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Littérature scientifique sur le sujet « Fluorescence Microscopy, Image Correlation Spectroscopy »

Auteur : Grafiati

Publié le 9 mars 2023

Mis à jour le 10 mars 2023

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Articles de revues sur le sujet "Fluorescence Microscopy, Image Correlation Spectroscopy"

Wiseman, Paul. "Introduction to Fluorescence and Image Correlation Spectroscopy." Microscopy and Microanalysis 10, S02 (2004): 246–47. http://dx.doi.org/10.1017/s1431927604886483.

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Wiseman, P. W., J. C. Bouwer, S. Peltier, and M. H. Ellisman. "High Speed Two Photon Excitation Microscopy in Live Cell Imaging using Image Correlation Spectroscopy (ICS)." Microscopy and Microanalysis 7, S2 (2001): 22–23. http://dx.doi.org/10.1017/s1431927600026180.

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For live-cell imaging, two-photon excitation microscopy (TPEM) is proving to be a significant technological advancement. The unique features offered by TPEM are the ability to image thick sections, excellent optical sectioning capabilities, low damage to living cells, and less out of focus fluorescence and out of focus photobleaching. of these features, the most useful for the biological microscopist, is optical sectioning. Optical sectioning is an intrinsic property of the two-photon process, whereby, two infrared (IR) photons are absorbed quickly to excite a single UV/blue transition. The probability for exciting a two photon transition is proportional to the instantaneous excitation intensity squared. Therefore, for a focused laser beam, only light at the focal point of the excitation beam excites a fluorescent transition. Thus, the need for confocal apertures and time consuming deconvolution algorithms are, for the most part, eliminated.We have continued to develop and enhance our ability to perform high-speed, two-photon excitation fluorescence microscopy. in 1998, we successfully deployed a prototype, video-rate twophoton laser scanning system (30 frames/sec or faster at reduced scan width) developed with support from Nikon Corporation. That system was built upon a Nikon RCM 8000 confocal microscope.

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Diaspro, Alberto, Giuseppe Chirico, and Maddalena Collini. "Two-photon fluorescence excitation and related techniques in biological microscopy." Quarterly Reviews of Biophysics 38, no. 2 (2005): 97–166. http://dx.doi.org/10.1017/s0033583505004129.

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1. Introduction 982. Historical background of two-photon effects 992.1 2PE 1002.2 Harmonic generation 1002.3 Fluorescence correlation spectroscopy 1003. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 1013.1 General considerations 1013.2 Fluorescence intensity under the 2PE condition 1033.3 Optical consequences of 2PE 1043.4 Saturation effects in 2PE 1083.5 Fluorescence correlation spectroscopy 1093.5.1 Autocorrelation analysis 1103.5.2 Photon-counting histogram analysis 1124. Two-photon-excited probes 1155. Design considerations for a 2PE fluorescence microscope 1195.1 General aspects 1195.2 Descanned and non-descanned 2PE imaging 1215.3 Lens objectives and pulse broadening 1225.4 Laser sources 1255.5 Example of a practical realization 1276. Applications 1346.1 Biological applications of 2PE 1346.1.1 Brain images 1346.1.2 Applications on the kidney 1396.1.3 Mammalian embryos 1396.1.4 Applications to immuno-response 1416.1.5 Myocytes 1416.1.6 Retina 1426.1.7 DNA imaging 1436.1.8 FISH applications 1446.2 2PE imaging of single molecules 1446.3 FCS applications 1486.4 Signals from nonlinear interactions 1517. Conclusions 1538. Acknowledgements 1549. References 155This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.

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Bates, Ian R., Paul W. Wiseman, and John W. Hanrahan. "Investigating membrane protein dynamics in living cellsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease." Biochemistry and Cell Biology 84, no. 6 (2006): 825–31. http://dx.doi.org/10.1139/o06-189.

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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.

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Laňková, Martina, Jana Humpolíčková, Stanislav Vosolsobě, et al. "Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy." Microscopy and Microanalysis 22, no. 2 (2016): 290–99. http://dx.doi.org/10.1017/s1431927616000568.

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AbstractA number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

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Friaa, Ouided, and Cécile Fradin. "Coincidence Measurements in Dual-Color Confocal Microscopy: A Combined Single-Particle and Fluorescence Correlation Approach." Biophysical Reviews and Letters 09, no. 03 (2014): 249–71. http://dx.doi.org/10.1142/s1793048014400074.

