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Добірка наукової літератури з теми "Fluorescent recovery after photobleaching (FRAP)"

Автор: Grafiati

Опубліковано: 25 січня 2025

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Статті в журналах з теми "Fluorescent recovery after photobleaching (FRAP)"

Cadar, Adrian G., Tromondae K. Feaster, Kevin R. Bersell, Lili Wang, TingTing Hong, Joseph A. Balsamo, Zhentao Zhang, et al. "Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes." American Journal of Physiology-Cell Physiology 318, no. 1 (January 1, 2020): C163—C173. http://dx.doi.org/10.1152/ajpcell.00107.2019.

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Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

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Combs, Christian A., and Robert S. Balaban. "Enzyme-Dependant Fluorescence Recovery After Photobleaching (ED-FRAP): Application to Imaging Dehydrogenase Activity in Living Single Cells." Microscopy and Microanalysis 7, S2 (August 2001): 18–19. http://dx.doi.org/10.1017/s1431927600026167.

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Fluorescent recovery from photobleaching coupled with confocal microscopy was explored as a potential high-resolution method of imaging the distribution of enzyme activity in single living cardiac myocytes without relying on steady state measurements of fluorescence. On a fundamental level, much remains to be determined regarding how local conditions within a cell affect metabolism. Many studies have suggested that energy metabolism in muscle cells cannot be accurately described assuming a homogenous system of enzymatic reactions [1,2]. The autofluorescence of NADH has been used in many studies as a quantitative assay of mitochondrial energy metabolism [3,4], but studies of steady state fluorescence cannot distinguish between changes in energy production or utilization.In this study conditions were created where the fluorescent recovery of a probe would be solely dependent on cellular enzymatic activity (Enzyme Dependent Fluorescence Recovery after Photobleaching (ED-FRAP)). Experiments examining the inherent fluorescence of NADH (351nm excitation, 450 nm emission) were conducted on small droplets (less than 325 μm diameter) containing NADH alone, in droplets containing an enzyme system capable of synthesis of NADH (Figure 1A) and on isolated rabbit cardiac myocytes (Figure 1D). Photobleaching of the entire cell or droplet eliminated diffusion or bulk transport of NADH from non-bleached regions. Droplets containing NADH alone did not recover, while droplets containing enzyme were shown to recover exponentially (Figure 1B) with a rate constant of fluorescent recovery (kf) that was proportional to enzyme concentration (Figure 1C).

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Srikantha, Nishanthan, Yurema Teijeiro-Gonzalez, Andrew Simpson, Naba Elsaid, Satyanarayana Somavarapu, Klaus Suhling, and Timothy L. Jackson. "Determining vitreous viscosity using fluorescence recovery after photobleaching." PLOS ONE 17, no. 2 (February 10, 2022): e0261925. http://dx.doi.org/10.1371/journal.pone.0261925.

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Purpose Vitreous humor is a complex biofluid whose composition determines its structure and function. Vitreous viscosity will affect the delivery, distribution, and half-life of intraocular drugs, and key physiological molecules. The central pig vitreous is thought to closely match human vitreous viscosity. Diffusion is inversely related to viscosity, and diffusion is of fundamental importance for all biochemical reactions. Fluorescence Recovery After Photobleaching (FRAP) may provide a novel means of measuring intravitreal diffusion that could be applied to drugs and physiological macromolecules. It would also provide information about vitreous viscosity, which is relevant to drug elimination, and delivery. Methods Vitreous viscosity and intravitreal macromolecular diffusion of fluorescently labelled macromolecules were investigated in porcine eyes using fluorescence recovery after photobleaching (FRAP). Fluorescein isothiocyanate conjugated (FITC) dextrans and ficolls of varying molecular weights (MWs), and FITC-bovine serum albumin (BSA) were employed using FRAP bleach areas of different diameters. Results The mean (±standard deviation) viscosity of porcine vitreous using dextran, ficoll and BSA were 3.54 ± 1.40, 2.86 ± 1.13 and 4.54 ± 0.13 cP respectively, with an average of 3.65 ± 0.60 cP. Conclusions FRAP is a feasible and practical optical method to quantify the diffusion of macromolecules through vitreous.

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Braga, José, Joana M. P. Desterro, and Maria Carmo-Fonseca. "Intracellular Macromolecular Mobility Measured by Fluorescence Recovery after Photobleaching with Confocal Laser Scanning Microscopes." Molecular Biology of the Cell 15, no. 10 (October 2004): 4749–60. http://dx.doi.org/10.1091/mbc.e04-06-0496.

