Biomedical subjects

Paul W Wiseman

Publications and source records attributed to Paul W Wiseman.

14 recordsLinked to original sources

PubMed2006

Probing the integrin-actin linkage using high-resolution protein velocity mapping.

Cell migration is regulated in part by the connection between the substratum and the actin cytoskeleton. However, the very large number of proteins involved in this linkage and their complex network of interactions make it difficult to assess their role in cell migration. We apply a novel image analysis tool, spatio-temporal image correlation spectroscopy (STICS), to quantify the directed movements of adhesion-related proteins and actin in protrusions of migrating cells. The STICS technique reveals protein dynamics even when protein densities are very low or very high, and works in the presence of large, static molecular complexes. Detailed protein velocity maps for actin and the adhesion-related proteins alpha-actinin, alpha5-integrin, talin, paxillin, vinculin and focal adhesion kinase are presented. The data show that there are differences in the efficiency of the linkage between integrin and actin among different cell types and on the same cell type grown on different substrate densities. We identify potential mechanisms that regulate efficiency of the linkage, or clutch, and identify two likely points of disconnect, one at the integrin and the other at alpha-actinin or actin. The data suggests that the efficiency of the linkage increases as actin and adhesions become more organized showing the importance of factors that regulate the efficiency in adhesion signaling and dynamics.

3T3 Cells↗

PubMed2006

A guide to accurate fluorescence microscopy colocalization measurements.

Biomolecular interactions are fundamental to the vast majority of cellular processes, and identification of the major interacting components is usually the first step toward an understanding of the mechanisms that govern various cell functions. Thus, statistical image analyses that can be performed on fluorescence microscopy images of fixed or live cells have been routinely applied for biophysical and cell biological studies. These approaches measure the fraction of interacting particles by analyzing dual color fluorescence images for colocalized pixels. Colocalization algorithms have proven to be effective, although the dynamic range and accuracy of these measurements has never been well established. Spatial image cross-correlation spectroscopy (ICCS), which cross-correlates spatial intensity fluctuations recorded in images from two detection channels simultaneously, has also recently been shown to be an effective measure of colocalization as well. Through simulations, imaging of fluorescent antibodies adsorbed on glass and cell measurements, we show that ICCS performs much better than standard colocalization algorithms at moderate to high densities of particles, which are often encountered in cellular systems. Furthermore, it was found that the density ratio between the two labeled species of interest plays a major role in the accuracy of the colocalization analysis. By applying a direct and systematic comparison between the standard, fluorescence microscopy colocalization algorithm and spatial ICCS, we show regimes where each approach is applicable, and more importantly, where they fail to yield accurate results.

Algorithms↗

PubMed2006

Measurement of monomer-oligomer distributions via fluorescence moment image analysis.

We present higher-order moment analysis of fluorescence intensity fluctuations from individual laser scanning microscopy images applied to study monomer-oligomer distributions. We demonstrate that the number densities and brightness ratios of a mixed population of monomers and oligomers can be determined by analyzing higher-order moments of the fluorescence intensity fluctuations from individual images for specific ranges of densities and particle brightness ratios. Computer simulations and experiments with fluorescent microspheres and cells were performed to illustrate the detection limits and accuracy of this statistical approach. The simulation results show that the concentration of the dimer or oligomer population should be less than or equal to the monomeric concentration for the method to provide accurate results, and that the upper density detection limit of the population of monomers is one order-of-magnitude higher than the concentration of the oligomers. We implemented this technique to resolve two populations of fluorescent microspheres with different brightness ratios and we also applied the moment-analysis method to examine the distribution of aggregation states of PDGF-beta receptors in human fibroblast cells. The method was able to resolve a tetrameric population of the PDGF-beta receptors relative to the background distribution of nonspecifically bound fluorophore.

Cells, Cultured↗

PubMed2006

k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics.

We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope. In contrast to r-space correlation techniques, we show kICS can recover accurate dynamics even in the presence of complex fluorophore photobleaching and/or "blinking". Furthermore, these quantities can be calculated without nonlinear curve fitting, or any knowledge of the beam radius of the exciting laser. The number densities calculated by kICS are less sensitive to spatial inhomogeneity of the fluorophore distribution than densities measured using image correlation spectroscopy. We use simulations as a proof-of-principle to show that number densities and transport coefficients can be extracted using this technique. We present calibration measurements with fluorescent microspheres imaged on a confocal microscope, which recover Stokes-Einstein diffusion coefficients, and flow velocities that agree with single particle tracking measurements. We also show the application of kICS to measurements of the transport dynamics of alpha5-integrin/enhanced green fluorescent protein constructs in a transfected CHO cell imaged on a total internal reflection fluorescence microscope using charge-coupled device area detection.