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In this paper we discuss how the coincident detection of mobile particles in dual-color confocal images can be improved. Optimal coincidence detection requires a careful choice of experimental conditions and image acquisition parameters in order to maximize the overlap between the two detection volumes. By measuring this overlap with fluorescence cross-correlation spectroscopy, we show in particular that a small confocal field of view is necessary in order to maintain good coincidence. Most importantly, coincidence detection also requires a dedicated image analysis strategy. Traditionally, two approaches have been adopted to assess coincidence of mobile particles: fluorescence fluctuation measurements, notably cross-correlation spectroscopy, and single particle detection. Here we propose to combine these two approaches by calculating a cross-correlation coefficient for each of the detected single particles. We show that this allows to remove accidental coincidence events from a data set, and thus to unambiguously identify particles that instead carry two different fluorophores. This strategy can help increase the available concentration range for confocal coincidence measurements and detect rare binding events. [Formula: see text]Special Issue Comments: This article about coincident detection of mobile particle in two-color confocal images is thematically related to several articles in this Special Issue, namely the review of FRET-based single-molecule fluorescence techniques by Ruedas-Rama et al.,1 the single particle detection work presented by de Keersmaecker et al.2 and the general considerations on the mathematical treatment of single molecule trajectories presented by Flomenbom3. Our study of single liposomes is also relevant to experiments involving proteins and liposomes, such as the enzyme experiments described in the review by Jørgensen and Hatzakis.4

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Pelicci, Simone, Laura Furia, Mirco Scanarini, Pier Giuseppe Pelicci, Luca Lanzanò, and Mario Faretta. "Novel Tools to Measure Single Molecules Colocalization in Fluorescence Nanoscopy by Image Cross Correlation Spectroscopy." Nanomaterials 12, no. 4 (2022): 686. http://dx.doi.org/10.3390/nano12040686.

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Super Resolution Microscopy revolutionized the approach to the study of molecular interactions by providing new quantitative tools to describe the scale below 100 nanometers. Single Molecule Localization Microscopy (SMLM) reaches a spatial resolution less than 50 nm with a precision in calculating molecule coordinates between 10 and 20 nanometers. However new procedures are required to analyze data from the list of molecular coordinates created by SMLM. We propose new tools based on Image Cross Correlation Spectroscopy (ICCS) to quantify the colocalization of fluorescent signals at single molecule level. These analysis procedures have been inserted into an experimental pipeline to optimize the produced results. We show that Fluorescent NanoDiamonds targeted to an intracellular compartment can be employed (i) to correct spatial drift to maximize the localization precision and (ii) to register confocal and SMLM images in correlative multiresolution, multimodal imaging. We validated the ICCS based approach on defined biological control samples and showed its ability to quantitatively map area of interactions inside the cell. The produced results show that the ICCS analysis is an efficient tool to measure relative spatial distribution of different molecular species at the nanoscale.

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Pandzic, E., and R. M. Whan. "A Practical Guide to Fluorescence Temporal and Spatial Correlation Spectroscopy." Biophysicist 2, no. 1 (2021): 40–69. http://dx.doi.org/10.35459/tbp.2019.000143.

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ABSTRACT The aim of this article is to introduce the basic principles behind the widely used microscopy tool: fluorescence fluctuation correlation spectroscopy (FFCS). We present the fundamentals behind single spot acquisition (FCS) and its extension to spatiotemporal sampling, which is implemented through image correlation spectroscopy (ICS). The article is an educational guide that introduces theoretic concepts of FCS and some of the ICS techniques, followed by interactive exercises in MATLAB. There, the learner can simulate data time series and the application of various FFCS techniques, as well as learn how to measure diffusion coefficients, molecular flow, and concentration of particles. Additionally, each section is followed by a short exercise to reinforce learning concepts by simulating different scenarios, seek verification of outcomes, and make comparisons. Furthermore, we invite the learner throughout the article to consult the literature for different extensions of FFCS techniques that allow measurements of different physicochemical properties of materials. Upon completion of the modules, we anticipate the learner will gain a good understanding in the field of FFCS that will encourage further exploration and adoption of the FFCS tools in future research and educational practices.

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Cainero, Isotta, Elena Cerutti, Mario Faretta, et al. "Measuring Nanoscale Distances by Structured Illumination Microscopy and Image Cross-Correlation Spectroscopy (SIM-ICCS)." Sensors 21, no. 6 (2021): 2010. http://dx.doi.org/10.3390/s21062010.

