Biomedical subjects

Warren R Zipfel

Publications and source records attributed to Warren R Zipfel.

14 recordsLinked to original sources

PubMed2006

Achieving uniform mixing in a microfluidic device: hydrodynamic focusing prior to mixing.

We describe a microfluidic mixer that is well-suited for kinetic studies of macromolecular conformational change under a broad range of experimental conditions. The mixer exploits hydrodynamic focusing to create a thin jet containing the macromolecules of interest. Kinetic reactions are triggered by molecular diffusion into the jet from adjacent flow layers. The ultimate time resolution of these devices can be restricted by premature contact between co-flowing solutions during the focusing process. Here, we describe the design and characterization of a mixer in which hydrodynamic focusing is decoupled from the diffusion of reactants, so that the focusing region is free from undesirable contact between the reactants. Uniform mixing on the microsecond time scale is demonstrated using a device fabricated by imprinting optical-grade plastic. Device characterization is carried out using fluorescence correlation spectroscopy (FCS) and two-photon microscopy to measure flow speeds and to quantify diffusive mixing by monitoring the collisional fluorescence quenching, respectively. Criteria for achieving microsecond time resolution are described and modeled.

Microfluidics↗

PubMed2005

Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution.

Water-soluble quantum dots (qdots) are now being used in life sciences research to take advantage of their bright, easily excited fluorescence and high photostability. Although the frequent erratic blinking and substantial dark (never radiant) fractions that occur in all available qdots may interfere with many applications, these properties of individual particles in biological environments had not been fully evaluated. By labeling Qdot-streptavidin with organic dyes, we were able to distinguish individual dark and bright qdots and to observe blinking events as qdots freely diffused in aqueous solution. Bright fractions were measured by confocal fluorescence coincidence analysis (CFCA) and two-photon cross-correlation fluorescence correlation spectroscopy (FCS). The observed bright fractions of various preparations were proportional to the ensemble quantum yields (QYs), but the intrinsic brightness of individual qdots was found to be constant across samples with different QYs but the same emission wavelengths. Increasing qdots' illuminated dwell time by 10-fold during FCS did not change the fraction of apparently dark qdots but did increase the detected fraction of blinking qdots, suggesting that the dark population does not arise from millisecond blinking. Combining CFCA with wide-field imaging of arrays of qdots localized in dilute agarose gel, the blinking of qdots was measured across five orders of magnitude in time: approximately 0.001-100 s. This research characterizes photophysical pathologies of qdots in biologically relevant environments rather than adhered on dielectric surfaces and describes methods that are useful for studying various bioapplicable nanoparticles.

Biophysical Phenomena↗

PubMed2005

Use of multiphoton imaging for studying cell migration in the mouse.

We describe a method for studying cell motility in the living mouse using multiphoton microscopy. The procedure consists of mouse anesthesia, labeling of target cells with enhanced green fluorescent protein by infection with recombinant adenovirus, implantation of beads carrying chemoattractant, preparation of the mouse for imaging, and imaging of individual cell motions via multiphoton microscopy. Two-photon fluorescence excitation of enhanced green fluorescent protein allows visualization of cells within the dermis, whereas second harmonic generation (a non-linear scattering process) allows a simultaneous detailed definition of the dermis structure.

Animals↗

PubMed2004

Interpreting second-harmonic generation images of collagen I fibrils.

Fibrillar collagen, being highly noncentrosymmetric, possesses a tremendous nonlinear susceptibility. As a result, second-harmonic generation (SHG) microscopy of collagen produces extremely bright and robust signals, providing an invaluable tool for imaging tissue structure with submicron resolution. Here we discuss fundamental principles governing SHG phase matching with the tightly focusing optics used in microscopy. Their application to collagen imaging yields several biophysical features characteristic of native collagen structure: SHG radiates from the shell of a collagen fibril, rather than from its bulk. This SHG shell may correspond to the supporting element of the fibril. Physiologically relevant changes in solution ionic strength alter the ratio of forward-to-backward propagating SHG, implying a resulting change in the SHG shell thickness. Fibrillogenesis can be resolved in immature tissue by directly imaging backward-propagating SHG. Such findings are crucial to the design and development of forthcoming diagnostic and research tools.

