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At least 1,603 records · Page 89Linked to original sources
PubMed1998

Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy.

Monomolecular films of polymerized dimethyl-bis[pentacosadiinoic-oxyethyl] ammonium bromide (EDIPAB) provide one- and two-photon excited fluorescence that is sufficiently high to quantify the axial resolution of 3-D fluorescence microscopes. When scanned along the optical axis, the fluorescence of these layers is bright enough to allow online observation of the axial response of these microscopes, thus facilitating alignment and fluorescence throughput control. The layers can be used for directly measuring and monitoring the axial response of 4Pi-confocal microscopes, as well as for their initial alignment and phase adjustment. The proposed technique has the potential to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Coverslips with EDIPAB-layers can be used as substrates for the cultivation of cells.

Journal Article↗
PubMed2003

Confocal total-internal-reflection fluorescence microscopy with a high-aperture parabolic mirror lens.

We present a theoretical study of a new total-internal-reflection fluorescence microscope for the detection of fluorescence at a water-glass interface. The system is designed for confocal imaging and spectroscopy of nanoparticles and single molecules. Focusing and fluorescence collection through standard glass coverslips is accomplished by a parabolic mirror lens. The large aperture of the element is used to excite fluorescence within the evanescent field of a diffraction-limited focus and to collect focal emission with high efficiency. Tight focusing and supercritical excitation reduce the detection volume for fluorescent analyte molecules well below that of an attoliter (10(-18) L), which can be advantageous for monitoring surface binding of single molecules without interference from fluorescence of the unbound bulk. Calculations of the electric fields in the focus region and simulated confocal imaging demonstrate the performance of the system.

Journal Article↗
PubMed2005

Spectral imaging and linear unmixing in light microscopy.

Fluorescence microscopy is an essential tool for modern biological research. The wide range of available fluorophores and labeling techniques allows the creation of increasingly complex multicolored samples. A reliable separation of the different fluorescence labels is required for analysis and quantitation, but it is complicated by the significant overlap of the emission spectra. This problem can be addressed on the acquisition and the processing side by the use of spectral imaging in conjunction with linear unmixing of the image data. This method allows the reliable separation of even strongly overlapping fluorescence signals and has become an important tool in colocalization and in FRET studies. In this chapter, the microscope techniques available for spectral imaging are presented and the theory of linear unmixing is explained. Possible limitations as well as approaches for image optimization are discussed to help to realize the full potential of this novel method. Biological applications that can be improved by spectral imaging and linear unmixing are presented.

Fluorescence Resonance Energy Transfer↗
PubMed2004

Correlation of syntaxin-1 and SNAP-25 clusters with docking and fusion of insulin granules analysed by total internal reflection fluorescence microscopy.

AIMS/HYPOTHESIS: The interaction of syntaxin-1 and SNAP-25 with insulin exocytosis was examined using the diabetic Goto-Kakizaki (GK) rat and a total internal reflection fluorescence (TIRF) imaging system. METHODS: Primary rat pancreatic beta cells were immunostained with anti-syntaxin-1A, anti-SNAP-25 and anti-insulin antibodies, and then observed by TIRF microscopy. The real-time image of GFP-labelled insulin granules motion was monitored by TIRF. RESULTS: The number of syntaxin-1A and SNAP-25 clusters, and the number of docked insulin granules on the plasma membrane were reduced in GK beta cells. When GK rats were treated with daily insulin injection for 2 weeks, the number of syntaxin-1 and SNAP-25 clusters was restored, along with the number of docked insulin granules. The infection of GK beta cells with Adex1CA SNAP-25 increased the number of docked insulin granules. TIRF imaging analysis demonstrated that the decreased number of fusion events from previously docked insulin granules in GK beta cells was restored when the number of docked insulin granules increased by insulin treatment or Adex1CA SNAP-25 infection. CONCLUSIONS/INTERPRETATION: There was a close correlation between the number of syntaxin-1 and SNAP-25 clusters and the number of docked insulin granules, which is associated with the fusion of insulin granules.