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Since the introduction of super-resolution microscopy, there has been growing interest in quantifying the nanoscale spatial distributions of fluorescent probes to better understand cellular processes and their interactions. One way to check if distributions are correlated or not is to perform colocalization analysis of multi-color acquisitions. Among all the possible methods available to study and quantify the colocalization between multicolor images, there is image cross-correlation spectroscopy (ICCS). The main advantage of ICCS, in comparison with other co-localization techniques, is that it does not require pre-segmentation of the sample into single objects. Here we show that the combination of structured illumination microscopy (SIM) with ICCS (SIM-ICCS) is a simple approach to quantify colocalization and measure nanoscale distances from multi-color SIM images. We validate the SIM-ICCS analysis on SIM images of optical nanorulers, DNA-origami-based model samples containing fluorophores of different colors at a distance of 80 nm. The SIM-ICCS analysis is compared with an object-based analysis performed on the same samples. Finally, we show that SIM-ICCS can be used to quantify the nanoscale spatial distribution of functional nuclear sites in fixed cells.

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Waharte, François, Karine Steenkeste, Romain Briandet, and Marie-Pierre Fontaine-Aupart. "Diffusion Measurements inside Biofilms by Image-Based Fluorescence Recovery after Photobleaching (FRAP) Analysis with a Commercial Confocal Laser Scanning Microscope." Applied and Environmental Microbiology 76, no. 17 (2010): 5860–69. http://dx.doi.org/10.1128/aem.00754-10.

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ABSTRACT Research about the reactional and structural dynamics of biofilms at the molecular level has made great strides, owing to efficient fluorescence imaging methods in terms of spatial resolution and fast acquisition time but also to noninvasive conditions of observation consistent with in situ biofilm studies. In addition to conventional fluorescence intensity imaging, the fluorescence recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring of local diffusion coefficients in biofilms and could become an alternative to fluorescence correlation spectroscopy (FCS). We present here an image-based FRAP protocol to improve the accuracy of FRAP measurements inside “live” biofilms and the corresponding analysis. An original kymogram representation allows control of the absence of perturbing bacterial movement during image acquisition. FRAP data analysis takes into account molecular diffusion during the bleach phase and uses the image information to extract molecular diffusion coefficients. The fluorescence spatial intensity profile analysis used here for the first time with biofilms is supported both by our own mathematical model and by a previously published one. This approach was validated to FRAP experiments on fluorescent-dextran diffusion inside Lactoccocus lactis and Stenotrophomonas maltophilia biofilms, and the results were compared to previously published FCS measurements.

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Thèses sur le sujet "Fluorescence Microscopy, Image Correlation Spectroscopy"

Nicovich, Philip R. "Widefield fluorescence correlation spectroscopy." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/33849.

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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.

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BOUZIN, MARGAUX. "Correlazione di Immagini per lo Studio di Processi Dinamici in Sistemi Biologici." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/94231.

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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.

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Gallagher, Joseph. "Adaptive optics for fluorescence correlation spectroscopy." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAY054/document.

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Ce projet de recherche conjugue deux aspects complémentaires : le développement d’un montage de microscopie intégrant un système d'Optique Adaptative (OA) et l’étude de masses cancéreuses (Sphéroïdes Multicellulaires) sous pression mécanique.Ces deux axes seront mutuellement bénéfiques puisque l’implémentation de l’OA rendra possible l’imagerie et les mesures physiques au sein des sphéroïdes ; d’un autre côté, l’étude des sphéroïdes permettra de caractériser les aberrations induites par ce type d’échantillons et de mieux comprendre les exigences sur le système d’OA qu’imposent l’observation de ces échantillons ainsi que les limites de la microscopie optique dans les tissus biologiques This research project combines two complementary aspects: the development of an assembly incorporating an Adaptive Optics microscope system and the study of cancerous masses (multicellular spheroids) under mechanical pressure.These two axes are mutually beneficial since the implementation of the adaptive optics will enable imaging and physical measurements in spheroids; On the other hand, the study of spheroids will characterize the aberrations induced by this type of samples and understand the requirements of the adaptive optics system imposed by the observation of these samples as well as the limits of optical microscopy in biological tissues

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Doroshenko, Mikheil [Verfasser]. "Diffusion in heterogeneous systems studied by laser scanning confocal microscopy and fluorescence correlation spectroscopy / Mikheil Doroshenko." Mainz : Universitätsbibliothek Mainz, 2014. http://d-nb.info/104870758X/34.

