR clusters is decreased compared with sham-operated controls, and their brightness is unchanged. The size of small/dim AChR clusters is unchanged by denervation, but their brightness is increased. Denervation results in a shift in the contribution of each AChR cluster class to the total measurable AChR pool-from one dominated by large/bright clusters to one dominated by small/dim clusters. These results show that the nerve terminals on cardiac ganglion neurons appear to exert a continual and reversible influence on the organization of the postsynaptic membrane.

Animals↗
PubMed1997

Microleakage evaluation of bonded amalgam restorations: confocal microscopy versus radioisotope.

The purpose of this study was to compare the degree and pathway of microleakage in bonded amalgam restorations utilizing fluorescent dyes with scanning laser confocal microscopy and 45Ca radioisotope with autoradiographs. Mesial slot preparations with enamel cervical margins and distal preparations with dentin margins were prepared in extracted human molars. Four liners, Copalite (control) All-Bond 2/Resinomer, Amalcoden, and Panavia 21, were used. Teeth were restored with Dispersalloy or Tytin. After thermocycling, 7 days' storage, and staining, the degree of microleakage was measured on an ordinal scale. Dental adhesive liners minimized microleakage; the ranking was, from most to least effective, All-Bond 2, Panavia 21, and Amalcoden. The control exhibited the greatest microleakage. Microleakage occurred most frequently at the liner-tooth interface. No significant difference was observed between amalgams. The high correlation between the results of the fluorescent and radioisotope studies indicated that these two microleakage tests can be directly compared.

Autoradiography↗
PubMed1994

In vivo scanning slit confocal microscopy of Acanthamoeba keratitis. A case report.

A 29-year-old woman presented with clinical signs and symptoms of Acanthamoeba keratitis. Scanning slit confocal microscopy revealed a 26-mu-diameter object, resembling an Acanthamoeba cyst, in the anterior stroma. Numerous ovoid objects (possibly inflammatory cells, trophozoites, or altered keratocytes) were present. Normal keratocyte nuclei and the anterior corneal mosaic, readily imaged by scanning slit confocal microscopy of the normal cornea, were noticeably absent. Subsequent corneal biopsy confirmed the diagnosis of Acanthamoeba keratitis.

Acanthamoeba↗
PubMed2001

Direct nitric oxide imaging in cultured hippocampal neurons with diaminoanthraquinone and confocal microscopy.

A method for the direct measurement of intracellular nitric oxide (NO) production stimulated by penicillin G (PG) in cultured hippocampal neurons with diaminoanthraquinone (DAA) using laser scanning confocal microscopy (LSCM) was developed. Intracellular DAA fluorescence could specifically represent NO production based on two facts: (1) 3-morpholinosydnonimine, a NO donor, could dose-dependently increase DAA fluorescence; and (2) haemoglobin, a NO scavenger, could inhibit the increase of DAA fluorescence. The PG dose-dependently increased the intercellular level of glutamate (Glu, 5 min after stimulation) and the intracellular NO production (30 min throughout stimulation). The increase of NO production could be reversed by N(w)-nitro- l -arginine (a NO synthase inhibitor), and also by d (-)2-amino-5-phosphonovaleric acid, a subtype of Glu receptor antagonist. These results revealed that DAA could be used to indicate real-time and kinetic intracellular NO production of hippocampal neurons with higher sensitivity, specificity and accuracy.

2-Amino-5-phosphonovalerate↗
PubMed1994

Application of the Cavalieri principle in volume estimation using laser confocal microscopy.

The Cavalieri principle, a well-established stereological technique, uses interpolation between samples to estimate volume of three-dimensional (3D) objects. Serial optical sectioning with the confocal microscope resembles certain aspects of the Cavalieri principle, albeit with no interpolation. However, reconstruction and analysis of finely spaced optical sections can be cumbersome and time consuming. Application of the Cavalieri principle to confocal sections may be advantageous in reducing the size of the data set required to obtain reliable estimates of volume. In the present study, somal volumes of phrenic motoneurons were estimated by applying the Cavalieri principle to confocal images. These estimates were compared to measurements of somal volume using on interpolation of confocal sections. Phrenic motoneurons in adult rats were retrogradely labeled with a fluorescent rhodamine dye. Confocal optical sections of 0.6 micron thickness were then obtained from 150-micron-thick spinal cord slices containing labeled neurons. These image sets were reoriented to represent transverse sections. The Cavalieri principle was applied to these confocal image sets at selected sampling intervals from 1.2 to 3.0 microns. Planimetric measurements of motoneuron somal cross-sectional area in the selected sections were made using a point-counting method. At sampling intervals less than 2.4 microns, individuals motoneuron somal volume estimates were similar for the noninterpolated confocal and the interpolated Cavalieri methods. At these sampling intervals, the distributions of motoneuron somal volumes were also similar for the two methods. At a sampling interval of 2.4 microns or greater, there was a greater variability in individual motoneuron somal volume estimates, although the population mean and median were similar to the noninterpolated confocal measurements. Therefore, a satisfactory agreement between noninterpolated confocal measurements and the Cavalieri estimates suggests that less-stringent optical sectioning parameters may suffice for individual cell volume measurements when using confocal microscopy, thus making it significantly more efficient.

