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Results for “Microscopy, Fluorescence, Multiphoton”

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PubMed2006

Visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells.

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria.

Animals↗

PubMed2005

In vivo imaging of lymphocyte trafficking.

Over the past decades, intravital microscopy (IVM), the imaging of cells in living organisms, has become a valuable tool for studying the molecular determinants of lymphocyte trafficking. Recent advances in microscopy now make it possible to image cell migration and cell-cell interactions in vivo deep within intact tissues. Here, we summarize the principal techniques that are currently used in IVM, discuss options and tools for fluorescence-based visualization of lymphocytes in microvessels and tissues, and describe IVM models used to explore lymphoid and non-lymphoid organs. The latter will be introduced according to the physiologic itinerary of developing and differentiating T and B lymphocytes as they traffic through the body, beginning with their development in bone marrow and thymus and continuing with their migration to secondary lymphoid organs and peripheral tissues.

Animals↗

PubMed2004

Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes.

Biological membranes decorated with suitable contrast agents give rise to nonlinear optical signals such as two-photon fluorescence and harmonic up-conversion when illuminated with ultra-short, high-intensity pulses of infrared laser light. Microscopic images based on these nonlinear contrasts were acquired at video or higher frame rates by scanning a focused illuminating spot rapidly across neural tissues. The scan engine relied on an acousto-optic deflector (AOD) to produce a fast horizontal raster and on corrective prisms to offset the AOD-induced dispersion of the ultra-short excitation light pulses in space and time. Two membrane-bound derivatives of the green fluorescent protein (GFP) were tested as nonlinear contrast agents. Synapto-pHluorin, a pH-sensitive GFP variant fused to a synaptic vesicle membrane protein, provided a time-resolved fluorescent read-out of neurotransmitter release at genetically specified synaptic terminals in the intact brain. Arrays of dually lipidated GFP molecules at the plasma membrane generated intense two-photon fluorescence but no detectable second-harmonic power. Comparison with second-harmonic generation by membranes stained with a synthetic styryl dye suggested that the genetically encoded chromophore arrangement lacked the orientational anisotropy and/or dipole density required for efficient coherent scattering of the incident optical field.

Animals↗

PubMed2006

Imaging the embryonic kidney.

The structural and functional development of the permanent mammalian kidney or metanephros is a complex process involving the actions of thousands of gene products, complex cell movements and tissue patterning in three dimensions (3D). This review focuses on the recent advances made in imaging technology, processing and analysis combined with mouse genetics and the generation of protein-reporter mice which has enabled us to monitor the development and movement of defined cell populations within the developing kidney in 3D and over time (4D).

Animals↗

PubMed2006

Influence of nanoencapsulation on human skin transport of flufenamic acid.

The effect of the inclusion of flufenamic acid in poly(lactide-co-glycolide) nanoparticles on the transport of flufenamic acid into excised human skin was investigated. Penetration and permeation data were acquired using two different in vitro test systems: the Saarbrucken penetration model, where the skin acts as its own receptor medium, and the Franz diffusion cell, where the receptor medium is a buffer solution. For the stratum corneum, no differences were found between nanoencapsulated and free drug. Drug accumulation in the deeper skin layers and drug transport across human epidermis were slightly delayed for the nanoencapsulated drug compared to the free drug after shorter incubation times (<12 h). In contrast, after longer incubation times (>12 h), the nanoencapsulated drug showed a statistically significantly enhanced transport and accumulation (p < 0.05). Additionally, nanoencapsulated flufenamic acid was visualized by multiphoton fluorescence microscopy. Particles were found homogeneously distributed on the skin surface and within the dermatoglyphs, but no nanoparticles were detected within or between the corneocytes.

Administration, Cutaneous↗

PubMed2000

Single granule pH cycling in antigen-induced mast cell secretion.

The pH cycling of individual granules in secreting (serotonin-loaded) mast cells is quantitatively examined using multicolor multiphoton fluorescence microscopy. A typical exocytosis event consists of maximal calcium rise at time zero, granule alkalization a few seconds later and, finally, complete contents release at a fraction of a second after alkalization. Membrane fusion is either transient, as indicated by subsequent granule reacidification, or 'full', as indicated by a granule disappearance with a collapse of its membrane into the plasma membrane. The relative frequency of these two coexisting behaviors (the 'kiss-to-collapse' ratio) is approximately 2:1. A typical transiently fusing granule experiences multiple alkalization/acidification cycles after addition of exogenous antigen. Between recycling granules, coalescence events are frequent, with 80% resulting in a collapse of the formed granule complex to the plasma membrane. The full dynamics of secretion encompass a complex combination of these granule activities.