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Xu, Lei. "Development and application of ultra-sensitive fluorescence spectroscopy and microscopy for biomolecular interaction studies." Doctoral thesis, KTH, Experimentell biomolekylär fysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-146181.

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This thesis describes the development of sensitive and high-resolution fluorescence spectroscopic and microscopic techniques and their application to probe biomolecules and their interactions in solution, lipid membrane model systems and in cells. Paper I-IV are largely focused on methodological developments. In paper I, a new fluorescence method based on fluorescence correlation spectroscopy (FCS) for detecting single particles was realized, requiring no fluorescent labeling of the particles. The method can yield information both about the diffusion properties of the particles as well as about their volumes. In paper II, a modified fluorescence cross correlation spectroscopy procedure with well characterized instrumental calibration was developed and applied to study cis interactions between an inhibitory receptor and its Major Histocompatibility Complex class I ligand molecule, both within the same cellular membranes. The quantitative analysis brought new insights into the Nature killer cell’s self-regulating of tolerance and aggressiveness for immune responses. Paper III describes a multi-color STED (STimulated Emission Depletion) microscopy procedure, capable of imaging four different targets in the same cells at 40nm optical resolution, which was developed and successfully demonstrated on platelets. In paper IV, a modified co-localization algorithm for fluorescence images analysis was proposed, which is essentially insensitive to resolutions and molecule densities. Further, the performance of this algorithm and of using STED microscopy for co-localization analysis was evaluated using both simulated and experimentally acquired images. Papers V-VII have their main emphasis on the application side. In paper V, transient state imaging was demonstrated on live cells to image intracellular oxygen concentration and successfully differentiated different breast cancer cell lines and the different metabolic pathways they adopted to under different culturing conditions. Paper VI describes a FCS-based study of proton exchange at biological membranes, the size-dependence of the membrane proton collecting antenna effect as well as effects of external buffer solutions on the proton exchange, in a nanodisc lipid membrane model system. These findings provide insights for understanding proton transport at and across membranes of live cells, which has a central biological relevance. In paper VII, STED imaging and co-localization analysis was applied to analyze cell adhesion related protein interactions, which are believed to have an important modulating role for the proliferation, differentiation, survival and motility of the cells. The outcome of efforts taken to develop means for early cancer diagnosis are also presented. It is based on single cells extracted by fine needle aspiration and the use of multi-parameter fluorescence detection and STED imaging to detect protein interactions in the clinical samples. Taken together, detailed studies at a molecular level are critical to understand complex systems such as living organisms. It is the hope that the methodologies developed and applied in this thesis can contribute not only to the development of fundamental science, but also that they can be of benefit to mankind in the field of biomedicine, especially with an ultimate goal of developing novel techniques for cancer diagnosis. QC 20140609

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Wang, Ruixing. "STED-fluorescence correlation spectroscopy for dynamic observations in cell biology : from theoretical to practical approaches." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0163/document.

