Thèses sur le sujet « Image Correlation Spectroscopy »

This thesis studies optical microscopy based high order autocorrelation approaches for measuring molecular aggregation of fluorescently labeled particles in fluid systems. As the particles randomly diffuse into and out of the volume defined by the focus of a confocal laser beam illumination, the collected fluorescence intensity fluctuates. Fluorescence Correlation Spectroscopy (FCS) and Image Correlation Spectroscopy (ICS) have been used as methods which analyse temporal and spatial intensity fluctuations, and provide quantitative information of the molecular transport processes. Theoretical expressions for the high order autocorrelation function magnitudes for a non-interactive model are derived as well as their fitting equations for single- and multicomponent diffusion.<br>We present an experimental verification of the model applied to simple systems. Solutions of fluorescent microspheres of well-defined size have been imaged using confocal laser scanning microscopy. It has been shown that translational diffusion coefficients were not very sensitive to molecular size dispersion, which made a first order autocorrelation approach to be somewhat ineffective for dealing with multicomponent systems. We demonstrate that the number densities of a mixture of two fluorescent particles can be determined analyzing the higher order autocorrelation function magnitudes. Numerical simulations have been analyzed for testing the experimental tools we use. The technique outlined may be developed to detect and characterize aggregates of fluorescently labeled biological molecules such as membrane proteins and cell surface receptors. Such quantitative aggregation measurements, therefore, can provide information about the mechanism of intercellular signaling which is believed to depend on the oligomerization of cell membrane protein receptors.

Fluorescence correlation spectroscopy has become a standard technique for modern biophysics and single molecule spectroscopy research. Here is presented a novel widefield extension of the established single-point technique. Flow in microfluidic devices was used as a model system for microscopic motion and through widefield fluorescence correlation spectroscopy flow profiles were mapped in three dimensions. The technique presented is shown to be more tolerant to low signal strength, allowing image data with signal-to-noise values as low as 1.4 to produce accurate flow maps as well as utilizing dye-labeled single antibodies as flow tracers. With proper instrumentation flows along the axial direction can also be measured. Widefield fluorescence correlation spectroscopy has also been utilized to produce super-resolution confocal microscopic images relying on the single-molecule microsecond blinking dynamics of fluorescent silver clusters. A method for fluorescence modulation signal extraction as well as synthesis of several novel noble metal fluorophores is also presented.

This thesis is about the use and development of new fluorescence correlation techniques to measure the dynamics, number density, and aggregation state of fluorescently labelled proteins in living cells. An extensive investigation of the accuracy and precision of temporal image correlation spectroscopy (TICS) is presented first. Using computer simulations of laser scanning microscopy image time series, the effect of spatiotemporal sampling, particle density, noise, and photobleaching of fluorophores on the recovery of transport coefficients and number densities by TICS is investigated. It is shown that photobleaching of the fluorophore can significantly perturb TICS measurements. The theory of k-space image correlation spectroscopy (kICS) is then developed in detail. kICS involves Fourier transforming each image in an image series, and then correlating these transforms, in time. This technique measures the number density, diffusion coefficient, and velocity of fluorescently labelled macromolecules in a cell membrane. 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." 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. The wide applicability of the technique is shown by imaging cells transfected with fluorescent protein, and quantum dot (QD) labelled cells on two-photon and total internal reflection fluorescence microscopes. Finally, kICS is used to measure immune T cell receptor (TCR) clustering in live cells using QDs as labels. kICS quantifies the aggregation of TCR by two different approaches. The first uses spatial intensity fluctuat<br>Cette thèse est à propos de l'utilisation et du développement de nouvelles techniques de corrélation de fluorescence afin de mesurer les dynamiques, la densité, et l'état d'agrégation de protéines marquées par fluorescence dans des cellules vivantes. Une vaste recherche de la précision de la spectroscopie temporelle par corrélations d'images (STCI) est premièrement présentée. En utilisant des simulations informatiques à balayage de laser de séries d'images de microscopie, l'effet de l'échantillon spatiotemporelle, densité de particules, le bruit, la fréquence de prises d'échantillons, et le photoblanchiment des fluorophores lors de la mesure des coefficients de transport et la densité par STCI sont examinés. C'est démontré que le photoblanchiment des fluorophores perturbent de manière significative les mesures STCI. La théorie de la spectroscopie de corrélations d'images d'espace-k (CIEk) est développée en détail. CIEk implique la transformation Fourier de chaque image dans une série d'images, et ensuite de faire la corrélation de ces transformations, dans le temps. Cette technique mesure la densité, le coefficient de diffusion, et la vélocité de macromolécules marquées fluorescentes dans une membrane de cellules. Contrairement aux techniques de corrélation espace-r, nous démontrons que CIEk peut mesurer les dynamiques précises, même en présence de complexes photoblanchiments de fluorophores et/ou "clignotement." Nous utilisons des simulations comme une preuve de principes pour démontrer que les densités et les coefficients de transport peuvent être extraits en utilisant cette technique. Nous présentons des mesures d'étalonnage avec des microsphères fluorescentes imagées sur un microscope confocal, qui mesurent la diffusion de coefficients Stokes-Einstein, et les vitesses d'écroulement qui correspondent avec les mesures de suivi de particules uniques. L'application vaste de cette technique est démontrée avec d