Animals↗
PubMed2006

Particle tracking using confocal microscopy to probe the microrheology in a phase-separating emulsion containing nonadsorbing polysaccharide.

Brownian diffusion of fluorescent microspheres (0.21, 0.5, and 0.89 microm diameter) in conjunction with confocal microscopy has been used to monitor the microrheology of phase-separated regions in a protein-stabilized oil-in-water emulsion containing various low concentrations of a nonadsorbing polysaccharide, xanthan gum. The sensitivity and reliability of the technique has been demonstrated in test experiments on (i) aqueous glycerol solutions and (ii) concentrated surfactant-stabilized emulsions (30-60 vol % oil, 1-2 wt % Tween 20). From particle tracking measurements on the caseinate-stabilized emulsions (30 vol % oil, 1.4 wt % sodium caseinate, pH 7) containing xanthan (0.03-0.07 wt %), the apparent viscosity in the oil-droplet-rich regions has been estimated to be up to 10(3) times higher than that in the phase-separated xanthan-rich regions. This means that our previously determined shape relaxation times for xanthan-containing blobs in the same systems can be attributed to the dominant viscoelasticity of the surrounding regions of concentrated oil droplets and not to the rheology of the xanthan-rich blobs themselves. These data provide clear and unequivocal evidence for the dominant role of the interconnected depletion-flocculated network of oil droplets in the physicochemical mechanism by which hydrocolloid thickeners control the creaming instability of concentrated oil-in-water emulsions.

Adsorption↗
PubMed2002

Quantitative image analysis of drug-induced lung fibrosis using laser scanning confocal microscopy.

Pulmonary fibrosis is a serious lung disorder that in certain cases may be difficult to quantify. It was our objective to evaluate the use of laser scanning confocal microscopy (LSCM) in quantifying fibrosis after exposure to amiodarone (AD) and bleomycin (BLM), two commonly used therapeutic drugs known to cause debilitating lung fibrosis in humans. Male F344 rats were intratracheally dosed with AD (6.25 mg/kg on days 0 and 2), BLM (0.25 and 1.0 mg/kg on day 0), or their respective vehicle controls. The right lung was assayed for hydroxyproline, a biochemical measure of collagen, at day 21 for the BLM groups and day 28 for the AD groups. The left lung was fixed, sectioned into blocks, dehydrated, stained with Lucifer yellow (LY, 0.1 mg/ml), and embedded in Spurr resin. The area of lung tissue stained by LY was quantified by LSCM. A fibrotic response in the AD and BLM groups was confirmed by histopathological assessment and a significant increase (p < 0.05) in total right lung hydroxyproline above control values. The area of connective tissue stained by LY of the two drug-treated groups appeared as bright linear bands in the alveolar septae and was significantly increased (p < 0.05) as measured by image analysis when compared with their respective controls. LSCM, with its advanced image analysis system, is an alternate method to quantify fibrotic lung disease. LSCM could be particularly useful when tissue quantity is limited, such as when tissue has been archived from previous studies, or when analyzing human lung biopsy samples for disease diagnosis, where biochemical analysis is difficult.

Amiodarone↗
PubMed1992

Subcellular distribution of cytosolic Ca2+ in isolated rat hepatocyte couplets: evaluation using confocal microscopy.

Ca2+ agonists induce Ca2+ waves and other non-uniform Ca2+ patterns in the cytosol of epithelial cells. To define subcellular Ca2+ transients in the cytosol of hepatocytes we examined Fluo-3-loaded isolated rat hepatocyte couplets using confocal microscopy. Optical sections of less than 1 micron in thickness were observed in couplets, and fluorescence from cytosolic Ca2+ signals was readily distinguished from nuclear, mitochondrial, and lysosomal fluorescence. The nature of the noncytosolic components of the fluorescent images was verified by double labelling with the mitochondrial dye DiOC6(3) and with the lysosomal marker acridine orange. Using the line scanning mode of confocal microscopy, measurements of cytosolic Ca2+ were made with a frequency of up to 250 Hz and without significant bleaching. It was found that phenylephrine-induced Ca2+ signals generally began at the basal pole of the hepatocytes, then spread to the canaliculus at average speeds of 80 micron/s. These findings demonstrate the utility of confocal line scanning microscopy for detecting rapid changes in the subcellular distribution of cytosolic Ca2+ in hepatocyte couplets, and suggest that phenylephrine-induced Ca2+ waves radiate in a basal-to-apical direction in this cell type.

Animals↗
PubMed2002

In vivo confocal microscopy through-focusing to measure corneal flap thickness after laser in situ keratomileusis.