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Les techniques de super-résolution offrent un nouvel aperçu de la description de l'organisation moléculaire dynamique de la membrane plasmique. Parmi ces techniques, la microscopie par déplétion d'émission stimulée (stimulated emission depletion, STED) dépasse la limite de diffraction optique et atteint une résolution de quelques dizaines de nanomètres. Il est une technique polyvalente qui peut être combinée avec d'autres techniques telles que la spectroscopie par corrélation de fluorescence (fluorescence correlation spectroscopy, FCS), fournissant des résolutions spatiales et temporelles élevées pour explorer les processus dynamiques qui se produisent dans les cellules vivantes. Ce projet de doctorat vise à mettre en œuvre un microscope STED, puis à combiner ce module STED avec la technique FCS pour les applications biologiques. Des études théoriques du STED et de la technique combinant STED et FCS ont permis dans les aspects spatio-temporels. Une solution analytique pour la fonction d'autocorrélation FCS a été dérivée dans l'état de déplétion STED incomplet. et un nouveau modèle d'ajustement FCS a été proposé. La méthode de variation du volume d’observation FCS (spot variation FCS, svFCS) a démontré sa capacité à identifier la présence de nanodomaines limitant la diffusion latérale des molécules dans la membrane plasmique. L’approche STED-FCS permet d’étendre l’application de la svFCS à l'échelle nanométrique afin d’évaluer la persistance plus ou moins importante de tels nanodomaines. Dans ce contexte, des simulations préliminaires de Monte Carlo ont été réalisées figurant des molécules diffusant en présence d'auto-assemblage/désassemblage dynamique des nanodomaines Super-resolution techniques offer new insight into the description of the dynamic molecular organization at the plasma membrane. Among these techniques, the stimulated emission depletion (STED) microscopy breaks the optical diffraction limit and reaches the resolution of tens of nanometer. It is a versatile setup that can be combined with other techniques such as fluorescence correlation spectroscopy (FCS), providing both high spatial and temporal resolutions to explore dynamic processes occurring in live cells. This PhD project aims at implementing a STED microscope, and then at combining this STED module with FCS technique for biological applications. Detailed theoretical studies on STED and the combined STED-FCS technique in spatio-temporal aspects were performed. An analytical solution for FCS autocorrelation function was derived in the condition of incomplete STED depletion and a new FCS fitting model was proposed to overcome this problem. The spot variation FCS (svFCS) method has demonstrated its capability to identify the presence of nanodomains constraining the lateral diffusion of molecules at the plasma membrane. The STED-FCS can extend the svFCS approach to the nanoscale evaluating the long-lasting existence of such nanodomains. Within this frame, preliminary Monte Carlo simulations were conducted mimicking molecules diffusing in the presence of dynamic self-assembling/disassembling nanodomains

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Le, Andy Vinh. "Blood Microflow Characterization Using Micro-Particle Image Velocimetry and 2-Beam Fluorescence Cross-Correlation Spectroscopy." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41535.

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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.

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Persson, Gustav. "Temporal Modulation in Fluorescence Spectroscopy and Imaging for Biological Applications." Doctoral thesis, KTH, Experimentell biomolekylär fysik, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-10243.

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This thesis explores the benefits of intensity modulation for the purpose of extending the range of applications of fluorescence spectroscopy and imaging in cellular and molecular biology and medicine. Long-lived transient states of fluorescent molecules can, because of their long lifetimes, be used to detect subtle changes in the microenvironment of the molecule. A method for determining the kinetic rates for transitions to and from such states by registration of changes in the average fluorescence intensity related to different modulation of the excitation source is introduced. It combines the detection sensitivity of fluorescence with the environmental sensitivity of the long-lived transient states and allows the use of slow detectors such as CCD cameras, making parallelization and wide-field imaging possible developments. An extension of this method, generating image contrast based on triplet state population using a standard laser scanning microscope, is also shown. A strategy to combine fluorescence correlation spectroscopy (FCS) with modulated excitation, in a way that allows extraction of correlation data for all correlation times, is presented. This enables the use of modulation to optimize measurement conditions with respect to photophysical properties of the dyes used. FCS with modulated excitation will probably prove useful in future studies involving multiple kinetic processes occurring in overlapping time ranges. One of the ideas from this project also constitutes a powerful method for generating artifact free correlation curves from data sets where sections have been removed. This is potentially very useful in biological studies where spikes in the measurements often cause problems. In the final project, cross-correlation and alternating excitation are combined in measurements on a pH-sensitive ratiometric dye to clearly distinguish the protonation–deprotonation dynamics from other processes. The presented approach makes the protonation related fluctuations manifest themselves as a very distinct anti-correlating component in the correlation curve. This enables robust data analysis using a simple model. QC 20100805

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Vaillancourt, Benoit. "Novel biophysical appliations [sic] of STICS." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111550.

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The object of this thesis is to present two novel applications of Spatiotemporal Image Correlation Spectroscopy (STICS) to biological systems. STICS is a technique which uses the correlations in pixel intensity fluctuations of an image time series, captured under fluorescence microscopy, to measure the speed and direction of a flowing population of fluorescently labeled molecules. The method was first applied to measure the dynamics of transport vesicles inside growing pollen tubes of lily flowers. The measured vector maps allowed to confirm the presence of actin filaments along the periphery of the tubes, as well as the presence of a reverse-fountain pattern in the apical region. In a second set of experiments, STICS was used to measure the retrograde flow of filamentous actin in migrating chick DRG neuronal growth cones. These results serve as proof of principle that STICS can be used to probe the response of the growth cone cytoskeleton to external chemical cues.

