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At least 955 records · Page 53Linked to original sources
PubMed2005

A simple method allowing DIC imaging in conjunction with confocal microscopy.

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique (Barone-Nugent et al., 2002). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.

Animals↗
PubMed1995

Aquaporin-CHIP-related protein in frog urinary bladder: localization by confocal microscopy.

Aquaporin CHIP, a 28 kDa channel forming protein, has been proposed to function as water channel in both erythrocyte and kidney proximal tubule. Recently, we have reported that in frog urinary bladder, a model of the kidney collecting tubule, polyclonal antibodies against human erythrocyte CHIP recognize and immunoprecipitate a 30 kDa protein from the epithelial cell homogenate. In the present work confocal fluorescence microscopy was used to determine the cellular and subcellular localization of CHIP28-like proteins in the urinary epithelium. A clear labeling of the apical border was found after Triton X-100 permeabilization. The labeling was distributed throughout the apical domain and not restricted to specific domains of the membrane. The staining was also present in the deeper confocal sections where the fluorescence seems to be localized at the cellular contour. No difference in the labeling patterns was observed between resting and ADH-treated bladder. Specificity of the staining was confirmed by the absence of the labeling pattern when antiserum was preadsorbed on CHIP28 protein immobilized on Immobilon P stripes. Our results suggest that CHIP-like proteins are not proteins inserted in the apical membrane during the antidiuretic response. Moreover, we do not know whether the labeling was due to the presence of CHIP28 itself or an as-yet-unidentified protein sharing immunological analogies with aquaporin CHIP.

Animals↗
PubMed1995

Confocal microscopy of thick sections from acrylamide gel embedded embryos.

The preparation of optically clear, thick sections of fragile embryonic tissues greatly aids the power of confocal scanning laser microscopy in imaging three-dimensional structures. We report here conditions for embedding, sectioning, and staining embryos in polyacrylamide gels for a variety of confocal imaging techniques. Infiltration of tissues in standard mixtures of 10-15% acrylamide monomer yields, upon polymerization, blocks that cut easily by vibratome between 50 and 1,000 microns. These conditions worked well for tissues previously stained or for staining gel sections with low molecular weight water-soluble fluorochromes (MW < 5 kD [e.g., propidium iodide, phalloidin]). For immunostaining of tissue after embedding and sectioning, the acrylamide concentration was reduced to 2-3% acrylamide to allow access of immunoglobulins to antigenic sites; such gels were supplemented with 1% agarose to facilitate sectioning and handling. Either method yielded abundant, optically clear, and easily handled sections for mounting and examination in water-miscible media.

Acrylic Resins↗
PubMed2003

Dual-axes confocal microscopy with post-objective scanning and low-coherence heterodyne detection.

We present a dual-axes confocal microscope that employs postobjective scanning and low-coherence heterodyne detection to collect vertical cross-sectional images from biological tissue with high axial resolution, reduced noise from scattered light, deep tissue penetration, and a large dynamic range. This architecture can be scaled down to millimeter dimensions with microelectromechanical systems technology for performance of in vivo optical biopsy.

Biopsy↗
PubMed1994

To boldly glow ... applications of laser scanning confocal microscopy in developmental biology.

The laser scanning confocal microscope (LSCM) is now established as an invaluable tool in developmental biology for improved light microscope imaging of fluorescently labelled eggs, embryos and developing tissues. The universal application of the LSCM in biomedical research has stimulated improvements to the microscopes themselves and the synthesis of novel probes for imaging biological structures and physiological processes. Moreover the ability of the LSCM to produce an optical series in perfect register has made computer 3-D reconstruction and analysis of light microscope images a practical option.

Animals↗
PubMed1991

Three-dimensional specimen reconstruction by confocal microscopy and digital image processing.

In a confocal microscope point-wise illumination of the specimen is used, often in the form of a focused laser beam that is scanned in a raster pattern. The reflected or fluorescent light from the specimen is focused onto a small aperture in front of the detector. This imaging method has advantages such as improved resolution and less stray light. In addition optical sectioning is possible, whereby thick specimens can be studied layer by layer without the need for mechanical sectioning. By combining information from a large number of optical sections, registered at different depths, a three-dimensional reconstruction of the specimen can be made. Digital image processing can be used both for producing images for visual display and for extracting quantitative information. Quantitative evaluation is hampered in many cases by factors such as absorption, scattering, and refraction of the light travelling through the specimen volume.

Image Processing, Computer-Assisted↗
PubMed1998

Extended confocal microscopy of myocardial laminae and collagen network.