Acridine Orange↗

PubMed2004

Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos.

We demonstrate the association of third-harmonic generation (THG) microscopy and particle image velocimetry (PIV) analysis as a novel functional imaging technique for automated micrometer-scale characterization of morphogenetic movements in developing embryos. Using a combined two-photon-excited fluorescence and THG microscope, we characterize the optical properties of Drosophila embryos and show that sustained THG imaging does not perturb sensitive developmental dynamics. Velocimetric THG imaging provides a quantitative description of the dynamics of internal structures in unstained wild-type and mutant embryos.

Algorithms↗

PubMed2004

Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy.

Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three- to fivefold resolution increase in 4Pi-fluorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at approximately 80 nm axial resolution.

Equipment Design↗

PubMed2004

Fluorescence imaging for monitoring the colocalization of two single molecules in living cells.

The interaction, binding, and colocalization of two or more molecules in living cells are essential aspects of many biological molecular processes, and single-molecule technologies for investigating these processes in live cells, if successfully developed, would become very powerful tools. Here, we developed simultaneous, dual-color, single fluorescent molecule colocalization imaging, to quantitatively detect the colocalization of two species of individual molecules. We first established a method for spatially correcting the two full images synchronously obtained in two different colors, and then for overlaying them with an accuracy of 13 nm. By further assessing the precision of the position determination, and the signal/noise and signal/background ratios, we found that two single molecules in dual color can be colocalized to within 64-100 nm (68-90% detectability) in the membrane of cells for GFP and Alexa633. The detectability of true colocalization at the molecular level and the erroneous inclusion of incidental approaches of two molecules as colocalization have to be compromised at different levels in each experiment, depending on its purpose. This technique was successfully demonstrated in living cells in culture, monitoring colocalization of single molecules of E-cadherin fused with GFP diffusing in the plasma membrane with single molecules of Alexa633 conjugated to anti-E-cadherin Fab externally added to the culture medium. This work established a benchmark for monitoring the colocalization of two single molecules, which can be applied to wide ranges of studies for molecular interactions, both at the levels of single molecules and collections of molecules.

Fluorescent Dyes↗

PubMed2003

Imaging the effects of anti-angiogenic treatments.

It is clear that various imaging modalities have given keen insight into the molecular mechanisms involved in anti-angiogenic treatments. A key to the advancement of anti-angiogenic therapy is not only the discovery of new drugs and treatments, but the analysis of the specific modes of action of these compounds in order to produce the next generation with greater effectiveness. While existing clinical methods incorporate the analysis of serum and urine to measure angiogenic factors, an imaging technique monitoring the effectiveness of anti-angiogenic therapy would be a convenient, noninvasive, cost effective technique to aid in treatment planning and disease management.

Angiogenesis Inhibitors↗

PubMed2004

Acidosis-induced apoptosis in human and porcine heart.

BACKGROUND: Acidosis-mediated injury to cardiac myocytes during surgery may lead to progressive heart failure. The nature of this injury, although not well defined, may be caused by induction of apoptosis in cardiac myocytes. We applied fluorescence imaging and biochemical techniques to assess apoptosis in cardiac myocytes excised from human patients and porcine subjects maintained on cardiopulmonary bypass to demonstrate the relationship between acidosis and apoptosis. METHODS: Multiphoton microscopy was used to image fluorescence signals generated in myocytes deep within atrial and ventricular biopsies for identification of apoptotic changes. The biopsies, obtained during cardiac surgery, were subjected to ex vivo or in vivo acidosis. Proapoptotic markers such as exposure of phosphatidyl serine, cytochrome c, apoptotic protease-activating factor-1, and caspase-3 were identified using fluorescence-based imaging and biochemical assays. RESULTS: Within 30 minutes of storage in low pH (<7) buffers, apoptosis was detected in human atrial samples, the severity of which correlated well with low pH. Apoptosis was also detected in atrial and ventricular biopsy samples obtained from three porcine subjects maintained on cardiopulmonary bypass and undergoing 110 minutes of aortic cross-clamp and 10 minutes of reperfusion, in which the cardiac pH was 6.36, 7.14, and 7.48. The apoptosis level detected in postacidotic reperfused cardiac tissue was pH dependent and approximately threefold greater than the precross-clamp levels. CONCLUSIONS: Using fluorescence multiphoton microscopy and biochemical techniques we have assessed a direct correlation between low pH and induction of apoptosis in cardiac samples obtained both from human patients undergoing cardiac surgery and porcine subjects maintained on cardiopulmonary bypass simulating cardiac surgery.