Algorithms↗

PubMed2004

Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis.

We have found that two-photon fluorescence imaging of nicotinamide adenine dinucleotide (NADH) provides the sensitivity and spatial three-dimensional resolution to resolve metabolic signatures in processes of astrocytes and neurons deep in highly scattering brain tissue slices. This functional imaging reveals spatiotemporal partitioning of glycolytic and oxidative metabolism between astrocytes and neurons during focal neural activity that establishes a unifying hypothesis for neurometabolic coupling in which early oxidative metabolism in neurons is eventually sustained by late activation of the astrocyte-neuron lactate shuttle. Our model integrates existing views of brain energy metabolism and is in accord with known macroscopic physiological changes in vivo.

Animals↗

PubMed2004

Multiphoton microscopy guides neurotrophin modification with poly(ethylene glycol) to enhance interstitial diffusion.

Brain-derived neurotrophic factor (BDNF) is a promising therapeutic agent for the treatment of neurodegenerative diseases. However, the limited distribution of this molecule after administration into the brain tissue considerably hampers its efficacy. Here, we show how multiphoton microscopy of fluorescently tagged BDNF in brain-tissue slices provides a useful and rapid screening method for examining the diffusion of large molecules in tissues, and for studying the effects of chemical modifications-for example, conjugating with polyethylene glycol (PEG)-on the diffusion constant. This single variable, obtained by monitoring short-term diffusion in real time, can be effectively used for rational drug design. In this study on fluorescently tagged BDNF and BDNF-PEG, we identify slow diffusion as a major contributing factor to the limited penetration of BDNF, and demonstrate how chemical modification can be used to overcome this barrier.

Animals↗

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↗

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

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↗

PubMed2003

Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum.

Calcium is a second messenger in virtually all cells and tissues. Calcium signals in the nucleus have effects on gene transcription and cell growth that are distinct from those of cytosolic calcium signals; however, it is unknown how nuclear calcium signals are regulated. Here we identify a reticular network of nuclear calcium stores that is continuous with the endoplasmic reticulum and the nuclear envelope. This network expresses inositol 1,4,5-trisphosphate (InsP3) receptors, and the nuclear component of InsP3-mediated calcium signals begins in its locality. Stimulation of these receptors with a little InsP3 results in small calcium signals that are initiated in this region of the nucleus. Localized release of calcium in the nucleus causes nuclear protein kinase C (PKC) to translocate to the region of the nuclear envelope, whereas release of calcium in the cytosol induces translocation of cytosolic PKC to the plasma membrane. Our findings show that the nucleus contains a nucleoplasmic reticulum with the capacity to regulate calcium signals in localized subnuclear regions. The presence of such machinery provides a potential mechanism by which calcium can simultaneously regulate many independent processes in the nucleus.

Active Transport, Cell Nucleus↗

PubMed2002

Regulation of the type III InsP(3) receptor by InsP(3) and calcium.

It has been proposed that the inositol 1,4,5-trisphosphate receptor (InsP(3)R) type III acts as a trigger for InsP(3)-mediated calcium (Ca(2+)) signaling, because this InsP(3) isoform lacks feedback inhibition by cytosolic Ca(2+). We tested this hypothesis in RIN-m5F cells, which express predominantly the type III receptor. Extracellular ATP increases Ca(2+) in these cells, and we found that this effect is independent of extracellular Ca(2+) but is blocked by the InsP(3)R antagonist heparin. There was a dose-dependent increase in the number of cells responding to ATP and two-photon flash photolysis of caged-Ca(2+) heightened the sensitivity of RIN-m5F cells to this increase. These findings provide evidence that Ca(2+) increases the sensitivity of the InsP(3)R type III in intact cells and supports the idea that this isoform can act as a trigger for hormone-induced Ca(2+) signaling.

Adenosine Triphosphate↗