Ventricular myocardium has a complex three-dimensional structure which has previously been inferred from two-dimensional images. We describe a technique for imaging the 3D organization of myocytes in conjunction with the collagen network in extended blocks of myocardium. Rat hearts were fixed with Bouin's solution and perfusion-stained with picrosirius red. Transmural blocks from the left ventricular free wall were embedded in Agar 100 resin and mounted securely in an ultramicrotome chuck. Confocal fluorescence laser scanning microscopy was used to obtain 3D images to a depth of 60 microns in a contiguous mosaic across the surface. Approximately 50 microns was then cut off the surface of the block with an ultramicrotome. This sequence was repeated 20 times. Images were assembled and registered in 3D to form an extended volume 3800 x 800 x 800 microns 3 spanning the heart wall from epicardium to endocardium. Examples are given of how digital reslicing and volume rendering methods can be applied to the resulting dataset to provide quantitative structural information about the 3D organization of myocytes, extracellular collagen matrix and blood vessel network of the heart.

Animals↗
PubMedDate not supplied

Confocal microscopy of the lizard motor nerve terminals.

Confocal imaging was performed on the ceratomandibularis nerve muscle preparation of the lizard Anolis carolinensis, using 4-Di-2-ASP as a fluorescent probe. The imaging system consisted of a Sarastro Phoibos 1000 (Molecular Dynamics) scanning system and a Zeiss Universal microscope. The data were analyzed using the VANIS set of programs on a Silicon Graphics Personal Iris computer. A three dimensional reconstruction of the nerve terminals was performed using look-through and depth-coded projections. The volume of the nerve terminal was estimated in 15 neuromuscular junctions and found to be 84,835 microns 3 (+/- 8558 SEM). The largest diameter in each one of the 178 individual boutons was estimated from the look-through projections of 17 nerve terminals in 9 preparations. It was found to be 4.71 microns (+/- 0.08 SEM). The diameter perpendicular to the largest diameter in the same projection at 0 degrees was 3.3 microns (+/- 0.054 SEM). Thus it seems that the synaptic boutons of the ceratomandibularis are suitable for combined optical and electrophysiological recordings.

Animals↗
PubMed2000

Time-sequence histologic imaging of laser-treated cherry angiomas with in vivo confocal microscopy.

OBJECTIVE: To chronicle the pathophysiologic changes that occur subsequent to laser treatment of vascular lesions, we used a confocal scanning laser microscope that yields high-resolution microscopic images of skin in vivo. METHODS: Cherry angiomas were treated with the 585-nm flashlamp-pumped pulsed-dye laser (PDL) and the 568-nm continuous-wave krypton laser. Repeated confocal reflectance imaging was performed before and immediately after treatment, as well as after several hours, 1 day, 2 days, 1 week, 2 weeks, 3 weeks, and 4 weeks. RESULTS: Before treatment, confocal images revealed dilated blood vessels ranging from 10 to 50 microm in caliber, closely spaced at 5 to 50 microm apart. After PDL treatment, amorphous cords of refractile material conformed to the shape of the original vessels, followed by dark nonrefractile spaces where the vessels once were. Inflammation and necrosis ensued, with eventual replacement after 3 weeks by normal-appearing skin. After krypton laser treatment, dark nonrefractile spaces appeared immediately, with subsequent inflammation, necrosis, and eventual healing by 4 weeks. CONCLUSION: Confocal laser microscopic imaging elucidates the dynamic pathophysiologic events that occur after laser treatment of vascular lesions and has added insight into the different mechanisms of vessel damage induced by the PDL and krypton laser.

Hemangioma↗
PubMed1997

Imaging and analysis of perivascular nerves in human mesenteric and coronary arteries: a comparison between epi-fluorescence and confocal microscopy.

Perivascular nerves supplying human arteries can be visualised after immunohistochemical staining for a variety of markers. The pattern and density of perivascular nerves vary with region, age and disease. Quantification of the nerve plexus, which may be performed by image analysis, is a prerequisite to assess differences in nerve density. The use of epi-fluorescence microscopy (EFM) presents difficulties in visualising the nerve plexus in certain tissues, which can affect the reliability with which specific staining can be localised and distinguished from non-specific staining. In this study, confocal scanning laser microscopy (CSLM) was used in parallel with EFM, in order to compare images from both techniques. In a comparison of identical areas of nerve plexuses of human mesenteric and coronary arteries stained for protein gene product (PGP) 9.5 and imaged using CSLM and EFM, higher values for area percent (area occupied by nerves), and intercept density (ID/mm, which reflects the number of nerve bundles detected) were found in CSLM images. Similar comparisons of unmatched epi-fluorescence and confocal images from a group of 45 mesenteric arteries revealed no significant difference for area percent, but significantly higher values for ID/mm in CSLM images. These findings illustrate that the better image quality in CSLM influences image analysis and can be very useful in studies of dynamic changes in nerve plexuses. We recommend CSLM for tissues that suffer from high background staining, such as human mesenteric and coronary arteries.