Acidosis↗

PubMed2004

Four-dimensional multiphoton confocal microscopy: the new frontier in cellular imaging.

This paper reviews new developments in microscopy that combine gene transfer technology, multiphoton confocal fluorescence microscopy, live cell imaging and digital imaging techniques that provide unique insights into the complex physiological processes involved in tissue function at the cellular and subcellular level. The evolution of this novel, new technology is discussed with particular attention to earlier achievements in noninvasive ocular surface imaging. The practical basis of confocal microscopy, multiphoton confocal fluorescence microscopy, and the vital fluorescent labeling of cells in living tissues are also discussed. Additionally, one application using retroviral gene transfer to express enhanced green fluorescent protein in living wound healing fibroblasts is presented as an example of how living biology can be studied in situ in four dimensions (x, y, z, time).

Journal Article↗

PubMed2004

Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions.

We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM). The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) alpha in carcinoma cells for both live- and fixed-cell experiments. The CXCR4-EGFP: PKCalpha-mRFP1 complex was found to be localized precisely to intracellular vesicles and cell protrusions when imaged by multiphoton fluorescence-FLIM. A comparison of the FRET efficiencies obtained using mRFP1-tagged regulatory domain or full-length PKCalpha as the acceptor revealed that PKCalpha, in the closed (inactive) form, is restrained from associating with the cytoplasmic portion of CXCR4. Live-cell FLIM experiments show that the assembly of this receptor:kinase complex is concomitant with the endocytosis process. This is confirmed by experimental evidence suggesting that the recycling of the CXCR4 receptor is increased on stimulation with phorbol ester and blocked on inhibition of PKC by bisindolylmaleimide. The EGFP-mRFP1 couple should be widely applicable, particularly to live-cell quantitative FRET assays.

Breast Neoplasms↗

PubMed2006

Multiphoton excitation of autofluorescence for microscopy of glioma tissue.

OBJECTIVE: Intraoperative detection of residual tumor tissue in glioma surgery remains an important challenge because the extent of tumor removal is related to the prognosis of the disease. Multiphoton excited fluorescence tomography of living tissues provides high-resolution structural and photochemical imaging at a subcellular level. In this conceptual study, we have used multiphoton microscopy and fluorescence lifetime imaging (4D microscopy) to image cultured glioma cell lines, solid tumor, and invasive tumor cells in an experimental mouse glioma model and human glioma biopsy specimens. MATERIAL AND METHODS: A laser imaging system containing a mode-locked 80 MHz titanium:sapphire laser with a tuning range of 710 to 920 nm, a scan unit, and a time correlated single photon counting board was used to generate autofluorescence intensity images and fluorescence lifetime images of cultured cell lines, experimental intracranial gliomas in mouse brain, and biopsies of human gliomas. RESULTS: Multiphoton microscopy of native tumor bearing brain provided structural images of the normal brain anatomy at a subcellular resolution. Solid tumor, the tumor-brain interface, and single invasive tumor cells could be visualized. Fluorescence lifetime imaging demonstrated significantly different decay of the fluorescent signal in tumor versus normal brain, allowing a clear definition of the tumor-brain interface based on this parameter. Distinct fluorescence lifetimes of endogenous fluorophores were found in different cellular compartments in cultured glioma cells. The analysis of the relationship between the laser excitation wavelength and the lifetime of excitable fluorophores demonstrated distinct profiles for cells of different histotypes. CONCLUSION: Multiphoton excited fluorescence of endogenous fluorophores allows structural imaging of tumor and central nervous system histo-architecture at a subcellular level. The analysis of the decay of the fluorescent signal within specific excitation volumes by fluorescent lifetime imaging discriminates glioma cells and normal brain, and the excitation/lifetime profiles may further allow differentiation of cellular histotypes. This technology provides a noninvasive optical tissue analysis that may potentially be applied to an intraoperative analysis of resection plains in tumor surgery.