PURPOSE: To measure flap thickness in laser in situ keratomileusis (LASIK) patients using in vivo confocal microscopy through-focusing (CMTF) and compare measured versus intended flap thickness achieved by 2 microkeratomes, the Automated Corneal Shaper(R) (ACS) (Chiron Bausch & Lomb) and the Hansatome (Bausch & Lomb). SETTING: Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA. METHODS: Twenty-seven eyes of 27 patients were examined by in vivo CMTF 3 to 12 months after LASIK was performed with the ACS (12 patients) or Hansatome (15 patients) microkeratome. The central cornea was scanned, and the epithelium, flap, stroma, and total corneal thickness were measured. Normalized flap thickness (NFT) was also calculated to account for changes in epithelial thickness that may have occurred postoperatively. RESULTS: The mean posterior stromal thickness was 341.1 microm +/- 53.9 (SD) (range 233 to 431 microm) in the ACS group and 320.3 +/- 42.3 microm (range 258 to 382 microm) in the Hansatome group. The mean nonnormalized flap thickness was 132.7 +/- 12.5 microm (range 11 to 151 microm) in the ACS group and 167.4 +/- 21.4 microm (range 141 to 209 microm) in the Hansatome group. The NFT was 129.6 +/- 9.5 microm and 158.4 +/- 22.1 microm, respectively. Both microkeratomes cut significantly less than intended (P <.05); however, the ACS cut a thinner-than-intended thickness in all cases, and the Hansatome cut thicker than intended in 13% of cases. The Hansatome also showed significantly greater variability in flap thickness than the ACS (P <.05). CONCLUSIONS: A significant difference in precision was noted between the 2 microkeratomes. The findings emphasize the importance of performing thickness measurements and the usefulness of in vivo CMTF in making these determinations to ensure the safety and effectiveness of LASIK.

Adult↗
PubMed1996

Confocal microscopy of idarubicin localisation in sensitive and multidrug-resistant bladder cancer cell lines.

Idarubicin is a highly lipophilic anthracycline and appears effective against tumours resistant to conventional anthracyclines. Confocal microscopy demonstrates predominantly cytoplasmic idarubicin accumulation. This distribution is unaltered by resistance status or the resistance reversing agent verapamil. Our results contrast with studies on conventional anthracyclines and suggest that nuclear accumulation may not be a prerequisite for anthracycline cytotoxicity.

Antibiotics, Antineoplastic↗
PubMed2002

Assessing the sub-basal nerve plexus of the living healthy human cornea by in vivo confocal microscopy.

The purpose of the study was to perform quantitative analysis of the sub-basal epithelial nerve plexus of healthy, living human cornea,using real time in vivo confocal microscopy and the analySIS software system. The study was based on in vivo confocalmicrostructural analysis of 50 eyes of 50 subjects, divided into two age groups: group 1 (n = 25)25 +/- 5 years of age, and group 2 (n = 25) 70 +/- 5 years of age. All subjects exhibited clinically healthy corneas. The overall nerve density was 632.35 +/- 287.57 microm/mm2 for group 1 and 582.39 +/- 327.13 microm/mm2 for group 2. The mean fibre dia-meter was measured at 0.52 +/- 0.23 microm for group 1 and at 0.56 +/- 0.27 microm for group 2. Beadings of the nerve fibres were recorded at a density of 213 +/- 123/mm for group 1 and 201 +/- 192/mm for group 2. Establishing standards for normal nerve density and morphology of the living human cornea at different ages may be beneficial, both in early detection and follow up of various corneal diseases and in post-surgical management following corneal surgery.

Adult↗
PubMed1999

Intracellular localization of 6- and 7-substituted 2-[2'-(dimethylamino)ethyl]-1,2-dihydro-3H-dibenz[de,h]isoquino line-1,3-diones (azonafides) is not the limiting factor for their cytotoxicity: an in vitro confocal microscopy study.

The intracellular localization of 14 structurally unique azonafide analogs was studied to determine if intracellular drug distribution is the limiting factor in azonafide cytotoxicity. Using scanning laser confocal microscopy, cytotoxicity of the azonafide analogs studies was observed in Chinese hamster ovary cells immediately after a 1 h exposure. The intracellular drug distribution patterns varied significantly for different analogs. Eight analogs showed primarily nuclear localization, five analogs showed primarily cytoplasmic localization and two analogs displayed perinuclear localization. In general, the type of chemical substitution on the anthracene nucleus determined the distribution pattern. For example, for each analog seven of eight nuclear-localizing analogs were amine-substituted agents, while four of five cytoplasmic-localized agents were ethoxy-substituted analogs. The individual exception within these groups was the 6-[(dimethylamino)ethoxy] agent that was nuclear localized. The two perinuclear-localized agents included the unsubstituted parent, azonafide, and its 6-methyl azonafide analog. Comparison of the cytotoxicity of the azonafides, based on intracellular localization, revealed that none of the localization patterns were associated with increased cytotoxicity. These results show that minor structural changes in the azonafide class of antitumor agents involving substitution along an anthracene chromophore result in substantially different intracellular drug distribution patterns. However, these distribution differences do not determine relative cytotoxic potency in vitro.

Analysis of Variance↗