Animals↗

PubMed2006

Membrane lateral diffusion and capture of CFTR within transient confinement zones.

The cystic fibrosis transmembrane conductance regulator (CFTR) channel interacts with scaffolding and other proteins that are expected to restrict its lateral movement, yet previous studies have reported predominantly free diffusion. We examined the lateral mobility of CFTR channels on live baby hamster kidney cells using three complementary methods. Channels bearing an extracellular biotinylation target sequence were labeled with streptavidin conjugated with fluorescent dyes (Alexa Fluor 488 or 568) or quantum dots (qDot605). Fluorescence recovery after photobleaching and image correlation spectroscopy of the dye-labeled channels revealed a significant immobile population ( approximately 50%), which was confirmed by direct single particle tracking (SPT) of qDot605-labeled CFTR. Adding 10 histidine residues at the C-terminus of CFTR to mask the postsynaptic density 95, Discs large, ZO-1 (PDZ) binding motif abolished its association with EBP50/NHERF1, reduced the immobile fraction, and increased mobility. Other interactions that are not normally detected on this timescale became apparent when binding of PDZ domain proteins was disrupted. SPT revealed that CFTR(His-10) channels diffuse randomly, become immobilized for periods lasting up to 1 min, and in some instances are recaptured at the same location. The impact of transient confinement on the measured diffusion using the three fluorescence techniques were assessed using computer simulations of the biological experiments. Finally, the impact of endosomal CFTR on mobility measurements was assessed by fluorescence correlation spectroscopy. These results reveal unexpected features of CFTR dynamics which may influence its ion channel activity.

Animals↗

PubMed2006

Investigating membrane protein dynamics in living cells.

Live cell imaging is a powerful tool for understanding the function and regulation of membrane proteins. In this review, we briefly discuss 4 fluorescence-microscopy-based techniques for studying the transport dynamics of membrane proteins: fluorescence-correlation spectroscopy, image-correlation spectroscopy, fluorescence recovery after photobleaching, and single-particle and (or) molecule tracking. The advantages and limitations of each approach are illustrated using recent studies of an ion channel and cell adhesion molecules.

Animals↗

PubMed2005

Two-photon fluorescent microlithography for live-cell imaging.

Fluorescent dyes added to UV-cure resins allow the rapid fabrication of fluorescent micropatterns on standard glass coverslips by two-photon optical lithography. We use this lithographic method to tailor fiduciary markers, focal references, and calibration tools, for fluorescence and laser scanning microscopy. Fluorescent microlithography provides spatial landmarks to quantify molecular transport, cell growth and migration, and to compensate for focal drift during time-lapse imaging. We show that the fluorescent patterned microstructures are biocompatible with cultures of mammalian cell lines and hippocampal neurons. Furthermore, the high-relief topology of the lithographed substrates is utilized as a mold for poly(dimethylsiloxane) stamps to create protein patterns by microcontact printing, representing an alternative to the current etching techniques. We present two different applications of such protein patterns for localizing cell adhesion and guidance of neurite outgrowth.

Animals↗

PubMed2005

Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy.

We present an extensive investigation of the accuracy and precision of temporal image correlation spectroscopy (TICS). Using simulations of laser scanning microscopy image time series, we investigate the effect of spatiotemporal sampling, particle density, noise, sampling frequency, and photobleaching of fluorophores on the recovery of transport coefficients and number densities by TICS. We show that the recovery of transport coefficients is usually limited by spatial sampling, while the measurement of accurate number densities is restricted by background noise in an image series. We also demonstrate that photobleaching of the fluorophore causes a consistent overestimation of diffusion coefficients and flow rates, and a severe underestimation of number densities. We derive a bleaching correction equation that removes both of these biases when used to fit temporal autocorrelation functions, without increasing the number of fit parameters. Finally, we image the basal membrane of a CHO cell with EGFP/alpha-actinin, using two-photon microscopy, and analyze a subregion of this series using TICS and apply the bleaching correction. We show that the photobleaching correction can be determined simply by using the average image intensities from the time series, and we use the simulations to provide good estimates of the accuracy and precision of the number density and transport coefficients measured with TICS.

