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PubMedDate not supplied

Method for 3D volumetric analysis of intranuclear fluorescence distribution in confocal microscopy.

The introduction of the confocal laser scanning microscope makes it possible to acquire fluorescent specimens in 3D. We present basic image processing tools to enhance the data and to quantitate the morphology and topography of the intravolume elements. The tools were applied to the description of the spatial distribution of DNA replication sites in mammalian cell nuclei as an example.

Algorithms↗
PubMed1994

Gelatinase A expression and localization in human breast cancers. An in situ hybridization study and immunohistochemical detection using confocal microscopy.

The gelatinase A (72 kDa type IV collagenase) is a matrix metallo-proteinase which degrades basement membrane collagens. Various studies emphasize its role in stromal invasion of cancers, but there is some controversy about its origin. Gelatinase A was localized by immunohistochemistry using confocal microscopy in 15 human mammary carcinomas. In addition, the cells responsible for the synthesis of this enzyme were detected by in situ hybridization. Most invasive and non-invasive tumour cells were labelled by immunohistochemistry. Of particular interest was the pattern observed in some pre-invasive areas. Gelatinase A was found in fibroblasts in close contact with pre-invasive tumour clusters. Confocal observation allowed a more precise localization of gelatinase A to the periphery of tumour clusters along the basement membranes and in peritumour fibroblasts. The malignant epithelial cells were negative by immunohistochemistry in these areas. By in situ hybridization, mRNAs encoding gelatinase A were detected only in fibroblasts in close contact with pre-invasive and well differentiated tumour clusters. These findings support the hypothesis that peritumour fibroblasts produce gelatinase A and that breast cancer cells may bind this enzyme to their cell surface and/or internalize it.

Amino Acid Sequence↗
PubMed1996

Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system.

The bilateral imaging approach known from confocal applications operating in the line mode was used to realize real-time two-photon imaging. It is shown that the sectioning inherent to two-photon imaging could be improved by the introduction of a confocal line aperture in the imaging path. Using a high-power, low-repetition-rate amplified Ti:sapphire system, various biological objects were visualized including live boar sperm.

Animals↗
PubMed2001

Test of canine sperm function in vitro using primary homologous oocytes with fluorescence and confocal microscopy.

In vitro capacitated canine spermatozoa can interact with both mature and immature homologous oocytes in vitro, but it is difficult to determine whether spermatozoa have actually penetrated the oocyte or have simply bound to the zona pellucida. The aim of the first part of this study was to determine accurately the location of spermatozoa in relation to the oocyte by comparing observations made using confocal and fluorescence microscopy. The fluorescence technique had a sensitivity of 66%, and correctly identified 98% of the spermatozoa that had penetrated or bound oocytes and could discriminate between penetrated and bound spermatozoa with a sensitivity and specificity of 97% and 100%, respectively. This finding demonstrates that evaluation of the assay by fluorescence microscopy is reliable for detecting the presence and location of spermatozoa in relation to homologous oocytes. The aim of the second part of the study was to attempt to simplify the assay using fluorescence microscopy and immature oocytes. No significant difference was found between sperm interaction with mature and immature oocytes, which demonstrates that culturing oocytes before the assay has no benefit, and that the assay can be performed quickly and easily using non-cultured oocytes to provide rapid evidence of the fertilizing potential of spermatozoa.

Animals↗
PubMed2006

Mapping electrochemiluminescence as generated at double-band microelectrodes by confocal microscopy under steady state.

A confocal microspectrometer was used to collect spatially resolved electrochemiluminescence (ECL) spectra from the annihilation reaction between Ru(bpy)3+ and Ru(bpy)3(3+), each species being generated at a platinum band microelectrode and separated by a micrometric insulating gap. The use of a confocal microspectrometer allows in situ photon detection of the ECL reaction with micrometric spatial resolution and observes the concentration distribution of the photon source in the vicinity of the interelectrode gap.

Journal Article↗
PubMed1992

Confocal microscopy and three-dimensional reconstruction of thick, transparent, vital tissue.

The three-dimensional visualization of the 400 micron thick, transparent, in situ cornea is described to demonstrate the use of confocal light microscopy for noninvasive imaging of living cells and thick tissues in their normal, vital conditions. Specimen preparation and physiological stability, as well as light attenuation corrections are critical to data acquisition. The technique to provide mechanical stability of the specimen during the duration of the image acquisition is explained. A laser scanning confocal light microscope (LSCM) was used to obtain optical serial sections from rabbit eyes that were freshly removed and placed in a physiological Ringer's solution. This study demonstrates the capability of the confocal light microscope to obtain a series of high contrast images, with a depth resolution of one micron, across the full thickness of living, transparent tissue. The problems of nonisotropic sampling and the limited eight-bit dynamic range are discussed. The three-dimensional reconstructions were obtained by computer graphics using the volume visualization projection technique. The three-dimensional visualization of the cornea in the in situ eye is presented as an example of image understanding of thick, viable biological cells and tissues. Finally, the criterion of image fidelity is explained. The techniques of confocal light microscopy with its enhanced lateral and axial resolution, improved image contrast, and volume visualization provides microscopists with new techniques for the observation of vital cells and tissues, both in vivo and in vitro.