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Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22°C reduces the dextran diffusion rates by ∼30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.

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Lorén, Niklas, Joel Hagman, Jenny K. Jonasson, Hendrik Deschout, Diana Bernin, Francesca Cella-Zanacchi, Alberto Diaspro, et al. "Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice." Quarterly Reviews of Biophysics 48, no. 3 (August 2015): 323–87. http://dx.doi.org/10.1017/s0033583515000013.

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AbstractFluorescence recovery after photobleaching (FRAP) is a versatile tool for determining diffusion and interaction/binding properties in biological and material sciences. An understanding of the mechanisms controlling the diffusion requires a deep understanding of structure–interaction–diffusion relationships. In cell biology, for instance, this applies to the movement of proteins and lipids in the plasma membrane, cytoplasm and nucleus. In industrial applications related to pharmaceutics, foods, textiles, hygiene products and cosmetics, the diffusion of solutes and solvent molecules contributes strongly to the properties and functionality of the final product. All these systems are heterogeneous, and accurate quantification of the mass transport processes at the local level is therefore essential to the understanding of the properties of soft (bio)materials. FRAP is a commonly used fluorescence microscopy-based technique to determine local molecular transport at the micrometer scale. A brief high-intensity laser pulse is locally applied to the sample, causing substantial photobleaching of the fluorescent molecules within the illuminated area. This causes a local concentration gradient of fluorescent molecules, leading to diffusional influx of intact fluorophores from the local surroundings into the bleached area. Quantitative information on the molecular transport can be extracted from the time evolution of the fluorescence recovery in the bleached area using a suitable model. A multitude of FRAP models has been developed over the years, each based on specific assumptions. This makes it challenging for the non-specialist to decide which model is best suited for a particular application. Furthermore, there are many subtleties in performing accurate FRAP experiments. For these reasons, this review aims to provide an extensive tutorial covering the essential theoretical and practical aspects so as to enable accurate quantitative FRAP experiments for molecular transport measurements in soft (bio)materials.

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Golebiewska, Urszula, Jason G. Kay, Thomas Masters, Sergio Grinstein, Wonpil Im, Richard W. Pastor, Suzanne Scarlata, and Stuart McLaughlin. "Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages." Molecular Biology of the Cell 22, no. 18 (September 15, 2011): 3498–507. http://dx.doi.org/10.1091/mbc.e11-02-0114.

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To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D ∼ 1 μm2/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes.

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Verma, Sanjay K., Pratibha Kumari, Shagufi Naz Ansari, Mohd Ovais Ansari, Dondinath Deori, and Shaikh M. Mobin. "A novel mesoionic carbene based highly fluorescent Pd(ii) complex as an endoplasmic reticulum tracker in live cells." Dalton Transactions 47, no. 44 (2018): 15646–50. http://dx.doi.org/10.1039/c8dt02778a.

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Wagner, Stefan, Simion Chiosea, Maria Ivshina, and Jeffrey A. Nickerson. "In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160." Journal of Cell Biology 164, no. 6 (March 15, 2004): 843–50. http://dx.doi.org/10.1083/jcb.200307002.

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We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)–labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)–dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splicing speckled domains by an ATP-dependent mechanism. A second EJC protein, RNPS1, also has an ATP-dependent mobility, but SRm300, a protein that binds to SRm160 and participates with it in RNA splicing, remains immobile after ATP supplementation. This finding suggests that SRm160-containing RNA export, but not splicing, complexes have an ATP-dependent mobility. We propose that RNA export complexes have an ATP-regulated mechanism for release from binding sites at splicing speckled domains. In vitro fluorescence recovery after photobleaching is a powerful tool for identifying cofactors required for nuclear binding and mobility.

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Wadsworth, P., and E. D. Salmon. "Analysis of the treadmilling model during metaphase of mitosis using fluorescence redistribution after photobleaching." Journal of Cell Biology 102, no. 3 (March 1, 1986): 1032–38. http://dx.doi.org/10.1083/jcb.102.3.1032.