Adult↗
PubMed2006

Image adaptive point-spread function estimation and deconvolution for in vivo confocal microscopy.

Visualizing deep inside the tissue of a thick biological sample often poses severe constraints on image conditions. Standard restoration techniques (denoising and deconvolution) can then be very useful, allowing one to increase the signal-to-noise ratio and the resolution of the images. In this paper, we consider the problem of obtaining a good determination of the point-spread function (PSF) of a confocal microscope, a prerequisite for applying deconvolution to three-dimensional image stacks acquired with this system. Because of scattering and optical distortion induced by the sample, the PSF has to be acquired anew for each experiment. To tackle this problem, we used a screening approach to estimate the PSF adaptively and automatically from the images. Small PSF-like structures were detected in the images, and a theoretical PSF model reshaped to match the geometric characteristics of these structures. We used numerical experiments to quantify the sensitivity of our detection method, and we demonstrated its usefulness by deconvolving images of the hearing organ acquired in vitro and in vivo.

Algorithms↗
PubMed2005

Adaptive penalty likelihood for reconstruction of multidimensional confocal microscopy images.

In this paper we devise a penalty likelihood with noise constraints method to restore 2D and 3D confocal microscope images. Regularization is a commonly used technique in image restoration to balance restored image quality and noise suppression, but despite this noise is usually amplified. Taking into account common confocal imaging system degradation, we develop an algorithm by using a gradient descent method (PLGDA) to approach the minimum solution of the penalty likelihood equation. A Lagrange parameter controls the balance between the penalty and likelihood terms and is estimated using an adaptive method. We show that the a priori information is key to the regularization and Lagrange parameter estimation. The convergence characteristics are analysed and discussed. PLGDA and a traditional maximum likelihood expectation maximization are used to restore 2D and 3D confocal images. The point spread function (PSF), used to restore the data is collected from an experiment and modelled by bi-cubic splines to give an accurate noise free representation. Our experimental results show that the restored images are significantly improved by PLGDA.

Algorithms↗
PubMed1989

Three-dimensional visualization methods for confocal microscopy.

Three-dimensional images of microscopic objects can be obtained by confocal scanning laser microscopy (CSLM). The imaging process in a CSLM consists of sampling a specific volume in the object and storing the result in a three-dimensional memory array of a digital computer. Methods are needed to visualize these images. In this paper three methods are discussed, each suitable in a specific area of application. For purposes where realistic rendering of solid or semi-transparent objects is required, an algorithm based on simulation of a fluorescence process is most suitable. When speed is essential, as for interactive purposes, a simple procedure to generate anaglyphs can be used. Both methods have in common that they require no previous interpretation or analysis of the image. When the study of an object imaged by CSLM involves analysis in terms of a geometrical model, sophisticated graphics techniques can be used to display the results of the analysis.

Algorithms↗
PubMed1998

Vital staining of the hearing organ: visualization of cellular structure with confocal microscopy.

Cells inside the intact organ of Corti were labelled with fluorescent probes reflecting various aspects of structure and function. The dyes were introduced into the perilymphatic space by perfusion of the scala tympani of the temporal bone from the guinea-pig maintained in isolation. The dyes were able to diffuse through the basilar membrane and into the organ of Corti where they were spontaneously absorbed by the sensory and supporting cells. Confocal microscopic observation was made through an opening in the apex of the cochlea. A number of different dyes were used; a carbocyanine dye which stains mitochondria; two styryl dyes which are absorbed by the cell membranes and calcein, a cytoplasmic marker that fluoresces in vital cells. Extracellular space was stained by a cell-impermeant Dextran fluorescein. The most striking finding was that the membrane dyes preferentially stained the sensory cells and neural elements whereas the staining of the supporting cells was faint. The cytoplasmic dye in general stained sensory and supporting cells to the same extent. By tilting the organ, a view could be obtained from the side like a radial section through the organ. Outer and inner hair cells with their sensory hairs, nerve fibres and nerve endings, especially under the inner hair cells, could be seen in profile. Introduction of a high molecular weight Dextran into the endolymphatic space outlined the tectorial membrane which was seen in negative contrast. The simultaneous perfusion with a membrane dye stained the hair cells and their sensory hairs. Merging of the two images gave the possibility to examine, in the living tissue, the cilia to tectorial membrane relationship. Of general interest is the finding that the membrane dyes preferentially stained the sensory and neural elements of the nervous system, represented here by the hair cells and nerve fibres of the inner ear.

Animals↗
PubMed1997

Direct measurement of endosomal pH in living cells of the rat yolk sac epithelium by laser confocal microscopy.