The object of this thesis is to develop a new extension of Image Correlation Spectroscopy (ICS) that can measure velocity vectors for flowing protein populations in living cells. This new technique, called Spatio-Temporal Image Correlation Spectroscopy (STICS), allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) from fluorescence microscopy image time series of fluorescently labeled cellular proteins via monitoring of the time evolution of the full space-time correlation function of the intensity fluctuations. By using filtering in Fourier space to remove frequencies associated with immobile or slow components, it is possible to measure the protein transport even in the presence of a large fraction of immobile species that are static in the image series. The STICS method can generate complete transport maps of proteins within sub-regions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements, and it can be applied to any type of fluorescence microscopy image time series. This thesis presents the background theory, computer simulations, and analysis of measurements on fluorescent microspheres and fixed cell samples to demonstrate proof of principle, capabilities, and limitations of the method. Visible fluorescent proteins (VFPs) were used to label a variety of the proteins involved in cell-to-extra-cellular-matrix adhesions, including focal adhesion kinase, paxillin, alpha-actinin, alpha5-integrin, talin, vinculin and actin. Various fusion protein pairs were transfected in living cells and imaged using both laser scanning microscopy and total internal reflection microscopes. Using STICS analysis, co-transport maps of proteins were generated within protruding sub-regions of the basal membrane. The new space time image correlation method can probe the mechanistic details of the hypothesized molecular clutch that regulates the extra cellular matrix/cytoskeletal interactions during migration. The technique was also applied to mapping fluid flow in migrating keratocytes in order to elucidate the role that fluid flow plays in migrating cells.

k-space image correlation spectroscopy (kICS) is a recently developed technique that can be used to measure the transport dynamics and number density of fluorescently labeled molecules in living cells, while being completely unbiased for transport measurements by fluorophore photobleaching or blinking. Whereas the precision of fluorescence correlation spectroscopy (FCS) and temporal image correlation spectroscopy (TICS) have been investigated in detail, no such study exists for kICS. In this thesis, we present a thorough characterization of the accuracy and precision of kICS for measurements of 2D diffusion over a range of imaging frame rates, spatial dimensions, and particle distributions. We use computer simulations as a primary tool to vary simulated imaging conditions and data analysis parameters, and thereby obtain a statistical description of kICS error.We find that kICS measurements of diffusion are consistently biased low for image regions smaller than ~100 μm2 and we examine two alternative methods for correcting the bias. We also report the surprising discovery that kICS can measure 2D particle diffusion that is at least ten times faster than can be measured with other methods that compute correlations between successive image frames; this is possible because kICS measures long-range correlations that persist after particles have exited the specific volume of the laser focus where they were found in a previous frame. In addition, we show that unlike FCS or TICS, kICS measurements are accurate even when analyzing highly nonuniform particle distributions, as would be found after local release or photoactivation of fluorescently-tagged biological molecules. Finally, we describe a method of estimating the uncertainty from a single kICS measurement of diffusion, which is useful when measurements cannot easily be repeated.We use experimental fluorescence microscopy image series of diffusing microspheres to confirm that bias in kICS depends on the size of the image region analyzed, and we test the two methods of correcting the bias. We also apply kICS to measure the diffusion of membrane biomolecules tagged with blinking quantum dots in living cells, and compare the results with single particle tracking analyses of the same data.<br>La spectroscopie par corrélation d'images dans l'espace vectoriel (kICS) est une nouvelle technique qui permet de mesurer la dynamique du transport moléculaire ainsi que le nombre de molécules fluorescentes à l'intérieur de cellules vivantes et de leurs membranes. Cette technique présente l'avantage de fournir des mesures de dynamique de transport non biaisées par le photo-blanchiment et le clignotement des fluorophores. Alors que la précision des techniques de spectroscopie de corrélation de fluorescence (FCS) et de spectroscopie par corrélation temporelle d'images (TICS) a déjà été étudiée en détail, aucune étude n'existe concernant la technique kICS. Dans cette thèse, je présente une caractérisation approfondie de l'exactitude et de la précision de kICS sur des mesures de diffusion 2D pour une large plage de fréquences d'acquisition d'image, de tailles d'image et de distributions spatiales du nombre de particules. J'ai principalement utilisé des simulations par ordinateur afin de pouvoir modifier les conditions d'acquisition et d'analyse d'image et ainsi d'obtenir une description statistique des erreurs de kICS.Il ressort de mes analyses que les mesures de diffusions effectuées par kICS donnent des valeurs systématiquement trop faibles lorsque les régions imagées ont une surface de moins de ~100 μm2, j'ai donc étudié deux méthodes alternatives afin de corriger ce biais. J'ai aussi pu constaté que kICS permet de mesurer des diffusions de particules en 2D pour des vitesses au moins dix fois supérieures à celles des diffusions mesurées par des méthodes corrélant des images successives. Ceci est rendu possible par le fait que kICS mesure des corrélations à longue distance qui persistent même lorsque la particule quitte le point focal d'illumination du laser qu'elle occupait lors de la prise de l'image précédente. En outre, je montre que, contrairement à FCS ou TICS, les mesures effectuées par kICS sont exactes y compris lorsque des régions ayant une distribution spatiale de particules fortement hétérogène sont analysées, cas rencontré notamment lors de la libération locale ou la photo-activation de molécules biologiques marquées par un fluorophore. Finalement, je décris une méthode permettant d'estimer l'incertitude de la mesure à partir d'une seule donnée de diffusion obtenue par kICS. Ceci est particulièrement utile par exemple lorsqu'il est difficile de répéter une acquisition.J'ai utilisé des mesures expérimentales de diffusion de microsphères pour confirmer que le biais de kICS dépend bien de la taille de la région analysée et j'ai testé l'efficacité des deux méthodes proposées pour corriger ce biais. J'ai également employé kICS afin de mesurer la diffusion dans des cellules vivantes de biomolécules membranaires marquées par point quantique et j'ai comparé ces résultats avec ceux effectués sur les mêmes images par suivi de particules isolées.