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One recent hypothesis for the mechanism of chromosome movement during mitosis predicts that a continual, uniform, poleward flow or "treadmilling" of microtubules occurs within the half-spindle between the chromosomes and the poles during mitosis (Margolis, R. L., and L. Wilson, 1981, Nature (Lond.), 293:705-711). We have tested this treadmilling hypothesis using fluorescent analog cytochemistry and measurements of fluorescence redistribution after photobleaching to examine microtubule behavior during metaphase of mitosis. Mitotic BSC 1 mammalian tissue culture cells or newt lung epithelial cells were microinjected with brain tubulin labeled with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF) to provide a fluorescent tracer of the endogenous tubulin pool. Using a laser microbeam, fluorescence in the half-spindle was photobleached in either a narrow 1.6 micron wide bar pattern across the half-spingle or in a circular area of 2.8 or 4.5 micron diameter. Fluorescence recovery in the spindle fibers, measured using video microscopy or photometric techniques, occurs as bleached DTAF-tubulin subunits within the microtubules are exchanged for unbleached DTAF-tubulin in the cytosol by steady-state microtubule assembly-disassembly pathways. Recovery of 75% of the bleached fluorescence follows first-order kinetics and has an average half-time of 37 sec, at 31-33 degrees C. No translocation of the bleached bar region could be detected during fluorescence recovery, and the rate of recovery was independent of the size of the bleached spot. These results reveal that, for 75% of the half-spindle microtubules, FRAP does not occur by a synchronous treadmilling mechanism.

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Kindermann, Stefan, and Štěpán Papáček. "On Data Space Selection and Data Processing for Parameter Identification in a Reaction-Diffusion Model Based on FRAP Experiments." Abstract and Applied Analysis 2015 (2015): 1–17. http://dx.doi.org/10.1155/2015/859849.

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Fluorescence recovery after photobleaching (FRAP) is a widely used measurement technique to determine the mobility of fluorescent molecules within living cells. While the experimental setup and protocol for FRAP experiments are usually fixed, data (pre)processing represents an important issue. The aim of this paper is twofold. First, we formulate and solve the problem ofrelevantFRAP data selection. The theoretical findings are illustrated by the comparison of the results of parameter identification when the full data set was used and the case when theirrelevant data set(data with negligible impact on the confidence interval of the estimated parameters) was removed from the data space. Second, we analyze and compare two approaches of FRAP data processing. Our proposition, surprisingly for the FRAP community, claims that the data set represented by the FRAP recovery curves in form of a time series (integrated data approachcommonly used by the FRAP community) leads to a larger confidence interval compared to thefull(spatiotemporal)data approach.

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Дисертації з теми "Fluorescent recovery after photobleaching (FRAP)"

Gaffield, Michael A. "FRAP measurements of synaptic vesicle mobility in motor nerve terminals /." Connect to abstract via ProQuest. Full text is not available online, 2007.

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Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2007.
Typescript. Includes bibliographical references (leaves 84-93). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

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Rodriguez-Enriquez, Ricardo. "Analysis of Bcl-2 family protein interactions in live cells by fluorescence recovery after photobleaching." Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/analysis-of-bcl2-family-protein-interactions-in-live-cells-by-fluorescence-recovery-after-photobleaching(aa5eb271-6e43-48f3-940d-f63763ea4629).html.

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The Bcl-2 family of proteins strictly regulates the intrinsic pathway of apoptosis. Direct physical interactions between Bcl-2 proteins regulate mitochondrial outerpermeabilisation (MOMP), which occurs in response to various cell stresses andapoptotic stimuli. How changes in Bcl-2 protein activity regulate apoptosiscommitment is still unclear, especially with regard to how they interact with eachother within the context of the mitochondrial membrane. Recent studies haveshown that Bcl-2 proteins exist in a dynamic equilibrium between the mitochondriaand the cytosol. In this thesis, by using FRAP, I have measured changes in Bcl-XLand Mcl-1 dynamics in single cells. Surprisingly, individual cells within a populationshow widely differing Bcl-XL and Mcl-1 dynamics. There is a corelation betweenBcl-XL and Mcl-1 dynamics with BH3-only protein expression. Anti-apoptotic andpro-apoptotic Bcl-2 proteins stabilise each other on the OMM. Together, theseresults indicate that cells constantly fine tune mitochondrial priming and thatanalysing anti-apoptotic Bcl-2 proteins by FRAP allows this to be measured at asingle cell level in real time before MOMP.

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Innhausen, u. Knyphausen Adrian zu [Verfasser], and Ralph [Akademischer Betreuer] Rupp. "A novel method for Fluorescence Recovery after Photobleaching (FRAP) analysis of chromatin proteins in pluripotent embryonic cells of the South African clawed frog X. laevis / Adrian zu Innhausen u. Knyphausen ; Betreuer: Ralph Rupp." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2020. http://d-nb.info/1221960563/34.

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Equy, Eloïse. "Polymersomes Janus : conception rationnelle, préparation et fonctionnalisation asymétrique pour le développement de systèmes auto-propulsés de délivrance ciblée de médicaments." Electronic Thesis or Diss., Bordeaux, 2024. http://www.theses.fr/2024BORD0465.