Animals↗
PubMed1994

Confocal microscopy in turbid media.

We examine the performance of confocal microscopes designed for probing structures embedded in turbid media. A heuristic scheme is described that combines a numerical Monte Carlo simulation of photon transport in a turbid medium with a geometrical ray trace through the confocal optics. To show the effects of multiple scattering on depth discrimination, we compare results from the Monte Carlo simulations and scalar diffraction theory. Experimental results showing the effects of the pinhole diameter and other variables on imaging performance at various optical depths in suspensions of polystyrene microspheres were found to correspond well with the Monte Carlo simulations. The major conclusion of the paper is that the trade-off between signal level and background scattered-light rejection places a fundamental limit on the sectioning capability of the microscope.

Light↗
PubMed1996

Efficient real-time confocal microscopy with white light sources.

The main advantage of confocal microscopes over their conventional counterparts arises from their ability to optically 'section' nearly transparent materials; the thin image slices thus obtained can be used to reconstruct three-dimensional images, a capability which is particularly useful for the study of biological specimens. Confocal microscopes have previously used either a single laser-illuminated point-source and single point-detector (which are scanned in tandem across the object) or white-light illumination with multiple point-sources and detectors. Single-point-source systems, however, do not usually form images in real time and are restricted to using available laser wavelengths. Multiple-point-source systems, on the other hand, produce images in real time but use light very inefficiently--typically 1% or less is used for imaging. Here we demonstrate a white-light, multiple-point-source method which can in principle produce images in real time with light efficiencies as high as 50%. This system is likely to find broad practical application, particularly in the imaging of weakly reflecting or weakly fluorescent specimens.

Animals↗
PubMed1994

Analysis of chick somite myogenesis by in situ confocal microscopy of desmin expression.

We explored the relationship in chick embryos between somitogenesis and the onset of somite myogenesis by immunodetection of the muscle-specific intermediate filament protein desmin. Early somite desmin expression was detected by whole-mount in situ confocal microscopy. No detectable somite desmin was observed in embryos of 15 somites (Stage 12) or younger. In embryos having between 16 and 26 somites (Stages 12-15), desmin could be detected in somites positioned increasingly more caudal in the embryo. Finally, in embryos of 27 somites (Stage 16) and older, somite desmin expression was consistently present in all but the caudal-most six somites. Although the rate of somite formation is fairly constant, the rate of observed somite desmin expression progressing caudally in the embryo is greater initially than the rate of segmentation. After an embryo has formed about 27 somites, the rate of desmin appearance parallels the rate of segmentation at a distance of about six somites. This result suggests that very early somite myogenesis is not linked to somitogenesis.

Animals↗
PubMed1995

Three-dimensional co-location of RNA polymerase I and DNA during interphase and mitosis by confocal microscopy.

The relative three-dimensional co-location of RNA polymerase I (RPI) and DNA was studied using confocal laser scanning microscopy during interphase and all the steps of mitosis in human cancerous cells. For each step of the cell cycle, immunolabeled RPI molecules and DNA specifically stained with chromomycin A3 were simultaneously imaged at high resolution through numerous optical sections. Then, all the data obtained were used to generate transverse sections, anaglyphs and volumic representations, which are all prerequisite approaches to a representative study of the three-dimensional organization of the nucleolus and the mitotic chromosomes. Our results indicated that in the interphasic nuclei, in which DNA is organized as a regular 3-D network, RPI was present within numerous irregular spheres arranged as several twisted necklaces. During metaphase, RPI labeling was segregated into pairs of spheres and typical crescent-shaped structures; both were centrally located within the set of chromosomes. During anaphase and telophase, a typical central and symmetric arrangement of labeled structures was systematically seen among the decondensing chromosomes, arranged as a regular cylinder and as a hollow half-sphere, respectively. This typical 3-D organization of structures containing RPI relative to DNA is another strong example of the non-random organization of the genome during interphase and mitosis.

Carcinoma, Squamous Cell↗
PubMed2004

GABAA receptor subunits identified in Paramecium by immunofluorescence confocal microscopy.