Animals↗
PubMed1998

Differentiation of muscle-specific proteins in chicken somites as studied by immunofluorescence microscopy.

Fluorescence microscopy of chicken cervical somites revealed that muscle-specific proteins began to appear at stage 11 (Hamburger and Hamilton numbering), and the onset of the expression of all the proteins examined in the present study had occurred by stage 17. Muscle proteins were classified into six groups according to the stage of their appearance. Since all these proteins were expressed before emergence of nerve fibers in myotomes, switching-on of their synthesis does not seem to require neuronal influence. However, since isoproteins other than adult muscle types disappeared and diversification of muscle fiber types occurred coordinately with the clustering of acetylcholine receptors in cervical muscles, switching-off of the synthesis of the nonadult isoforms might have been accelerated by the formation of functional neuromuscular junctions. The absence of nebulin and C-protein in early stages seems to indicate that these proteins are not required for the initial assembly of myofilaments and/or myofibrils. Further, this absence might be considered to facilitate exchangeabilities of proteins in nascent myofibrils, thereby changing the isoforms to adult types.

Animals↗
PubMed2006

Three-dimensional imaging of monogenoidean sclerites by laser scanning confocal fluorescence microscopy.

A nondestructive protocol for preparing specimens of Monogenoidea for both alpha-taxonomic studies and reconstruction of 3-dimensional structure is presented. Gomori's trichrome, a stain commonly used to prepare whole-mount specimens of monogenoids for taxonomic purposes, is used to provide fluorescence of genital spines, the copulatory organ, accessory piece, squamodisc, anchors, hooks, bars, and clamps under laser scanning confocal microscopy.

Animals↗
PubMed2003

Primary spherical aberration in two-color (two-photon) excitation fluorescence microscopy with two confocal excitation beams.

We study the effects of primary spherical aberration on the three-dimensional point spread function (PSF) of the two-color (two-photon) excitation (2CE) (2PE) fluorescence microscope with two confocal excitation beams that are separated by an angle theta. The two excitation wavelengths lambda1 and lambda2 are related to the single-photon excitation wavelength lambda(e) by: 1/lambda(e) = 1/lambda1 + 1/lambda2. The general case is considered where both focused beams independently suffer from spherical aberration. For theta = 0, pi/2, and pi, the resulting deterioration of the PSF structure is evaluated for different values of the spherical aberration coefficients via the Linfoot's criteria of fidelity, structural content, and correlation quality. The corresponding degradation of the peak 2CE fluorescence intensity is also determined. Our findings are compared with that of the 2PE fluorescence (lambda1 = lambda2) under the same aberration conditions. We found that the 2CE microscope is more robust against spherical aberration than its 2PE counterpart, with the pi/2 configuration providing the clearest advantage. The prospect of aberration correction in the two-beam 2CE microscope is also discussed.

Journal Article↗
PubMed2005

Probing the structure of supported membranes and tethered oligonucleotides by fluorescence interference contrast microscopy.

Fluorescence interference contrast microscopy (FLIC) is a powerful method to structurally characterize fluorescent objects with nanometer-scale resolution in the z direction. Here we use FLIC to characterize the water layer underlying supported membranes and membrane-tethered double-stranded oligonucleotides. FLIC measurements of supported membranes containing the lipid-anchored fluorescent dye DiI in both leaflets indicate the thickness of the water layer separating the solid support and the lower lipid leaflet is 1.3 +/- 0.2 nm. Addition of cobalt(II) chloride to a DiI-supported membrane quenches the fluorescence in the top leaflet of the supported membrane; FLIC measurements of this system precisely locate the DiI to the bottom leaflet. These experiments confirm the accuracy of the model and parameters used to determine the water layer thickness, demonstrate the ability to differentiate between fluorescent objects whose average position differs by approximately 1.9 nm, and provide a widely applicable method to test the resolution of other high-z-resolution fluorescent microscopies. FLIC measurements of Alexa-labeled double-stranded oligonucleotides tethered to a supported membrane indicate that the DNA double helix is oriented perpendicular to the surface. Complications that arise from uncertainly in the orientation of the fluorophore are discussed. Several improvements in FLIC methodology are described. These stringent tests of the resolution of FLIC and the ability to unambiguously determine fluorescent lipid distribution provide structural insight on assemblies at membrane interfaces and permit the detection of even subtle changes at such interfaces.