The object of this thesis is to present work done using spatio-temporal image correlation spectroscopy (STICS), a technique that uses fluorescence intensity fluctuations in a microscopy image time series to calculate a complete space-time correlation function in order to measure transport dynamics in cells. The time evolution of this correlation function gives information on the magnitude and direction of a flow of fluorescent particles sampled in the image series. First, a new application of STICS to plant cell biology is shown. In dividing plant cells, delivery of new cell wall material to the forming cell plate requires intricate coordination of secretory vesicle trafficking and delivery. In this work, STICS is used to measure vesicle dynamics during plant cell division. It was discovered that vesicle transport to the plane of division occurs in three phases, each with its characteristic flow patterns and range of velocities, which directly reflect the rate of growth of the forming cell plate. The second part of this thesis presents the extension of the STICS technique to a third spatial dimension. The development of this new technique, called 3D STICS, allows the study of transport dynamics in three dimensions, which is more relevant in tissues and non adherent cells which are inherently 3D. Computer simulations were performed to test the accuracy and precision of the technique under a range of parameters such as particle density of immobile and moving populations; and number of images, velocity and resolution in the third spatial dimension. A comparison between values of velocities in a 2D plane recovered using STICS and its new 3D version is also presented.<br>L'objet de cette thèse est de présenter des travaux faits à l'aide de la spectroscopie par corrélation spatiotemporelle d'images (STICS), une technique qui utilise les fluctuations d'intensité dans une série d'images capturées à l'aide d'un microscope par fluorescence pour calculer la fonction complète de corrélation spatiotemporelle, et ainsi mesurer la dynamique du transport de protéines à l'intérieur de cellules vivantes. L'évolution temporelle de cette fonction de corrélation donne de l'information sur la direction et la vitesse d'un flot de particules fluorescentes présentes dans la série d'images. Tout d'abord, une nouvelle application de la technique en biologie végétale est présentée. Lors de la division cellulaire végétale, le transport du matériel membranaire nécessaire à la formation de la plaque cellulaire requiert une grande précision dans la coordination du transport et de la livraison des vésicules de sécrétion. Dans cette thèse, STICS est utilisée pour mesurer la dynamique de ces vésicules pendant la division cellulaire végétale. Les résultats obtenus révèlent l'existence de trois phases dans le transport des vésicules de sécrétion au site de division cellulaire, chacune présentant une échelle de vitesse et des motifs de mouvement caractéristiques qui se reflètent dans le taux de croissance de la plaque cellulaire. Dans un deuxième temps, le développement de STICS pour inclure l'analyse de la troisième dimension spatiale est présenté. Cette nouvelle technique, appelée STICS 3D, permet l'étude de dynamiques en trois dimensions, ce qui est plus pertinent que la version deux-dimensionnelle pour les tissus et les cellules non adhérentes, qui ont un environnement intrinsèquement 3D. Des simulations par ordinateur ont été effectuées pour déterminer l'exactitude, la précision et les limites de la technique pour un éventail de paramètres comme la vitesse, le nombre d'images et la résolution dans la troisième dimension spatiale ainsi que la densité des populations immobiles et en mouvement. Une comparaison entre les résultats obtenus avec STICS et la nouvelle version 3D de la technique est également présentée.

