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Results for “Confocal microscopy”

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At least 289 records · Page 16Linked to original sources
PubMed2002

Comparing telomere length of sister chromatids in human lymphocytes using three-dimensional confocal microscopy.

BACKGROUND: The length of the terminal sequences of linear chromosomes changes dynamically during cellular proliferation. A crucial element in the study of telomere-related regulation mechanisms is the ability to measure telomere lengths of individual chromosomes. Individual telomere lengths can be measured using digital imaging fluorescence microscopy-based techniques. We extended this method using confocal microscopy for the acquisition of three-dimensional (3D) images. Consequently, variations in measured signal intensities due to erroneous focusing are avoided. METHODS: We employed our 3D telomere sizing method to compare telomere lengths of sister chromatids within metaphase preparations from human lymphocytes. The samples were treated following a quantitative fluorescence in situ hybridization (Q-FISH) protocol using fluorescein isothiocyanate (FITC)-labeled telomeric peptidic nucleic acid (PNA) probes and propidium iodide (PI) counterstain. RESULTS: We demonstrated that the telomere lengths of two sister chromatids are not necessarily equal in human lymphocytes. Profound statistical analysis demonstrated significant differences in the distribution of the sister chromatid telomere lengths, but we were not able to prove a discrete distribution of telomere sister ratios. These telomere length differences were more apparent in older individuals. CONCLUSION: Whereas the majority of sister telomere pairs have equal lengths, surprisingly, a minority was significantly different in each individual studied. We are convinced that these observations are not linked to the methodology or the protocol applied. We suggest that a biological phenomenon might be involved.

Adult↗
PubMed2002

Identification of immunological reagents for use in the study of freshwater planarians by means of whole-mount immunofluorescence and confocal microscopy.

In recent years, interest in planarians as a model system for the study of metazoan regeneration, adult stem cell biology, and the evolution of metazoan body plans has been growing steadily. The availability of RNA interference (RNAi), BrdU-labeling of planarian stem cells, and thousands of planarian cDNA sequences soon to be released into public databases has opened planarians to molecular dissection. However, the successful application of large-scale RNAi-based screens, for example, will depend in part on the availability of markers to characterize the resulting phenotypes. Given the paucity of antibodies available for the study of planarian biology, we have screened various public and commercial antibody resources to identify immunoreagents capable of cross-reacting with planarian tissues. Here we report the identification and characterization of 33 such antibodies recognizing a wide variety of tissues in freshwater planarians.

Animals↗
PubMed2004

Radial migration of developing microglial cells in quail retina: a confocal microscopy study.

Microglial cells spread within the nervous system by tangential and radial migration. The cellular mechanism of tangential migration of microglia has been described in the quail retina but the mechanism of their radial migration has not been studied. In this work, we clarify some aspects of this mechanism by analyzing morphological features of microglial cells at different steps of their radial migration in the quail retina. Microglial cells migrate in the vitreal half of the retina by successive jumps from the vitreal border to progressively more scleral levels located at the vitreal border, intermediate regions, and scleral border of the inner plexiform layer (IPL). The cellular mechanism used for each jump consists of the emission of a leading thin radial process that ramifies at a more scleral level before retraction of the rear of the cell. Hence, radial migration and ramification of microglial cells are simultaneous events. Once at the scleral border of the IPL, microglial cells migrate through the inner nuclear layer to the outer plexiform layer by another mechanism: they retract cell processes, become round, and squeeze through neuronal bodies. Microglial cells use radial processes of s-laminin-expressing Müller cells as substratum for radial migration. Levels where microglial cells stop and ramify at each jump are always interfaces between retinal strata with strong tenascin immunostaining and strata showing weak or no tenascin immunoreactivity. When microglial cell radial migration ends, tenascin immunostaining is no longer present in the retina. These findings suggest that tenascin plays a role in the stopping and ramification of radially migrating microglial cells.

Animals↗
PubMed2006

Issues in confocal microscopy for quantitative FRET analysis.

Previously, we have carried out extensive quantitative analysis of Förster (or fluorescence) resonance energy transfer (FRET) data to show that polymeric IgA receptors and their ligands cluster in endocytic membranes in the process of sorting and trafficking in polarized cells. Here, we use a similar technique to assay the organization and distribution of another membrane-bound receptor: transferrin receptor (TFR) and its ligand, holo-transferrin (Tfn), while explaining the step-by-step measures to be taken for successful quantitative analysis of the FRET data. In particular, methodological issues in FRET quantitative imaging, such as spectral bleed-through and background correction, optimal selection of regions of interest, how to deal with outliers and pooling data and statistical analysis of FRET data, are addressed. Our results indicating a clustered organization of TFR-Tfn complexes fit the well-known homodimeric structure of TFR. These quantitative approaches can be adapted for other biological applications of FRET.

Algorithms↗
PubMed1996

In vivo confocal microscopy of human skin: a new design for cosmetology and dermatology.