Endocytosis of dual-fluorescent dextran by epithelial cells was observed in the perfused rat yolk sac using a confocal laser scanning microscope. Endosomal pH was quantified from confocal images using a dual-fluorescence ratiometry technique. Changes in endosomal pH were followed over time up to 32 min with and without Brefeldin A treatment. After 4 min of endocytosis without Brefeldin A, endosomes had a pH of 6.1 +/- 0.3. After 16 min and 32 min, their pH levels varied widely from 4.0 to 6.6. When Brefeldin A (10 microM) was added to the perfusion medium, endosomal pH remained fairly stable in the range of 6.0 to 6.2 from 4 min to 32 min. The corresponding endosomal structures were examined by electron microscopy after endocytic labeling with horseradish peroxidase. After 4 min, small endocytic vesicles and large endosomal vacuoles with tubular extensions were labeled. After 16 min and 32 min, multivesicular bodies and dense lysosomal structures were progressively labeled, but their labeling was almost undetectable after Brefeldin A treatment. These results suggested that the pH within the sorting compartment of early endosomes is 6.1 +/- 0.3. This is the first quantitative measurement of pH within sorting endosomes in intact cells of the living yolk sac epithelium.

Animals↗
PubMed2000

Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid.

OBJECTIVES: The use of high-resolution in vivo confocal imaging may offer a clinically useful adjunct to standard methods for the diagnosis and screening of epithelial precancers. This study assesses the feasibility of real-time confocal reflectance imaging of cervical tissue and the use of acetic acid as a contrast agent to increase visualization of cell nuclei. STUDY DESIGN: A confocal microscope was used to image cervical cells and colposcopically normal and abnormal cervical biopsy specimens. Images were obtained before and after the application of 6% acetic acid. RESULTS: The confocal imaging system resolved subcellular detail throughout the entire epithelial thickness. Normal and abnormal tissues were clearly able to be differentiated. Addition of acetic acid enhanced nuclear signal in all acquired images. CONCLUSION: High-contrast reflected light images of cervical tissue are attainable in near real time. Acetic acid significantly increases light scattering from cell nuclei, which may partially explain why acetowhitening occurs.

Acetic Acid↗
PubMed2001

Evidence of pituitary adenylate cyclase activating polypeptide (PACAP) in pancreatic islet cells by confocal microscopy.

Several studies have shown that pituitary adenylate cyclase activating polypeptide (PACAP) stimulates at very low concentration insulin release from pancreatic beta cells. In addition, PACAP has been evidenced in pancreatic nervous fibers surrounding the islets, the core of the islet, and the capillaries. The aim of the present study was to demonstrate internalization of PACAP in pancreatic islet cells. Pancreatic islets were obtained from Wistar rat pancreata by modified Lacy's isolation method. The isolated islets were incubated in the presence of Fluo-PACAP 27, a fluorescent ligand specific for PACAP receptors. At the end of incubation the islets were fixed in paraformaldehyde and then observed by confocal microscope. Fluo-PACAP 27 was internalized into pancreatic islet cells, and this process was time- and temperature-dependent (37 degrees C). The fluorescent molecules converged toward the nucleus where an intense fluorescence was evidenced after 60 minutes. Incubation with phenyl arsine oxide as well as with PACAP 6-38, a receptor antagonist, prevented the internalization process. Further studies are required to explain the internalization process of PACAP 27 into the nucleus of pancreatic islet cells.

Animals↗
PubMed1997

Mesoderm-derived cells proliferate in the embryonic central nervous system: confocal microscopy and three-dimensional visualization.

In the chick and quail embryo, two cell populations migrate into the neural tube from the surrounding mesodermal tissues during the fourth day of incubation: individual cells which represent macrophages, and endothelial cells which remain continuous with the extraneural vessels. We report here on the proliferative capacity of these mesoderm-derived cells. A double-immunofluorescence protocol for two monoclonal antibodies of subtype IgG1, the endothelial cell/macrophage marker QH1, and the S-phase marker bromodeoxyuridine, was developed. With confocal laser scanning microscopy of thick microtome sections, labeling indices of intraneural individual QH1-positive cells (12%) and of endothelial cells (10%) were determined. In contrast, the labeling index of extraneural endothelial cells was 25%. With three-dimensional visualization of confocal data, the variable morphology of macrophages was shown. Our results indicate that: (1) proliferative activity of intraneural capillary endothelial cells is less than expected and that it is absent from sprouts; (2) both spheroidal and ramified macrophages proliferate inside the neural tissues; and (3) ramified macrophages frequently make contact with capillary endothelial cells. We conclude that most embryonic microglia may be derived from the early invasive QH1+ macrophages.

Animals↗