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Kohram, Maryam. "A Combined Microscopy and Spectroscopy Approach to Study Membrane Biophysics." University of Akron / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=akron1436530389.

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Chapitres de livres sur le sujet "Fluorescence Microscopy, Image Correlation Spectroscopy"

Lacoste, Judith, Charles Vining, Dongmei Zuo, Aleksandrs Spurmanis, and Claire M. Brown. "Optimal Conditions for Live Cell Microscopy and Raster Image Correlation Spectroscopy." In Reviews in Fluorescence 2010. Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4419-9828-6_12.

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Kohl, Tobias, and Petra Schwille. "Fluorescence Correlation Spectroscopy with Autofluorescent Proteins." In Microscopy Techniques. Springer Berlin Heidelberg, 2005. http://dx.doi.org/10.1007/b102212.

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Brock, Roland, and Thomas M. Jovin. "Fluorescence Correlation Microscopy (FCM): Fluorescence Correlation Spectroscopy (FCS) in Cell Biology." In Springer Series in Chemical Physics. Springer Berlin Heidelberg, 2001. http://dx.doi.org/10.1007/978-3-642-59542-4_7.

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Moreno, David F., and Martí Aldea. "Coincidence Analysis of Molecular Dynamics by Raster Image Correlation Spectroscopy." In Computer Optimized Microscopy. Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9686-5_17.

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Mazza, Davide, Timothy J. Stasevich, Tatiana S. Karpova, and James G. McNally. "Monitoring Dynamic Binding of Chromatin Proteins In Vivo by Fluorescence Correlation Spectroscopy and Temporal Image Correlation Spectroscopy." In Methods in Molecular Biology. Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-477-3_12.

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Dumas, D., B. Riquelme, H. Castellini, L. Basciano, N. de Isla, and J. F. Stoltz. "Calibration in fluorescence correlation spectroscopy for measurements of stem cell differentiation kinetic." In EMC 2008 14th European Microscopy Congress 1–5 September 2008, Aachen, Germany. Springer Berlin Heidelberg, 2008. http://dx.doi.org/10.1007/978-3-540-85228-5_86.

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"Fluorescence Correlation Spectroscopy." In Nanoscopy and Multidimensional Optical Fluorescence Microscopy. Chapman and Hall/CRC, 2010. http://dx.doi.org/10.1201/9781420078893-12.

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Shi, Xianke, and Thorsten Wohland. "Fluorescence Correlation Spectroscopy." In Nanoscopy and Multidimensional Optical Fluorescence Microscopy. Chapman and Hall/CRC, 2010. http://dx.doi.org/10.1201/9781420078893-c6.

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Anthony, Neil, and Keith Berland. "Global Analysis in Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Microscopy." In Methods in Enzymology. Elsevier, 2013. http://dx.doi.org/10.1016/b978-0-12-388422-0.00007-8.

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Elson, Elliot Lawrence. "Imaging the Cell: Light Microscopy – Fluorescence Correlation Spectroscopy: A Tool for Measuring Dynamic and Equilibrium Properties of Molecules in Cells." In Reference Module in Life Sciences. Elsevier, 2022. http://dx.doi.org/10.1016/b978-0-12-821618-7.00086-9.

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Actes de conférences sur le sujet "Fluorescence Microscopy, Image Correlation Spectroscopy"

Webb, Watt W. "Multiphoton Microscopy MPM: Imaging Spectra and Dynamics of Molecular Function Deep in Living Tissues." In In Vivo optical Imaging at the NIH. Optica Publishing Group, 1999. http://dx.doi.org/10.1364/ivoi.1999.msi3.

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Multiphoton Excitation (MPE) of fluorescence provides the optimum photophysics for microscopic imaging deep in living tissue with minimal photodamage, to depths so far ~ 400 µm. Tissue autofluorescence excited by two-photon or three-photon absorption to ultra-violet energies can provide specific indications of disease. Useful autofluorescence of serotonin (5HT), melatonin, indolamine breakdown products, NADH, collagen, elastin, and a number of yet-to-be-identified molecular species, some of which identify disease states are already being imaged routinely. For research in model animals, genetic constructs that label specific molecules with mutants of Green Fluorescent Protein (GFP) can be imaged deep in tissue with MPM. MPM excitation of GFP mutants at nanomolar concentrations for Fluorescence Correlation Spectroscopy (FCS) provides a robust, internally calibrated, new measure of pH in cells and tissues. Fluorescent labels that penetrate tissue can be usefully imaged in living animals and thick tissue cultures; for example, thioflavins in the beta amyloid plaques of Alzheimer’s Disease are being imaged deep in living transgenic mouse brains. Multiphoton imaging spectroscopy and fluorescence lifetime imaging (FLIM) provides useful molecular identification diagnostics. Some applications are shown in order to illustrate capability. However, the potential of MPM for in vivoimaging has barely been explored, and this technology should be regarded as providing a fertile opportunity that is yet to be fully exploited for biomedical research and for clinical applications.