Membranes, Artificial↗
PubMed1987

Quantitative fluorescence microscopy on single capillaries: alpha-lactalbumin transport.

We have extended the use of a microscope densitometric technique [Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H495-H505, 1983] to measure the solute permeability coefficients (Pa) of fluorescently labeled solutes in single perfused capillaries of frog mesentery. The method enables the transcapillary flux of solutes larger than 10,000 mol wt to be measured under conditions where the forces that determine both solute and water flows across the capillary wall are known. The Pa for alpha-lactalbumin (mol wt 14,176, Stokes radius 2.02 nm) increased from a mean value of 2.1 X 10(-6) cm/s when capillary pressure was 3.0 cmH2O (no net filtration) to greater than 4.0 X 10(-6) cm/s when capillary pressure was 15 cmH2O. Taking a value of 0.35 for the solvent drag reflection coefficient for alpha-lactalbumin, we conclude that the increased solute flux represents solvent drag through a water pathway with a hydraulic conductivity of 3.6 X 10(-7) cm X s-1 X cmH2O-1. Our data conforms to the hypothesis that alpha-lactalbumin is transported across the capillary wall by restricted diffusion and solvent drag in a pathway that carries 90% of the transcapillary water flow (the principle water pathway). In vitro and in vivo calibration experiments have been carried out to test the assumption that the measured fluorescent light intensity is proportional to the number of fluorescent molecules in the measuring window of the photometer.

Animals↗
PubMed2001

Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules.

Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.

Actin Cytoskeleton↗
PubMed2005

Localization and movement of mineral oil in plants by fluorescence and confocal microscopy.

Fluorescence and confocal laser scanning microscopy were explored to investigate the movement and localization of mineral oils in citrus. In a laboratory experiment, fluorescence microscopy observation indicated that when a 'narrow' distillation fraction of an nC23 horticultural mineral oil was applied to adaxial and opposing abaxial leaf surfaces of potted orange [Citrus x aurantium L. (Sapindales: Rutaceae)] trees, oil penetrated steadily into treated leaves and, subsequently, moved to untreated petioles of the leaves and adjacent untreated stems. In another experiment, confocal laser scanning microscopy was used to visualize the penetration into, and the subsequent cellular distribution of, an nC24 agricultural mineral oil in C. trifoliata L. seedlings. Oil droplets penetrated or diffused into plants via both stomata and the cuticle of leaves and stems, and then moved within intercellular spaces and into various cells including phloem and xylem. Oil accumulated in droplets in intercellular spaces and within cells near the cell membrane. Oil entered cells without visibly damaging membranes or causing cell death. In a field experiment with mature orange trees, droplets of an nC23 horticultural mineral oil were observed, by fluorescence microscopy, in phloem sieve elements in spring flush growth produced 4-5 months and 16-17 months after the trees were sprayed with oil. These results suggest that movement of mineral oil in plants is both apoplastic via intercellular spaces and symplastic via plasmodesmata. The putative pattern of the translocation of mineral oil in plants and its relevance to oil-induced chronic phytotoxicity are discussed.

Biological Transport↗
PubMed1997

FLUORESCENCE MICROSCOPY OF LIVING PLANT CELLS.

Since its inception, light microscopy has shown the elegance and subtlety with which function is expressed in the form of the cells, tissues, and organs of the plant. Recently, light microscopy has seen a resurgence in use fueled by advances in microscope design and computer-based image analysis. The structural resolution afforded by static, fixed samples is being increasingly supplemented by approaches using fluorescent analogs and selective fluorescent indicators, which visualize the dynamic processes in living, functioning cells. This review describes some of these approaches and discusses how they are taking us a step closer to viewing the intricate complexity with which plants organize and regulate their functions down to the subcellular level.

Journal Article↗