This thesis presents the application of k-space Image Correlation Spec- troscopy (kICS) to the analysis of fluorescence microscopy image time series for the measurement of particle diffusion in heterogeneous membranes, composed of micro- domains. The extension, testing and application of kICS for such measurements is developed both in silico with simulation and with in vivo cellular experiments.Connections between kICS analysis and other existing fluorescent microscopy techniques used in the study of heterogeneous membranes, such as single particle tracking (SPT) and spot vary Fluorescence Correlation Spectroscopy (FCS) are introduced. This is followed by the development of kICS theory of fluorescent particle diffusion within a heterogeneous two dimensional (2D) environment. Two possible membrane heterogeneities, isolated lipid micro-domains and actin proximal meshwork, are considered separately. The emergent models suggest that the kICS correlation function (CF) can be fit by a sum of two Gaussians in the case of particle diffusion in the presence of isolated micro-domains. These two fit components, called 'fast' and 'slow', with the fast associated with the rapid decay of the kICS CF at small spatial frequencies due to particle motion on large spatial scales outside domains while the slow component refers to the confined particle motion on large spatial frequencies or small spatial scales in domains. On the other hand, the meshwork confinement is well fit with a single Gaussian model for the analysis of kICS CF. These models suggest that the exponents and amplitudes of the fits embed the characteristic system parameters such as diffusion coefficients outside and inside domains, the partitioning rates, micro-domains radii and mesh pore size.Furthermore, systematic simulations to study different confinement scenarios were conducted and the calculated kICS correlation functions were fit and the output interpreted for recovery of self system parameters. The characterization of the simulated data suggests that kICS CFs exhibit various confinement dependent features, such as decays due to effective slow and fast dynamics populations and effective domain sizes. The in silico characterization of different confinement scenarios, suggests a connection between the apparent measured confinement properties, and the set system defining parameters. We explore the range and limits where confinement effects can be detected and accurately measured by kICS analysis. Possible systematic errors in the values of the fit extracted parameters due to background noise is discussed with possible alternative solutions.Finally, we apply this extension of kICS to the heterogeneous membrane en- vironment to explore the confinement dynamics of GPI-GFP anchored proteins in the basal plasma membrane of COS-7 cells. We employ a novel labelling approach of GPI-GFP using anti-GFP-Alexa594 and image the protein in COS-7 cell mem- branes with TIRF microscopy. Cells were exposed to enzymatic treatments, using the Cholesterol Oxidase (COase) and Sphingomyelinase (SMase), in order to dis- rupt membrane domains and change GPI-GFP confinement dynamics. We observe that GPI-GFP mobility and the effective domain size measured correlates with the enzymatic exposure time. We attribute it to the conversion of the membrane domain constituents, cholesterol and sphingomyelin, upon the enzymatic reactions, leading to membrane domain that are effectively larger and leakier. Finally, we conclude with possible improvements and future directions.<br>La thèse qui suit est a propos de l'adaptation de la technique de la spectroscopie par la corrélation des images dans l'espace de Fourier, appelle kICS. La nouveauté consiste en utilisation de kICS pour analyser les séries temporelles d'images fluorescentes afin de caractériser la diffusion des particules en présence des membranes hétérogénes, composées de micro-domaines.Tout d'abord, une parallèle est exposée entre l'analyse fondée sur kICS pro- posé ci-dessus et d'autres techniques de microscopie à fluorescence existantes et utilisées dans l'étude des membranes hétérogénes. Ensuite, on expose le développement de la théorie de kICS dans les cas de la diffusion des particules fluorescentes dans un espace hétérogène bidimensionnel (2D). Les deux hétérogénéités membranaires possibles, micro-domaines lipidiques isolés et le réseau de l'actine proximale, sont considérés séparément. Les modèles émergents suggèrent que la fonction de corrélation de kICS doit être caractérisé par une somme de deux Gaussiennes dans le cas de la dynamique des particules en présence de micro-domaines isolés. Ces deux éléments, appelés 'rapide' et 'lent', représentent les composantes dynamiques a deux échelles d'espace différentes. La rapide est associé à la décroissance rapide de la fonction de corrélation de kICS à petites fréquences spatiales dues au mouvement des particules sur de grandes échelles spatiales. La composante lente réfère au mouvement des particules confinées à des petites échelles spatiales, observées sur de grandes fréquences spatiales de kICS. D'autre part, la fonction de corrélation de kICS due au confinement par le réseau du cytosquelette peut être caractérise par unique décroissance Gaussienne. Ces modèles suggèrent que les exposants et les amplitudes obtenus par la caractérisation de la fonction kICS dépend des paramètres caractéristiques du système tels que les coefficients de diffusion à l'extérieur et à l'intérieur de domaines, les taux de migration de particules vers intérieur ou extérieur de micro-domaines ou des tailles de porosités du réseaux du cytosquelette.Les études systématiques par les simulations des scénarios différents de confinement et leurs effets sur la fonction de corrélation de kICS ont été explorés. La caractérisation des données simulées suggèrent que les fonctions de corrélation ont des caractéristiques qui dépendent de confinement et les propriétés spécifiques, tels que la dynamique des populations lents et rapides et la tailles effective de micro-domaines. La caractérisation des scénarios de confinement différents, représente les liens entre les propriétés apparentes mesurées de confinement, et un ensemble de paramètres définissant hétérogénéité. Nous explorons les limites pour lesquelles des effets de confinement ne sont pas observées dans la fonction de corrélation kICS. Les éventuelles erreurs systématiques dans les valeurs des paramètres extraits à cause du bruit de fond est discuté avec des possibles solutions. Finalement, nous utilisons l'analyse afin d'explorer la dynamique de confinement de la protéine ancrée à GPI-GFP dans la membrane plasmique basale des cellules COS-7. Nous explorons une approche nouvelle de la conjugaison entre le GPI-GFP et les anti-GFP-Alexa594 et imagé par la microscopie TIRF. Les cellules ont été exposées à des traitements enzymatiques, par Coase et SMase, afin de perturber domaines membranaires et changer la dynamique de confinement de GPI-GFP. Les réactions enzymatiques augmentent la mobilité et la taille effective des domaines de GPI-GFP. Nous attribuons cela à la conversion des constituants des domaines, le cholestérol et la sphingomyéline, par les réactions enzymatiques, ce qui conduit aux plus grandes et moins étanches domaines membranaires.

Blood flow through microcirculation in both simple and complex geometry has been difficult to predict due to the composition and complex behavior of blood at the microscale. Blood is a dense suspension of deformable red blood cells that is comparable in dimensions to the microchannels that it flows through. As a result, rheological properties at the microscale can vastly differ from bulk rheological properties due to non-continuum effects. To further develop our understanding of blood microflow; experimental techniques should be explored. In this work, we explore micro-particle image velocimetry (μPIV) and two-beam fluorescence cross-correlation spectroscopy (2bFCCS) in the application of characterizing blood in microflow conditions. For the development of the μPIV analysis, a polydimethylsiloxane co-flow channel is used to observe blood flow in controlled conditions. Flow conditions (velocity profile and blood layer thickness) are selected based on an analytical model and compared to experimental measurement. The experimental results presented indicate that current flow conditions are inadequate in providing a controlled rate of shear on the blood layer in the co-flow channel and further optimization are required to improve the measurement of the velocity profile. For the development of the 2bFCCS application for blood flow analysis, a wide glass capillary microfluidic device is used to complete the verification of fluorescence fluid admissibility, the effect of laser intensity on inducing photobleaching and the velocity measurement performance. The experimental measurement of the velocity profile is validated against the theoretical profile for a rectangular channel. Results of the velocity profile of high concentration red blood cells show promise in the technique’s ability to measure blood microflows closer to physiological conditions.