Animals↗

PubMed2005

Self-assembled photoactive polyelectrolyte/perylene-diimide composites.

A new class of polyelectrolyte-surfactant (PE-surf) composites having potential applications as thin film organic semiconductors is introduced. These materials are comprised of cationic asymmetrically substituted perylene diimides and oppositely charged poly(acrylate) polyanions. Thin films of the composite materials are prepared by mixing and drop casting aqueous solutions of the two precursors onto appropriate substrates. The resulting materials yield photovoltages of >140 mV for approximately equal to 0.6 W/cm(2) illumination intensities, when incorporated in p-n heterojunction devices. Solution-phase spectra obtained from the PE-surf complexes exhibit excimer-like emission and evidence for formation of weakly coupled aggregates in the ground state. Wide-angle X-ray scattering data show the composite films are locally amorphous, while small-angle X-ray data are consistent with a mixture of polymorphic structures that incorporate planar PE-surf bilayers of 3.9-nm repeat distances. Images obtained by conventional far-field light microscopy and multiphoton-excited fluorescence microscopy (MPEFM) indicate that the films are heterogeneous, incorporating submicrometer sized clusters dispersed among much thinner film regions that also incorporate dye. Polarization-dependent MPEFM studies prove the clusters are semiorganized, yielding order parameters (s and P(4)) of 0.09 and 0.01 for in-plane alignment of the chromophores, consistent with a relatively high degree of disorder.

Journal Article↗

PubMedDate not supplied

Photobleaching of reduced nicotinamide adenine dinucleotide and the development of highly fluorescent lesions in rat basophilic leukemia cells during multiphoton microscopy.

Endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence provides an intrinsic indicator of the cellular metabolic state, but prolonged monitoring is limited by photobleaching and/or phototoxicity. Multiphoton excitation of NADH by ultrashort, 740-nm laser pulses provides a significant improvement over UV excitation by eliminating peripheral photobleaching; however, molecules within the subfemtoliter excitation volume remain susceptible. We have investigated the photophysical mechanisms responsible for multiphoton photobleaching of NADH in living cells to permit the imaging technique to be optimized. The loss of fluorescence because of multiphoton photobleaching was measured by repetitively imaging individual planes within rat basophilic leukemia cells. The photobleaching rate was proportional to the fourth power of the laser intensity. Based on these measurements, we propose a double-biphotonic, four-photon photobleaching mechanism and estimate the quantum yield of photobleaching of intracellular NADH to be 0.0073 +/- 0.0002 by this mechanism. In addition to photobleaching, the development of bright, punctate fluorescent lesions can also be observed. The frequency of lesion formation also increased approximately as the fourth power of the laser intensity after an intensity-dependent threshold number of images had been exceeded. The consequences for two-photon metabolic imaging are discussed.

Animals↗

PubMed2003

Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation.

Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer's disease and cancer.

Alzheimer Disease↗

PubMedDate not supplied

Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar.

Multiphoton microscopy has evolved into a powerful bioimaging tool in three dimensions. However, the ability to image biological specimens in-depth can be hindered by sample spherical aberration and scattering. These two phenomena can result in the degradation of image resolution and the loss of detected multiphoton signal. In this work, we use the correction collar (for cover glass thickness) associated with a water immersion objective in an attempt to improve multiphoton imaging. In the two samples we examined (human skin and rat tail tendon), we found that while the improvement in image resolution was not visible qualitatively, the measured axial fluorescence or second harmonic generation signal profiles indicate that the use of the correction collar can help to improve the detected multiphoton signals. The maximum increases are 36% and 57% for the skin (sulforhodamine B fluorescence) and tendon (second harmonic generation) specimens, respectively. Our result shows that for in-depth multiphoton imaging, the correction collar may be used to correct for spherical aberration. However, each tissue type needs to be examined to determine the optimal correction collar setting to be used.

Animals↗