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Watt W Webb

Publications and source records attributed to Watt W Webb.

35 records · Page 2Linked to original sources
PubMed2004

Focal volume confinement by submicrometer-sized fluidic channels.

Microfluidic channels with two lateral dimensions smaller than 1 microm were fabricated in fused silica for high-sensitivity single-molecule detection and fluorescence correlation spectroscopy. The effective observation volumes created by these channels are approximately 100 times smaller than observation volumes using conventional confocal optics and thus enable single-fluorophore detection at higher concentrations. Increased signal-to-noise ratios are also attained because the molecules are restricted to diffuse through the central regions of the excitation volume. Depending on the channel geometries, the effective dimensionality of diffusion is reduced, which is taken into account by simple solutions to diffusion models with boundaries. Driven by electrokinetic forces, analytes could be flowed rapidly through the observation volume, drastically increasing the rate of detection events and reducing data acquisition times. The statistical accuracy of single-molecule characterization is improved because all molecules are counted and contribute to the analysis. Velocities as high as 0.1 m/s were reached, corresponding to average molecular residence times in the observation volume as short as 10 micros. Applications of these nanofabricated devices for high-throughput, single-molecule detection in drug screening and genomic analysis are discussed.

Kinetics↗
PubMed2004

Spontaneous nucleotide exchange in low molecular weight GTPases by fluorescently labeled gamma-phosphate-linked GTP analogs.

Regulated guanosine nucleotide exchange and hydrolysis constitute the fundamental activities of low molecular weight GTPases. We show that three guanosine 5'-triphosphate analogs with BODIPY fluorophores coupled via the gamma phosphate bind to the GTPases Cdc42, Rac1, RhoA, and Ras and displace guanosine 5'-diphosphate with high intrinsic exchange rates in the presence of Mg(2+) ions, thereby acting as synthetic, low molecular weight guanine nucleotide exchange factors. The accompanying large fluorescence enhancements (as high as 12-fold), caused by a reduction in guanine quenching of the environmentally sensitive BODIPY dye fluorescence on protein binding, allow for real-time monitoring of this spontaneous nucleotide exchange in the visible spectrum with high signal-to-noise ratios. Binding affinities increased with longer aliphatic linkers connecting the nucleotide and BODIPY fluorophore and were in the 10-100 nM range. Steady-state and time-resolved fluorescence spectroscopy showed an inverse relationship between linker length and fluorescence enhancement factors and differences in protein-bound fluorophore mobilities, providing optimization criteria for future applications of such compounds as efficient elicitors and reporters of nucleotide exchange. EDTA markedly enhanced nucleotide exchange, enabling rapid loading of GTPases with these probes. Differences in active site geometries, in the absence of Mg(2+), caused qualitatively different reporting of the bound state by the different analogs. The BODIPY analogs also prevented the interaction of Cdc42 with p21 activated kinase. Together, these results validate the use of these analogs as valuable tools for studying GTPase functions and for developing potent synthetic nucleotide exchange factors for this important class of signaling molecules.

Boron Compounds↗
PubMed2004

Optical recording of action potentials with second-harmonic generation microscopy.

Nonlinear microscopy has proven to be essential for neuroscience investigations of thick tissue preparations. However, the optical recording of fast (approximately 1 msec) cellular electrical activity has never until now been successfully combined with this imaging modality. Through the use of second-harmonic generation microscopy of primary Aplysia neurons in culture labeled with 4-[4-(dihexylamino)phenyl][ethynyl]-1-(4-sulfobutyl)pyridinium (inner salt), we optically recorded action potentials with 0.833 msec temporal and 0.6 microm spatial resolution on soma and neurite membranes. Second-harmonic generation response as a function of change in membrane potential was found to be linear with a signal change of approximately 6%/100 mV. The signal-to-noise ratio was approximately 1 for single-trace action potential recordings but was readily increased to approximately 6-7 with temporal averaging of approximately 50 scans. Photodamage was determined to be negligible by observing action potential characteristics, cellular resting potential, and gross cellular morphology during and after laser illumination. High-resolution (micrometer scale) optical recording of membrane potential activity by previous techniques has been limited to imaging depths an order of magnitude less than nonlinear methods. Because second-harmonic generation is capable of imaging up to approximately 400 microm deep into intact tissue with submicron resolution and little out-of-focus photodamage or bleaching, its ability to record fast electrical activity should prove valuable to future electrophysiology studies.

Action Potentials↗
PubMed2003

In vivo multiphoton microscopy of deep brain tissue.

Although fluorescence microscopy has proven to be one of the most powerful tools in biology, its application to the intact animal has been limited to imaging several hundred micrometers below the surface. The rest of the animal has eluded investigation at the microscopic level without excising tissue or performing extensive surgery. However, the ability to image with subcellular resolution in the intact animal enables a contextual setting that may be critical for understanding proper function. Clinical applications such as disease diagnosis and optical biopsy may benefit from minimally invasive in vivo approaches. Gradient index (GRIN) lenses with needle-like dimensions can transfer high-quality images many centimeters from the object plane. Here, we show that multiphoton microscopy through GRIN lenses enables minimally invasive, subcellular resolution several millimeters in the anesthetized, intact animal, and we present in vivo images of cortical layer V and hippocampus in the anesthetized Thy1-YFP line H mouse. Microangiographies from deep capillaries and blood vessels containing fluorescein-dextran and quantum dot-labeled serum in wild-type mouse brain are also demonstrated.