Cell penetrating peptides have been the focus of drug delivery research for 15 years due to their apparent ability to deliver cargoes inside cells more readily than many other carriers. Using two of the most commonly studied peptides (tat47-57 and R9), the present study differs from previous work by deliberately choosing to observe uptake with lower peptide concentrations closer to potential therapeutic doses, and by implementing raster image correlation spectroscopy (RICS) on a commercial microscope to quantify uptake in parallel to other techniques such as fluorescence correlation spectroscopy (FCS), confocal microscopy, and mass spectroscopy.An initial study using mass spectrometry and ExPASy (Expert Protein Analysis System) revealed that the peptides are stable for at least one hour in PBS. Based on this initial information and other experimental conditions, the study took two main directions with regards to the peptide: the membrane interaction and accumulation in the cell.The peptides interaction with the cell membrane revealed that neither tat-TAMRA nor R9-TAMRA disrupts the membrane of cells: incorporation of FM2-10 in the membrane was not modified in K562 cells whilst it was in presence of the control lytic peptides GALA and mellitin. Based on this information confocal microscopy was utilised to assess the localisation on the cell membrane. Peptide binding to the membrane appeared to be heterogeneous in distribution at 1µM bulk concentration.Accumulation in cells of the peptides was observed incubated at 37°C, confocal microscopy showed punctuated distribution with intracellular aggregations of fluorescence measuring between 2.5-3.5µm in diameter. Co-staining with a nuclear dye revealed these aggregations to be focused around the nucleus of the cell. Initial FCS experiments indicated a concentration dependent accumulation of the peptide in the cells and a decrease of the intracellular diffusion coefficients at high concentration possibly corresponding with molecular crowding. Interestingly the anomalous diffusion model did not statistically improve the results.RICS was implemented to study the kinetics of entry of TAMRA labelled cell penetrating peptides in both Caco-2 and HeLa cells lines at concentrations between 500nM and 2µM. Concentrations above 1µM exhibited higher final intracellular concentrations, yet the measured diffusion coefficients were similarly independent of extracellular concentration. Both peptides appeared to enter the cell quickly with a fast initial uptake over the first 10 minutes, reaching a concentration maxima after 30 minutes.Overall, the study reveals that many published studies may be incorrect as they may only be reporting the presence of a fluorescent dye inside the cell not the peptide. The fast binding of the peptide to the membrane is likely to cause false positive results when traditionally studying internalisation kinetics such as using flow cytometry and confocal microscopy. Correlation spectroscopy techniques such as FCS, provide useful information on internalisation of the peptides, but the single spot measurement is limited when providing information on the entire cell. RICS is a progression of correlation spectroscopy and provides a more representative picture of the cell.

Dynamic processes are ubiquitous in biological systems: the transport of organelles, proteins and cargoes in the micron-sized heterogeneous cellular environment mainly occurs by Brownian diffusion, while directional flow or drift phenomena contribute to enhance the diffusion-mediated intracellular trafficking and are responsible for the delivery of blood, nutrients and signaling molecules on the larger scale of whole tissues and organs. Motivated by the relevance of transport phenomena in several fields ranging from cell biology to immunology, I adopt and extend the approach of Fluorescence Image Correlation Spectroscopy to investigate diffusive and directional transport processes from the single-cell level up to whole microcirculatory systems. Dynamic transport parameters are quantified by the spatial and/or temporal correlation, in the direct or the reciprocal Fourier space, of the raster-scanned images acquired in-vivo by fluorescence (or reflectance) confocal microscopy. In this work, I focus at first on the measurement of flow velocities in geometrically complex microcirculatory networks, with the development of a novel image-processing method that I have called FLICS or FLow Image Correlation Spectroscopy. FLICS has the peculiarity of exploiting a single raster-scanned xy-image, acquired by detecting the signal of bright, sparse flowing objects (e.g., erythrocytes): by the Cross Correlation Function (CCF) of the fluorescence fluctuations detected in pairs of columns of the image, both the modulus and the direction of the flow velocity can be recovered and mapped, with single-capillary sensitivity and sub-second time resolution, in the whole vessel pattern within the imaged field of view. I derive the explicit analytical expression of the CCF for both two- and three- dimensional flow velocity vectors and, by the approximation of negligible Brownian diffusion, I refine the data-analysis protocol to optimize the flow speed measurement over extended circulatory networks. I validate the FLICS theoretical framework in systems of increasing complexity and I finally apply the method to the characterization of the sinusoidal blood flow in the intricate murine hepatic microcirculation. On the smaller single-cell spatial scale, I successively employ live-cell time-lapse confocal reflectance microscopy and image correlation to investigate the intracellular transport of branched, star-like nanoparticles (GNSs, or Gold NanoStars). Different transport mechanisms, spanning from Brownian diffusion to (sub-)ballistic super-diffusion, are revealed by Temporal and Spatio-Temporal Image Correlation Spectroscopy on the tens-of-seconds timescale. By combining these findings with numerical simulations and with a Bayesian (Hidden Markov Model based) analysis of single particle tracking data, I ascribe the super-diffusive, sub-ballistic behavior of the GNSs dynamics to a two-state switch between diffusion in the cytoplasm and molecular motor-mediated active transport along cytoskeletal filaments. I derive therefore a novel analytical theoretical framework for the investigation of intermittent transport by Fourier-space Image Correlation Spectroscopy (kICS). Besides evaluating on simulated kICS correlation functions the influence of all the dynamic parameters and of the transition rates between the diffusive and the active transport regimes, I derive whole-cell maps for the parameters underlying the GNSs intracellular dynamics. Notably, the method is capable of identifying the simplest transport mode that accurately describes the experimental data, without any prior assumption on its Brownian or super-diffusive nature. The results obtained here for the subcellular trafficking of gold nanostars will be of help in the rational design of the drug delivery and photo-thermal therapy applications of anisotropic gold nanoparticles.