The presence of opioid, beta-adrenergic and cholinergic receptors has been demonstrated in ciliated protozoa, but little is known about gamma-aminobutyric acid (GABA) receptors. In this study we have analyzed the distribution of GABA(A)-type receptor subunits in Paramecium. Confocal laser microscopy using antibodies specific for alpha(1)-, alpha(2)-, alpha(3)-, alpha(6)-, beta(2/3)-, gamma(2)-, epsilon-, lambda-, and theta-subunits showed that most receptors are aggregated in clusters and are distributed both on cell surface and in the cytoplasm. The intensity of labelling of the alpha(6)-, beta(2/3)- and gamma(2)-subunits was more intense than the alpha(1)-, epsilon-, and theta-subunits, suggesting that the former are present in higher concentrations than the latter.

Animals↗
PubMed1997

Enhanced resolution of specific chromosome and nuclear regions by reflectance laser scanning confocal microscopy.

DNA sequences digested by HaeIII and reconstructed by in situ nick translation employing digoxigenin-labelled nucleotides are usually revealed either by horseradish peroxidase or FITC fluorescence. To obtain a significant improvement in terms of resolution, sensitivity and specificity, colloidal gold has been used instead of FITC (as the reporter molecule) to reveal the labelled DNA. Colloidal gold and propidium iodide were visualised by employing the reflectance mode and the 488-nm laser line of a confocal laser scanning microscope. In chromosomes, the fluorescent reaction pattern showed diffuse areas of labelling in which it was impossible to identify any specific kind of banding along the arms. In some chromosomes and, in particular, 1 and 9, a C-negative banding due to the negativity of the centromeric areas was seen. A more accurate localisation on chromosomes, including telomeric regions, often organised in spot pairs that resembled an R-like banding, was detected using 1-nm colloidal gold. A fine labelling was also demonstrated in nuclei, especially at their peripheral heterochromatin. The non-fading properties of colloidal gold combined with visualisation by reflectance confocal laser scanning microscopy demonstrated the possibility of obtaining a higher spatial resolution than when using conventional fluorophores or higher laser wavelength. This improved way to study the localization of HaeIII digestion sites in single chromosomes and in interphase nuclei made the reaction a valuable tool for the detection of antigens or of specific DNA sequences in biological preparations.

Cell Nucleus↗
PubMed2004

The musculature and associated innervation of adult and intramolluscan stages of Echinostoma caproni (Trematoda) visualised by confocal microscopy.

Gross anatomy of muscle and sensory/motor innervation of adult and intramolluscan developmental stages of Echinostoma caproni have been investigated to ascertain the organisation and the functional correlates of any stage-specific patterns of staining. Using indirect immunocytochemistry to demonstrate neuroactive substances and the phalloidin-fluorescence technique for staining myofibril F-actin, the muscle systems and aminergic and peptidergic innervation of daughter rediae, cercariae, metacercariae, and pre- and post-ovigerous adults were examined and compared using confocal scanning laser microscopy. A complex arrangement of specific muscle fibre systems occurs within the body wall (composed of circular, longitudinal and diagonal fibres), suckers (radial, equatorial, meridional), pharynx (radial, circular), gut caeca (mainly circular), cercarial tail (circular, pseudo-striated longitudinal), and ducts of the reproductive system (circular, longitudinal), presumed to serve locomotor, adhesive, alimentary and reproductive functions. Immunostaining for serotonin (5-HT) and FMRFamide-related peptides (FaRPs) was evident throughout the central (CNS) and peripheral (PNS) nervous systems of all stages, and use of dual-labelling techniques demonstrated separate neuronal pathways for 5-HT and FaRP in both CNS and PNS. FaRP expression in the innervation of the ootype wall was demonstrated only in post-ovigerous worms and not in pre-ovigerous worms, suggesting an involvement of FaRP neuropeptides in the process of egg assembly. Comparison of the present findings with those recorded for other digeneans suggests that muscle organisation and innervation patterns in trematodes are highly conserved.

Actins↗
PubMed1994

Laser scanning confocal microscopy and quantitative microscopy with a charge coupled device camera improve detection of human papillomavirus DNA revealed by fluorescence in situ hybridization.

Epithelial cervical CaSki, SiHa and HeLa cells containing respectively 600 copies of human papillomavirus (HPV) DNA type 16, 1-2 copies of HPV DNA type 16 and 10-50 copies of HPV DNA type 18 were used as model to detect different quantities of integrated HPV genome. The HPV DNA was identified on cell deposits with specific biotinylated DNA probes either by enzymatic in situ hybridization (EISH) or fluorescence in situ hybridization (FISH) involving successively a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and streptavidin-alkaline phosphatase complex or streptavidin-fluorescein isothiocyanate complex. With brightfield microscopy and EISH, hybridization spots were observed in CaSki and HeLa cells but hardly any in SiHa cells. With fluorescence microscopy and FISH, hybridization spots were clearly seen only on CaSki cell nuclei. In an attempt to improve the detection of low quantities of HPV DNA signals revealed by FISH, laser scanning confocal microscopy (LSCM) and quantitative microscopy with an intensified charge coupled device (CCD) camera were used. With both LSCM and quantitative microscopy, as few as 1-2 copies of HPV DNA were detected and found to be confined to cell nuclei counterstained with propidium iodide. Under Nomarski phase contrast, a good preservation of the cell structure was observed. With quantitative microscopy, differences in the number, size, total area and integrated fluorescence intensity of hybridization spots per nucleus were revealed between CaSki, SiHa and HeLa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Nucleus↗
PubMed1992

Coordination of hormone-induced calcium signals in isolated rat hepatocyte couplets: demonstration with confocal microscopy.