Algorithms↗

PubMed2005

Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy.

We present a comprehensive study of the accuracy and dynamic range of spatial image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS). We use simulations to model laser scanning microscopy imaging of static subdiffraction limit fluorescent proteins or protein clusters in a cell membrane. The simulation programs allow us to control the spatial imaging sampling variables and the particle population densities and interactions and introduce and vary background and counting noise typical of what is encountered in digital optical microscopy. We systematically calculate how the accuracy of both image correlation methods depends on practical experimental collection parameters and characteristics of the sample. The results of this study provide a guide to appropriately plan spatial image correlation measurements on proteins in biological membranes in real cells. The data presented map regimes where the spatial ICS and ICCS provide accurate results as well as clearly showing the conditions where they systematically deviate from acceptable accuracy. Finally, we compare the simulated data with standard confocal microscopy using live CHO cells expressing the epidermal growth factor receptor fused with green fluorescent protein (GFP/EGFR) to obtain typical values for the experimental variables that were investigated in our study. We used our simulation results to estimate a relative precision of 20% for the ICS measured receptor density of 64 microm(-2) within a 121 x 98 pixel subregion of a single cell.

Algorithms↗

PubMed2005

Measuring fast dynamics in solutions and cells with a laser scanning microscope.

Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds-to-seconds timescale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.

Algorithms↗

PubMed2005

Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure.

Images obtained with a laser-scanning microscope contain a time structure that can be exploited to measure fast dynamics of molecules in solution and in cells. The spatial correlation approach provides a simple algorithm to extract this information. We describe the analysis used to process laser-scanning images of solutions and cells to obtain molecular diffusion constant in the microsecond to second timescale.

Algorithms↗

PubMed2005

Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells.

We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laser-scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of alpha-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mum/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mum/min) and diffusion of interacting alpha5 integrin/enhanced cyan fluorescent protein and alpha-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.

Actinin↗

PubMed2004

Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microscopy.

Image correlation microscopy methodology was extended and used to determine retrospectively the density, dynamics and interactions of alpha5-integrin in migrating cells. Alpha5-integrin is present in submicroscopic clusters containing 3-4 integrins before it is discernibly organized. The integrin in nascent adhesions, as identified by the presence of paxillin, is approximately 1.4 times more concentrated, approximately 4.5 times more clustered and much less mobile than in surrounding regions. Thus, while integrins are clustered throughout the cell, they differ in nascent adhesions and appear to initiate adhesion formation, despite their lack of visible organization. In more mature adhesions where the integrin is visibly organized there are approximately 900 integrins microm(-2) (about fivefold higher than surrounding regions). Interestingly, alpha5-integrin and alpha-actinin, but not paxillin, reside in a complex throughout the cell, where they diffuse and flow together, even in regions where they are not organized. During adhesion disassembly some integrins diffuse away slowly, alpha-actinin undergoes a directed movement at speeds similar to actin retrograde flow (0.29 microm min(-1)), while all of the paxillin diffuses away rapidly.

Actinin↗

PubMed2003

Isolation of bright aggregate fluctuations in a multipopulation image correlation spectroscopy system using intensity subtraction.

Image correlation spectroscopy allows sensitive measurement of the spatial distribution and aggregation state of fluorescent membrane macro molecules. When studying a single population system (i.e., aggregates of similar brightness), an accurate measure can be made of the aggregate number per observation area, but this measurement becomes much more complex in a distributed population system (i.e., bright and faint aggregates). This article describes an alternate solution that involves extraction of the bright aggregate population information. This novel development for image correlation spectroscopy, termed intensity subtraction analysis, uses sequential uniform intensity subtraction from raw confocal images. Sequential intensity subtraction results in loss of faint aggregate fluctuations that are smaller in magnitude than fluctuations due to the brightest aggregates. The resulting image has correlatable fluctuations originating from only the brightest population, permitting quantification of this population's distribution and further cross-correlation measurements. The feasibility of this technique is demonstrated using fluorescent microsphere images and biological samples. The technique is further used to examine the spatial distribution of a plasma-membrane-labeled fluorescent synthetic ganglioside, and to cross-correlate this probe with various membrane markers. The evidence provided demonstrates that bright aggregates of the fluorescent ganglioside are associated with clathrin-coated pits, membrane microvilli, and detergent-resistant membranes.

Animals↗