At present, structural health monitoring efforts focus primarily on the sensors and sensing systems for detecting instances and locations of damage through techniques such as X-ray, micro CT, acoustic emission, infrared thermography, lamb wave etc., which only detect cracks at relatively large length scales and rely heavily on sensors and sensing systems which are external to the material system. As an alternative to conventional commercially available SHM techniques, the current work explores processing-structure-property relationships starting from carbon nanotube (CNT) based nanocomposites to particulate composites with nanocomposite binder/matrix materials, i.e. hybrid particulate composites to investigate deformation and damage sensing capabilities of inherently sensing materials and structures through their piezoresistive (coupled electro-mechanical) response. Initial efforts focused on controlling the dispersion of CNTs and orientation of CNT filaments within nanocomposites under dielectrophoresis to guide design and fabrication process of nanocomposites by tuning CNT concentration, applied AC electric field intensity, frequency and exposure time. It is observed that a combination of exposure time to AC electric field and the AC field frequency are the key drivers of filament width and spacing and that the network for filament formation is much more efficient for pristine CNTs than for acid treated functionalized CNTs. With the knowledge obtained from controlling the morphological features, AC field-induced long range alignment of CNTs within bulk nanocomposites was scaled up to form structural test coupons. The morphology, electrical and mechanical properties of the coupons were investigated. The anisotropic piezoresistive response both for parallel and transverse to CNT alignment direction within bulk composite coupons under various loading conditions was obtained. It is observed that control of the CNT network allows for the establishment of percolation paths and piezoresistive response well below the nominal percolation threshold observed for random, so called well-dispersed CNT network distributions. The potential for use of such bulk nanocomposites in SHM applications to detect strain and microdamage accumulation is further demonstrated, underscoring the importance of microscale CNT distribution/orientation and network formation/disruption in governing the piezoresistive sensitivities. Finally, what may be the first experimental study in the literature is conducted for real-time embedded microscale strain and damage sensing in energetic materials by distributing the CNT sensing network throughout the binder phase of inert and mock energetic composites through piezoresistive response for SHM in energetic materials. The incorporation of CNTs into inert and mock energetic composites revealed promising self-diagnostic functionalities for in situ real-time SHM applications under quasi-static and low velocity impact loading for solid rocket propellants, detonators and munitions to reduce the stochastic nature of safety characterization and help in designing insult tolerant energetic materials.<br>Ph. D.

This thesis work aims to present a novel approach to reconstruct Structured Illumination Microscopy, SIM, raw data and to analyze SIM reconstructed images. These new approaches will be demonstrated in the study of chromatin organization. The dissertation will be articulated as follows: Chapter 1 provides an introduction to chromatin nanoscale organization and optical fluorescence microscopy, which is one of the main tools involved in life sciences studies. Indeed, optical microscopy allowed investigating, with high specificity and sensitivity, living samples such as cells, and even tissues. The reader will be presented with a summary on the fluorescence optical microscopy and on the super-resolution, SR, techniques available today including SIM, which is the microscope used in this thesis work. In Chapter 2 the focus is on the introduction of a new reconstruction tool for specific SR-SIM microscopy powered by the Separation of Photons by Lifetime Tuning, SPLIT, method. The introduction of the concept, applied in other works to different SR techniques, will be followed by the practical implementation of the method on the SIM microscope. Then, the applicability of the technique, which we called SPLIT-SIM, will be demonstrated on several different samples. Indeed, it will be used on Simulated data, on test experimental beads, on biological samples both in one and two-color staining. In Chapter 3 the focus will move on the coupling of SIM reconstructed data to colocalization analysis. In particular, for the first time, SIM was coupled to Image Cross-Correlation Spectroscopy, ICCS, in the study of two-color images of a model sample. DNA origami-based structures were chosen as a model sample with precise distances allowing for evaluation of the analysis results. Moreover, all the images analyzed by the pixel-based technique, SIM-ICCS, were analyzed also with an object-based technique as a comparison to evaluate which could be the best choice in SIM acquisitions. Finally, Chapter 4 will be focused on the application of the analysis, performed in chapter 3, to two-color SIM images of nuclear structure. The analysis will be performed on ‘positive control’ in which the target structures will be colocalized and on a negative control in which the structured are spatially segregated within the nucleus. Both object-based and pixel-based analysis will be able to extract coherent results thus showing how SIM-ICCS can become an interesting and useful tool to analyze SIM multicolor acquisitions.

L'objet de cette thèse est l'étude, à des échelles de temps de l'ordre ou inférieure à la femtoseconde, des corrélations dans la dynamique de photoionisation d'atomes et de molécules. Dans un premier temps, nous mettons en évidence l’influence des corrélations vibroniques donnant une asymétrie dans la dynamique d’ionisation de molécules diatomiques. Au travers de simulations numériques impliquant la résolution de l’équation de Schrödinger dépendant du temps, nous pouvons identifier l'influence de l’échange d’énergie se produisant entre le photoélectron et les noyaux sur cette dynamique. Plus encore, nous avons développé une approche stationnaire permettant de rendre compte de ces résultats, et de fournir une interprétation fondée sur la définition d’une géométrie effective associée à un canal vibrationnel. Cette approche est testée pour différentes molécules modèles, exhibant une asymétrie de charge ou encore de masse des noyaux. Nous validons cette approche par des simulations d’ionisation à un photon, mais aussi par des mesures interférométriques à deux photons. Le lien entre les deux phénomènes est ainsi mis en évidence. Dans une deuxième étude, nous sondons la dynamique de photoionisation d’atomes modèle en présence d’une résonance de Fano. Nous étudions, à l'aide d'un modèle multicanaux, la formation dans le temps des spectres de photoélectrons, puis validons une conjecture utilisée implicitement par différents groupes pour leurs interprétations d'expériences de spectroscopie résolue en temps. Nous montrons la robustesse de cette conjecture, et poussons plus loin la comparaison avec l’expérience en soulignant les effets de l’impulsion «sonde» sur la dynamique. Ces travaux ont été effectués en collaboration avec des groupes d’expérimentateurs, dont un au CEA Saclay<br>The main topic of this thesis is the subfemtosecond study of correlations during the photoionisation dynamics of atoms and molecules. First, we observe vibronic correlations on asymmetric ionisation of diatomic molecules. Solving the time-dependent Schrödinger equation numerically, we are able to highlight the influence of photoelectron-nuclei energy exchange on such processes. Moreover, we have developped a stationary approach enabling to retrieve those results, and to define a specific optimal geometry for each vibrational channel. Such an approach is tested for various model molecules, involving charge or mass asymmetry. We finally compare and establish the link between two-photons interferometric measurement simulations to one-photon results. Besides, we also study the photoionisation dynamics through a Fano resonance, using a multichannel model developped for this work. The time-dependent built-up of the photoelectron spectrum enables us to validate experimental interpretations conjectured in CEA Saclay group. We show how robust is this conjecture, et go further into details by highlighting the influence of probe pulse on the dynamics