Animals↗
PubMed2003

Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension.

Lipid bilayer membranes--ubiquitous in biological systems and closely associated with cell function--exhibit rich shape-transition behaviour, including bud formation and vesicle fission. Membranes formed from multiple lipid components can laterally separate into coexisting liquid phases, or domains, with distinct compositions. This process, which may resemble raft formation in cell membranes, has been directly observed in giant unilamellar vesicles. Detailed theoretical frameworks link the elasticity of domains and their boundary properties to the shape adopted by membranes and the formation of particular domain patterns, but it has been difficult to experimentally probe and validate these theories. Here we show that high-resolution fluorescence imaging using two dyes preferentially labelling different fluid phases directly provides a correlation between domain composition and local membrane curvature. Using freely suspended membranes of giant unilamellar vesicles, we are able to optically resolve curvature and line tension interactions of circular, stripe and ring domains. We observe long-range domain ordering in the form of locally parallel stripes and hexagonal arrays of circular domains, curvature-dependent domain sorting, and membrane fission into separate vesicles at domain boundaries. By analysing our observations using available membrane theory, we are able to provide experimental estimates of boundary tension between fluid bilayer domains.

Cell Membrane↗
PubMed2003

Direct measurement of Gag-Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy.

During retrovirus assembly, the polyprotein Gag directs protein multimerization, membrane binding, and RNA packaging. It is unknown whether assembly initiates through Gag-Gag interactions in the cytosol or at the plasma membrane. We used two fluorescence techniques-two-photon fluorescence resonance energy transfer and fluorescence correlation spectroscopy-to examine Rous sarcoma virus Gag-Gag and -membrane interactions in living cells. Both techniques provide strong evidence for interactions between Gag proteins in the cytoplasm. Fluorescence correlation spectroscopy measurements of mobility suggest that Gag is present in large cytosolic complexes, but these complexes are not entirely composed of Gag. Deletion of the nucleocapsid domain abolishes Gag interactions and membrane targeting. Deletion of the membrane-binding domain leads to enhanced cytosolic interactions. These results indicate that Gag-Gag interactions occur in the cytosol, are mediated by nucleocapsid domain, and are necessary for membrane targeting and budding. These methods also have general applicability to in vivo studies of protein-protein and -membrane interactions involved in the formation of complex macromolecular structures.

Animals↗
PubMed2003

Water-soluble quantum dots for multiphoton fluorescence imaging in vivo.

The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales.

Adipose Tissue↗
PubMed2003

Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy.

Microtubule (MT) ensemble polarity is a diagnostic determinant of the structure and function of neuronal processes. Here, polarized MT structures are selectively imaged with second-harmonic generation (SHG) microscopy in native brain tissue. This SHG is found to colocalize with axons in both brain slices and cultured neurons. Because SHG arises only from noninversion symmetric structures, the uniform polarity of axonal MTs leads to the observed signal, whereas the mixed polarity in dendrites leads to destructive interference. SHG imaging provides a tool to investigate the kinetics and function of MT ensemble polarity in dynamic native brain tissue structures and other subcellular motility structures based on polarized MTs.

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↗
PubMed2003

Spatial profiles of store-dependent calcium release in motoneurones of the nucleus hypoglossus from newborn mouse.

Hypoglossal motoneurones (HMN) are selectively damaged in both human amyotrophic lateral sclerosis (ALS) and corresponding mouse models of this neurodegenerative disease, a process which has been linked to their low endogenous Ca2+ buffering capacity and an exceptional vulnerability to Ca2+-mediated excitotoxic events. In this report, we investigated local Ca2+ profiles in low buffered HMNs by utilizing multiphoton microscopy, CCD imaging and patch clamp recordings in slice preparations. Bath application of caffeine induced highly localized Ca2+ release events, which displayed an initial peak followed by a slow 'shoulder' lasting several seconds. Peak amplitudes were paralleled by Ca2+-activated, apamin-sensitive K+ currents (IKCa), demonstrating a functional link between Ca2+ stores and HMN excitability. The potential involvement of mitochondria was investigated by bath application of CCCP, which collapses the electrochemical potential across the inner mitochondrial membrane. CCCP reduced peak amplitudes of caffeine responses and consequently IKCa, indicating that functionally intact mitochondria were critical for store-dependent modulation of HMN excitability. Taken together, our results indicate localized Ca2+ release profiles in HMNs, where low buffering capacities enhance the role of Ca2+-regulating organelles as local determinants of [Ca2+]i. This might expose HMN to exceptional risks during pathophysiological organelle disruptions and other ALS-related, cellular disturbances.

Amyotrophic Lateral Sclerosis↗
PubMed2003

Diffusion of nerve growth factor in rat striatum as determined by multiphoton microscopy.