Excitable cells often display rapid coordination of hormone-induced intracellular calcium signals. Calcium elevations that begin in a single epithelial cell also may spread to adjacent cells, but coordination of hormone-induced signals among epithelial cells has not been described. We report the use of confocal microscopy to determine the inter- and intracellular distribution of cytosolic calcium in isolated rat hepatocyte couplets, an isolated epithelial cell system in which functional polarity is maintained. Both vasopressin and phenylephrine evoked sequential coordinated calcium signals in the couplets, even during cytosolic calcium oscillations. The coupling was abolished by closure of intercellular gap junction channels by treatment with octanol. These observations demonstrate that hormone-induced intracellular calcium signals are coordinated among hepatocytes and suggest that gap junction channels mediate this intercellular integration of tissue responsiveness.

Animals↗
PubMed1999

Terminal end of the human odontoblast process: a study using SEM and confocal microscopy.

Towards the middle of the eighteenth century, Tomes described the presence of membranous structures of cellular origin inside the dentinal tubules. Subsequent studies have been controversial regarding the terminal end of the odontoblasts. According to Fusayama, this cellular process reaches even the dentinal-enamel junction; others, such as Brännström, believed that this cellular process is present only in the inner third of the dentin. The aim of the present study was to determine the exact area up to which the terminal ends of the odontoblasts extend. With the aid of advanced confocal laser scanning microscopy (CLSM) cylindrical structures were demonstrated inside the tubules even in the absence of odontoblasts. This would confirm that the structures previously described as cellular processes can be identified with the lamina limitans of the peritubular dentin. High resolution field-emission scanning electron microscopy (FE-SEM) provided further evidence that tubular structures are only seen in the inner third of the dentin, towards the pulp.

Adult↗
PubMed1994

Digital confocal microscopy allows measurement and three-dimensional multiple spectral reconstruction of Neisseria gonorrhoeae/epithelial cell interactions in the human fallopian tube organ culture model.

A strategy for measuring Neisseria gonorrhoeae attachment and invasion in the human Fallopian tube organ culture (FTOC) model via computerized image analysis (CIA) combined with "digital" confocal microscopy (DCM) was tested. DCM on serial image stacks of fluorescent latex beads reduced out-of-focus light propagation in the Z-axis (p < 0.005) and improved the shape factor of lateral three-dimensional reconstructions of the beads (p < 0.001). Sections of tissue infected for 44 hr with piliated, Opa+ gonococci were stained with fluorescein-labeled monoclonal anti-gonococcal antibodies, rhodamine-labeled phalloidin, and Hoechst 33342. Serial images collected at identical focal planes for each fluorochrome were subjected to DCM. Epithelial cytoplasmic regions of interest defined by rhodamine-stained actin were superimposed on the corresponding fluorescein-stained and Hoechst-stained images. Fluorescent objects defined by gray-scale threshold were measured by computerized image analysis using different border treatments to differentiate attached from intracellular gonococci or count cell nuclei. Compared with raw images, measurement of DCM images was less dependent on threshold choice (p < 0.05). DCM augments conventional microscopy in removing out-of-focus light from fluorescent images, in reconstructing three-dimensional images, and in quantitatively differentiating extracellular from intracellular gonococci in a natural target tissue.

Antibodies, Monoclonal↗
PubMed1999

Confocal Microscopy Studies of Trypsin Immobilization on Porous Glycidyl Methacrylate Beads.

The immobilization of trypsin on porous glycidyl methacrylate (GMA-GDMA) beads has been investigated. In particular, the distribution within the beads of trypsin and of dextran used for hydrophilizing the bead surface prior to protein immobilization was investigated with confocal microscopy. For the system investigated, the fluorescence intensity profiles obtained when using borate buffer as an ambient solution displayed a distinct minimum at the center of the beads, irrespective of the observation depth. However, by reduction of the refractive index difference between the solution and the beads through the addition of glucose to the aqueous solution, artifacts relating to optical length differences could be reduced. For both low molecular weight fluorescein isothiocyanate (FITC), FITC-labeled trypsin, and FITC-labeled dextran, an essentially homogeneous distribution throughout the beads was observed. This simple "contrast matching" method seems therefore to be an interesting tool when investigating the distribution of immobilized protein in porous chromatography media. Copyright 1999 Academic Press.

Journal Article↗