Orientadores: Fernando Cendes, Marcio Luiz Figueredo Balthazar<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Cências Médicas<br>Made available in DSpace on 2018-08-20T20:16:05Z (GMT). No. of bitstreams: 1 Lopes_TatilaMartins_M.pdf: 971844 bytes, checksum: c5993413c16c861553c1a2994f3aba69 (MD5) Previous issue date: 2012<br>Resumo: A doença de Machado Joseph ou ataxia espinocerebelar tipo 3 (DMJ/SCA3) se caracteriza pela marcha atáxica e diversos sinais extracerebelares. Manifestações neuropsiquiátricas e cognitivas já foram descritas, porém não há um consenso quanto aos domínios cognitivos alterados. Portanto nosso objetivo foi investigar os aspectos neuropsicológicos na SCA3 e correlacionar os domínios cognitivos comprometidos com volume de substância cinzenta (SC), integridade de substância branca (FA), metabólitos cerebelares, repetição de CAG, tempo de duração e gravidade da doença. Participaram 32 pacientes com SCA3 pareados por idade, gênero e escolaridade com 32 controles saudáveis. Adquirimos IRM-3T- 3D ponderadas em T1 para análise de morfometria baseada em voxel, processadas e analisadas pelo software SPM8; imagens por tensor de difusão, processadas e analisadas pelo software FSL e espectroscopia por ressonância magnética (single-voxel em cerebelo) processadas pelo software LCModel. Todos os participantes realizaram o mesmo protocolo de avaliação neuropsicológica (NPs). Somente os pacientes com SCA3 se submeteram a escala para avaliação da gravidade da ataxia e pelo inventário neuropsiquiátrico. Utilizamos o teste Mann-Witney para comparação entre grupos quanto à avaliação NPs e correlação de Spearman entre os achados cognitivos e os metabólitos cerebelares, FA, número de CAG, tempo de duração e gravidade da doença. Consideramos p<0.05 não corrigido para múltiplas comparações. Para correlação entre os achados cognitivos e SC usamos cluster-size=50 voxel, p<0.001 (não corrigidos). Encontramos diferença significativa entre grupos nos testes: Rey auditory verbal learning test (RAVLT)-codificação [p=0.001], evocação tardia [p=0.001] e reconhecimento [p=0.027], matrizes progressivas: escala geral [p=0.003]; blocos de Corsi- ordem direta [p=0.002]; blocos de Corsi- ordem inversa [p=0.001]; span de dígitos- ordem direta [p=0.024]; fluência verbal categórica [p=0.029]; sintomas indicativos de depressão [p<0.001] e ansiedade [p<0.001]. A análise qualitativa mostrou prejuízo leve no RAVLT- codificação e médio inferior nos demais achados cognitivos. Houve correlação positiva entre RAVLT-codificação e áreas do córtex temporal, frontal, ínsula e cúlmen cerebelar; RAVLT-evocação tardia e giro précentral, cúlmen, áreas do córtex frontal, temporal e parietal; RAVLTreconhecimento e giro frontal; matrizes progressivas e precúneo e giro temporal; blocos de Corsi- ordem direta e lobo parietal; dígitos- ordem direta e giro temporal e fluência verbal com giro precentral. Todos os p<0.001. Houve correlação entre valor de FA do tronco cerebral de pacientes SCA3 e o teste dígitos- ordem direta [rs=0.485]. Encontramos diferença significativa entre grupos quanto aos metabólitos cerebelares: N-Acetil-Aspartato [p<0.001], N-Acetil-Aspartato-total [p<0.001], glutamato [p=0.017], glutamina [p=0.005], fosfocolina [p=0.019] e compostos de colina [p=0.014], exceto creatina [p=0.522] e inositol [p=0.475]. Houve correlação entre o teste blocos de Corsi- ordem direta e glutamato [rs=- 0.405] e glutamina [rs=-0.419]; fluência verbal semântica fosfocolina [rs= 0.669] e compostos de colina [rs=0.692] e dígitos- ordem direta e N-Acetil-Aspartato [rs=0.418] e N-Acetil-Aspartato-total [rs=0.432]. Não encontramos correlação entre os achados cognitivos e aspectos genético e clínicos. Os resultados sugerem que pacientes com SCA3 apresentam comprometimento de memória episódica e operacional e habilidades visuoespaciais associados a alterações encefálicas. As alterações neuropsiquiátricas apontam para heterogeneidade na doença também referente a esse aspecto e a falta de correlação entre os aspectos cognitivos e os aspectos genéticos e clínicos sugerem que essas variáveis ocorrem independentemente<br>Abstract: The Machado- Joseph disease or spinocerebellar ataxia type 3 (MJD/SCA3) is characterized by ataxic gait and extracerebellar signs. Neuropsychiatric and cognitive impairment have demonstrated, however there is no consensus on the cognitive domains altered. Therefore, the aim of this study was to investigate the neuropsychological features in SCA3 and correlate the cognitive findings with the gray matter (GM) volume, integrity of white matter, by means of the fractional anisotropy (WM-FA), cerebellar metabolites, CAG lengths, disease duration and severity. Participated 32 SCA3 patients matched by age, gender and educational level with 32 healthy control group. We acquired MRI-3T-3D, T1- weighted for voxel-based-morfometry, processed and analyzed using the SPM8 software, diffusion tensor images (DTI) processed and analyzed by FSL software and magnetic resonance spectroscopy (RMS- single voxel cerebellum) analyzed using LCModel software. All participants underwent the same neuropsychological evaluation. Only SCA3 patients underwent the scale for the assessment and rating of ataxia and Neuropsychiatric inventory. We used the Mann-Whitney test to compare the differences between the groups regarding the NPs and Spearman correlation between the cognitive findings and the cerebellar metabolites, FA values, CAG repeats, disease duration and severity. We used p<0.05 noncorrected for multiple comparison. For GM analysis used cluster-size=50 voxel, p<0.001 (uncorrected). We found significant differences between groups in the tests: Rey auditory verbal learning test (RAVLT)-coding [p=0.001], delayed recall [p=0.001] and recognition [p=0.027], progressive matrices [p=0.003]; Corsi block span-forward [p=0.002]; Corsi block span-backward [p=0.001]; digit span-forward [p=0.024]; semantic verbal fluency [p=0.029]; and symptoms indicative of depression [p<0.001] and anxiety [p<0.001]. The qualitative analysis showed mild impairment in the test RAVLT-coding and inferior medium impairment in the other tests. There were positive correlations between RAVLT-coding and temporal cortex areas, frontal, insula and culmen cerebellar; RAVLT-delayed recall and precentral gyrus, culmen, frontal cortex areas, temporal and parietal lobes; RAVLTrecognition and frontal gyrus; progressive matrices and precuneus and temporal gyrus; Corsi block span- forward and parietal lobe; digit span-forward and temporal gyrus; semantic verbal fluency with precentral gyrus (p<0.001). We found correlation between FA value of brainsteam in SCA3 patients and digits spanforward [rs=0.485]. There were difference between groups related to cerebellar metabolites: N-Acetyl-Aspartate [p<0.001], N-Acetyl-aspartyl-glutamate [p<0.001], glutamate [p=0.017], glutamine [p=0.005], phosphorylcholine [p=0.019] e glycerophosphorylcholine [p=0.014], except creatine [p=0.522] and inositol [p=0.475]. A significant correlation was found between: Corsi block span-forward with glutamate (rs=-0.419) and glutamine (rs=-0.405); verbal fluency with phosphorylcoline (rs=0.661) and glycerophosphorylcholine (rs=0.692); digits spanforward with N-acetyl-Aspartate (rs=0.418) and N-Acetyl-Aspartyl-glutamate (rs=0.432). We found no significant correlation between cognitive findings and clinical variables and CAG lengths. We found no significant correlation between the cognitive findings and clinical and genetic variables. The results suggest there are episodic and working memory dysfunction and visuospatial disabilities in the SCA3 associated with encephalic alterations. The lack of correlation between the neuropsychiatric abnormalities and genetic and clinics features suggest this variables occur independently of primary disease progression<br>Mestrado<br>Neurociencias<br>Mestre em Fisiopatologia Médica