Neurotrophins such as nerve growth factor (NGF) may be useful for treating diseases in the central nervous system; our ability to harness the potential therapeutic benefit of NGF is directly related to our understanding of the fate of exogenously supplied factors in brain tissue. We utilized multiphoton microscopy to quantify the dynamic behavior of NGF in coronal, 400- micro m thick, fresh rat brain tissue slices. We administered a solution containing bioactive rhodamine nerve growth factor conjugate via pressure injection and monitored the dispersion in the striatal region of the coronal slices. Multiphoton microscopy facilitated repeated imaging deep ( approximately 200 micro m) into tissue slices with minimal photodamage of tissue and photobleaching of label. The pressure injection paradigm approximated diffusion from a point source, and we therefore used the corresponding solution to the diffusion equation to estimate an apparent diffusion coefficient in brain tissue (D(b)(34 degrees C)) of 2.75 +/- 0.24 x 10(-7) cm(2)/s (average +/- SE). In contrast, we determined a corresponding free diffusion coefficient in buffered solution (D(f)(34 degrees C)) of 12.6 +/- 0.9 x 10(-7) cm(2)/s using multiphoton fluorescence photobleaching recovery. The tortuosity, defined as the square root of the ratio of D(f) to D(b), was 2.14 and moderate in magnitude.

Algorithms↗
PubMed2003

Nonlinear magic: multiphoton microscopy in the biosciences.

Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.

Biological Science Disciplines↗
PubMed2002

DNA fragment sizing by single molecule detection in submicrometer-sized closed fluidic channels.

The fabrication of fluidic channels with dimensions smaller than 1 microm is described and characterized in respect to their use for detection of individual DNA molecules. The sacrificial layer technique is used to fabricate these devices as it provides CMOS-compatible materials exhibiting low fluorescence background. It also allows creating microfluidics circuitry of submicrometer dimensions with great control. The small dimensions facilitate single molecule detection and minimize events of simultaneous passage of more than one molecule through the measurement volume. The behavior of DNA molecules inside these channels under an applied electrical field was first studied by fluorescence correlation spectroscopy using M13 double-stranded DNA. A linear relationship between the flow speed and applied electric field across the channel was observed. Speeds as high as 5 mm/s were reached, corresponding to only a few milliseconds of analysis time per molecule. The channels were then used to characterize a mixture of nine DNA fragments. Both the distribution and relative proportions of the individual fragments, as well as the overall concentration of the DNA sample, can be deduced from a single experiment. The amount of sample required for the analysis was approximately 10,000 molecules, or 76 fg. Other potential applications of these submicrometer structures for DNA analysis are discussed.

DNA↗
PubMed2002

Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) can provide a wealth of information about biological and chemical systems on a broad range of time scales (<1 micros to >1 s). Numerical modeling of the FCS observation volume combined with measurements has revealed, however, that the standard assumption of a three-dimensional Gaussian FCS observation volume is not a valid approximation under many common measurement conditions. As a result, the FCS autocorrelation will contain significant, systematic artifacts that are most severe with confocal optics when using a large detector aperture and aperture-limited illumination. These optical artifacts manifest themselves in the fluorescence correlation as an apparent additional exponential component or diffusing species with significant (>30%) amplitude that can imply extraneous kinetics, shift the measured diffusion time by as much as approximately 80%, and cause the axial ratio to diverge. Artifacts can be minimized or virtually eliminated by using a small confocal detector aperture, underfilled objective back-aperture, or two-photon excitation. However, using a detector aperture that is smaller or larger than the optimal value (approximately 4.5 optical units) greatly reduces both the count rate per molecule and the signal-to-noise ratio. Thus, there is a tradeoff between optimizing signal-to-noise and reducing experimental artifacts in one-photon FCS.

Biophysical Phenomena↗
PubMed2002

Precise nanometer localization analysis for individual fluorescent probes.

Calculation of the centroid of the images of individual fluorescent particles and molecules allows localization and tracking in light microscopes to a precision about an order of magnitude greater than the microscope resolution. The factors that limit the precision of these techniques are examined and a simple equation derived that describes the precision of localization over a wide range of conditions. In addition, a localization algorithm motivated from least-squares fitting theory is constructed and tested both on image stacks of 30-nm fluorescent beads and on computer-generated images (Monte Carlo simulations). Results from the algorithm show good agreement with the derived precision equation for both the simulations and actual images. The availability of a simple equation to describe localization precision helps investigators both in assessing the quality of an experimental apparatus and in directing attention to the factors that limit further improvement. The precision of localization scales as the inverse square root of the number of photons in the spot for the shot noise limited case and as the inverse of the number of photons for the background noise limited case. The optimal image magnification depends on the expected number of photons and background noise, but, for most cases of interest, the pixel size should be about equal to the standard deviation of the point spread function.

Fluorescent Dyes↗
PubMed2002

Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein.

Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma2P) increase rapidly at wavelengths below 800 nm, and the maximum sigma2P of LipDH is approximately 5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., approximately 410-490 nm and approximately 510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique.

Animals↗