In-depth exploration of cellular structures in living human skin in situ is possible with the tandem scanning microscope (TSM). However, the rigid design of the microscope limited observations to the arms, hands, and fingers. A mobile version allowing the investigation of any parts of the body has been designed. The head containing the Nipkow disk and the optical path were the only part saved from the original TSM. This prototype can be used to observe, in real time, the different skin structures down to a depth of 200 microns and to measure the thickness of the different layers with micron precision level. The hydration of the stratum corneum (SC) could be assessed. For example, lengthy immersion of the hand in water led to an increase in SC thickness without affecting that of the living epidermis. Occlusive patch tests also showed that water and, even more so. propylene glycol, led to transient swelling of the SC. In dermatology, the example of psoriasis illustrated the value of the TSM for describing, measuring, and assessing pathologic skin changes. The availability of this noninvasive method for observing changes with time in a given skin site should prove useful for monitoring treatment efficacy. This tool opens up new insight for the investigation of cutaneous pathophysiology.

Cosmetics↗
PubMedDate not supplied

Multiphoton confocal microscopy using a femtosecond Cr:forsterite laser.

With its output wavelength covering the infrared penetrating window of most biological tissues at 1,200-1,250 nm, the femtosecond Cr:forsterite laser shows high potential to serve as an excellent excitation source for the multiphoton fluorescence microscope. Its high output power, short optical pulse width, high stability, and low dispersion in fibers make it a perfect replacement for the currently widely used Ti:sapphire laser. In this paper, we study the capability of using a femtosecond Cr:forsterite laser in multiphoton scanning microscopy. We have performed the multiphoton excited photoluminescence spectrum measurement on several commonly used bioprobes using the 1,230 nm femtosecond pulses from a Cr:forsterite laser. Efficient fluorescence can be easily observed in these bioprobes through two-photon or three-photon excitation processes. These results will assist in the selection of dichroic beam splitter and band pass filters in a multiphoton microscopic system. We have also performed the autofluorescence spectrum measurement from chlorophylls in live leaves of the plant Arabidopsis thaliana excited by 1,230 nm femtosecond pulses from the Cr:forsterite laser. Bright luminescence from chlorophyll, centered at 673 and 728 nm, respectively, can be easily observed. Taking advantage of the bright two-photon photoluminescence from chlorophyll, we demonstrated the two-photon scanning paradermal and cross-sectional images of palisade mesophyll cells in live leaves of Arabidopsis thaliana.

Arabidopsis↗
PubMed1996

Coiled mechanoreceptors in Aplysia revealed by sensorin immunofluorescence and confocal microscopy.

Identified mechanosensory neurons of Aplysia are established model neurons for studies on learning and memory, and for examining responses to axonal injury. Although many characteristics of these sensory neurons have received intensive study, the nature of the peripheral mechanoreceptive endings remains unknown. Identification of a peptide, sensorin, specific in Aplysia for mechanosensory neurons, led to the development of an antibody which proved useful in studying the peripheral morphology of these neurons. Immunostaining for sensorin in tail body wall revealed that sensorin is present in peripheral arborizations. Examination of sensorin-positive fibers in the periphery revealed that they terminate as coiled structures in the muscle layer of the body wall. These coiled structures (approximately 0.5 microns diameter processes, 2-3 microns across the coil, approximately 60 microns long) run parallel to muscle fibers and have a pitch of about one turn per 4 microns. Sensorin immunostaining was particularly intense in varicosities, both along peripheral fibers and along the coiled structure. The localization of sensorin suggests that it may be released peripherally where it could have various paracrine and/or autocrine neuromodulatory actions.

Animals↗
PubMed2001

Reliability and reproducibility of corneal endothelial image analysis by in vivo confocal microscopy.

BACKGROUND: The aim of our study was to evaluate the reliability and reproducibility of in vivo slit-scanning corneal microscopy by endothelial analysis. METHODS: We examined 12 normal eyes of 12 persons. We analysed intraobserver differences in group I and interobserver differences in group II by manual and automatic evaluation of endothelial images. We also compared the methods of analysis in the two groups. We observed endothelial density, mean cell area, coefficient of variation and number of cells within the frame. RESULTS: In group I we found no significant difference between the observed parameters either by automatic or manual analysis. In group II no significant difference between the two observers was found using automatic analysis. On manual analysis, however, we observed a significant difference between endothelial density and the number of evaluated cells (P=0.0034 and P=0.0028). We observed a significant difference (P<0.001) between automatic and manual analysis concerning each parameter, with a rather tight linear correlation (Pearson correlation between 0.76 and 0.94). CONCLUSION: Automatic endothelial analysis was reliable and well reproducible in both--intraobserver and interobserver--groups. By manual evaluation, the clinical significance of interobserver differences can be disregarded. The differences between automatic and manual methods of analysis can be traced back to measurement technical reasons. We observed tight linear correlation between parameters. The data can be described well by linear regression. In vivo slit-scanning corneal microscopy may be an alternative to specular microscopic analyses for clinical use.

Adolescent↗
PubMed1999

A digital three-dimensional atlas of the honeybee antennal lobe based on optical sections acquired by confocal microscopy.

We present a digital atlas of the glomeruli in the antennal lobe of the honeybee, Apis mellifera, accessible to the scientific community via the Internet. The atlas allows the identification of glomeruli in preparations in which the glomeruli can be recognized, be it in sections or in whole-mounts. The high resolution of the anatomical data upon which the atlas is based and its electronic form should prove to be an important tool for anyone involved in the study of the honeybee antennal lobe. Its accessibility via the Internet is a step towards interactive and freely accessible databases of animal brains.

Animals↗