The objective of the project is to use the RICS analysis technique in complement with confocal microscopy to determine the diffusion coefficient of the selectively labeled green fluorescent protein (GFP), GFP-EGFR and GFP-p53 in cervical cancer cells. This is a collaboration work with MD Anderson Cancer Center. The application of the study is to lay the foundation for further study in understanding the cell metabolism, subcellular morphologic and dynamic biochemical processes to aid in the diagnosis and to potentially screen cancers. Fluorescence microscopy techniques have been developed for the study of cellular processes and molecular signal pathway. However, the spatial resolution to distinguish and resolve the interactions of single molecular complexes or molecule in cells is limited by the wavelength. Hence, indirect image correlation methods complementary to the imaging techniques were developed to obtain the dynamic information within the cell. RICS is one such mathematical image processing method to determine the dynamics of the cell. HeLa cells are transfected with GFP to highlight the protein of interest. These samples were imaged with confocal microscope, Olympus FV1000 with a 60 x 1.2 NA water objective in the pseudo photon counting mode with an excitation of 488 nm argon ion laser. About 100 frames of scan area 256x256 pixels were collected from each sample at scan speed of 12.5 seconds per pixel. The stacks of images were processed with SimFCS software. The images were subjected to immobile subtraction algorithm to remove the immobile features. Consequently, each frame in the stack is subjected to 2D-correlation; then, the average 2D-spatial correlation is calculated. This 2D-spatial correlated data constitutes as RICS data which is then displayed and analyzed by fitting it to specific models. This generates a spatial temporal map of the molecular dynamics of fluorescently labeled probes within the cell. In summary, we apply RICS techniques based on correlation spectroscopy to the image data and quantify diffusion coefficient of protein of interest in cancerous cells with different treatments. This is expected to better understand cellular dynamics of cancerous cells and build better diagnostic biosensor devices for early screening.

In the cancer research, it is important to understand protein dynamics which are involved in cell signaling. Therefore, particular protein detection and analysis of target protein behavior are indispensable for current basic cancer research. However, it usually performed by conventional biochemical approaches, which require long process time and a large amount of samples. We have been developed the new applications based on microfluidics and Raster image Correlation spectroscopy (RICS) techniques. A simple microfluidic 3D hydrodynamic flow focusing device has been developed for quantitative determinations of target protein concentrations. The analyte stream was pinched not only horizontally, but also vertically by two sheath streams by introducing step depth cross junction structure. As a result, a triangular cross-sectional flow profile was formed and the laser was focused on the top of the triangular shaped analyte stream. Through this approach, the target protein concentration was successfully determined in cell lysate samples. The RICS technique has been applied to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. P53 tagged with Green Fluores-cent Protein (GFP) were incubated with and without DNA damaging agents, cisplatin or eptoposide. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. This result demonstrates that RICS is able to detect protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening. As another application of microfluidics, an integrated microfluidic platform was developed for generating collagen microspheres with encapsulation of viable cells. The platform integrated four automated functions on a microfluidic chip, (1) collagen solution cooling system, (2) cell-in-collagen microdroplet generation, (3) collagen microdroplet polymerization, and (4) incubation and extraction of the microspheres. This platform provided a high throughput and easy way to generate uniform dimensions of collagen microspheres encapsulating viable cells that were able to proliferate for more than 1 week.