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Gregor, Ingo, Niels Rademacher, Max Tillmann, Matthias Patting, Jörg Enderlein, and Felix Koberling. "Fluorescence lifetime image scanning microscopy." In Single Molecule Spectroscopy and Superresolution Imaging XV, edited by Ingo Gregor, Rainer Erdmann, and Felix Koberling. SPIE, 2022. http://dx.doi.org/10.1117/12.2625458.

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Liu, Yafeng, Tongsheng Chen, and Qingming Luo. "Fluorescence correlation spectroscopy based upon two-photon excitation." In Biomolecular photonoics and Multidimensional Microscopy, edited by Qingming Luo and Min Gu. SPIE, 2003. http://dx.doi.org/10.1117/12.546244.

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Gregor, Ingo, Niels Radmacher, and Jörg Enderlein. "Fluorescence lifetime image scanning microscopy (Conference Presentation)." In Single Molecule Spectroscopy and Superresolution Imaging XIII, edited by Ingo Gregor, Rainer Erdmann, and Felix Koberling. SPIE, 2020. http://dx.doi.org/10.1117/12.2546532.

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Sarkar, Anirban, Irène Wang, Aditya Katti, Jörg Enderlein, Jacques Derouard, and Antoine Delon. "Fluorescence speckle image correlation spectroscopy (Conference Presentation)." In Unconventional Optical Imaging II, edited by Corinne Fournier, Marc P. Georges, and Gabriel Popescu. SPIE, 2020. http://dx.doi.org/10.1117/12.2558129.

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Krmpot, Aleksandar J., Stanko N. Nikolić, Marco Vitali, et al. "Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy." In European Conference on Biomedical Optics. OSA, 2015. http://dx.doi.org/10.1364/ecbo.2015.95360o.

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Krmpot, Aleksandar J., Stanko N. Nikolić, Marco Vitali, et al. "Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy." In European Conferences on Biomedical Optics, edited by Emmanuel Beaurepaire, Peter T. C. So, Francesco Pavone, and Elizabeth M. Hillman. SPIE, 2015. http://dx.doi.org/10.1117/12.2183935.

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Li, Yilun. "Variance lower bound on fluorescence microscopy image denoising." In High-Speed Biomedical Imaging and Spectroscopy VIII, edited by Keisuke Goda and Kevin K. Tsia. SPIE, 2023. http://dx.doi.org/10.1117/12.2647750.

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Qing, De-Kui, M. Pinar Mengu¨c¸, Fred A. Payne, and Mary-Grace C. Danao. "Fluorescence Correlation Spectroscopy for Detection of Trace Amount of Biological Agents." In ASME 2002 International Mechanical Engineering Congress and Exposition. ASMEDC, 2002. http://dx.doi.org/10.1115/imece2002-32357.

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A procedure to the trace amount of biological agents in water, based on fluorescence correlation spectroscopy and confocal microscopy, is explored. A special flow system is developed to increase the probability of detecting Escherichia coli (E. coli), and confocal microscopy is used to increase the sensitivity of the detection system. It is observed that concentrations higher than 1.5×105 E. coli per milliliter (2.5×10−16 M) are detectable with the present setup. This concentration corresponds to about 1.0 nM level of Rhodamine 6-G dyes. For detection of lower concentrations, further improvements need to be implemented to increase signal/background ratio. A detailed analysis of the optical system is presented and further improvements for the procedure are discussed.

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Iketaki, Yoshinori. "Fluorescence correlation spectroscopy for analysis of atto-liter space using three-dimensional super-resolution microscopy." In Optical Manipulation and Structured Materials Conference, edited by Takashige Omatsu, Hajime Ishihara, Keiji Sasaki, and Kishan Dholakia. SPIE, 2020. http://dx.doi.org/10.1117/12.2573793.

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