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Mimer les propriétés des cellules vivantes dans des protocellules artificielles suscite un intérêt considérable, notamment pour reproduire la motilité et le mouvement directionnel dans des applications de thérapies « intelligentes ». En raison de leur morphologie vésiculaire et de leur stabilité, les polymersomes présentent un grand potentiel pour la délivrance de médicaments, et l'introduction d'une asymétrie est essentielle pour permettre leur auto-propulsion. Bien que plusieurs approches, telles que la séparation de phase au sein de la membrane, aient été utilisées pour créer des polymersomes asymétriques, le choix des polymères appropriés reste un défi. Cette thèse de doctorat vise à concevoir des polymersomes asymétriques, de type Janus, capables de s'auto-propulser grâce à la décomposition enzymatique du glucose. Nous décrivons le développement de vésicules géantes unilamellaires de type Janus (JGUVs) par séparation de phase au sein de la membrane de deux copolymères à blocs distincts composés de blocs hydrophobes chimiquement incompatibles. En utilisant la théorie de Flory-Huggins, nous démontrons que les copolymères peuvent être rationnellement sélectionnés et conçus pour s'auto-assembler en polymersomes asymétriques, avec une séparation de phase modulable selon des paramètres tels que la composition, la masse molaire et la température. Notre méthode prédictive s'est avérée efficace pour les techniques d'auto-assemblage avec et sans solvant, permettant l'élaboration de diagrammes de phase génériques corrélant l'énergie libre de mélange à la morphologie des polymersomes, fournissant ainsi des indications clés pour la conception de JGUVs. Nous montrons également que la présence de solvant lors de la formation des vésicules permet d'étendre la gamme des polymères incompatibles pouvant être utilisés. De plus, nous avons réussi à contrôler, grâce à l'extrusion, la taille des vésicules tout en préservant leur morphologie Janus et avons montré que les JGUVs ainsi obtenus pouvaient être stables pendant plusieurs mois. Enfin, nous avons fonctionnalisé asymétriquement les JGUVs avec l'enzyme glucose oxydase par chimie click, et une étude préliminaire sur leur dynamique en présence de glucose est présentée, fournissant des indications pour leur utilisation comme micromoteurs
Mimicking the properties of living cells in artificial protocells has attracted significant interest, particularly for replicating motility and directional swimming for applications in smart therapeutics. Due to their vesicular and stable morphology, polymersomes hold great promise for drug delivery, and the introduction of asymmetry is crucial to enable self-propulsion. While several approaches, such as phase separation within the membrane, have been used to create asymmetric polymersomes, the selection of appropriate polymers remains a challenge. This PhD thesis aims at designing asymmetric, Janus-like polymersomes capable of self-propulsion, and powered by enzymatic glucose decomposition. We describe the development of Janus Giant Unilamellar Vesicles (JGUVs) through phase separation within the membrane of two distinct block copolymers comprising chemically incompatible hydrophobic blocks. We demonstrate, using the Flory-Huggins theory, that copolymers can be rationally selected and designed to self-assemble into asymmetric polymersomes, with tunable phase separation driven by parameters such as composition, molecular weight, and temperature. Our predictive method proves to be effective for both solvent-free and solvent-switch self-assembly processes, enabling the elaboration of generic phase diagrams correlating mixing free energy with polymersome morphology, providing valuable insights for JGUVs design. We also evidence that the presence of solvent during the vesicle formation broadens the range of incompatible polymers that can be used. Additionally, we successfully control, thanks to extrusion, the vesicle size while preserving their Janus morphology and evidence that the resulting JGUVs could be stable for several months. Furthermore, we asymmetrically functionalized JGUVs with glucose oxidase enzymes via click-chemistry, and a preliminary study on their dynamic behavior in the presence of glucose is presented, looking forward to their potential use as micromotors

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Irrechukwu, Onyi Nonye. "Role of matrix composition and age in solute diffusion within articular cartilage." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/19699.

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Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2008.
Committee Chair: Levenston, Marc; Committee Member: Garcia, Andres; Committee Member: Koros, William; Committee Member: Sambanis, Athanassios; Committee Member: Temenoff, Johnna; Committee Member: Vidakovic, Brani.

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Piette, Nathalie. "Micropatterning subcellulaire pour étudier la connectivité neuronale." Electronic Thesis or Diss., Bordeaux, 2024. http://www.theses.fr/2024BORD0034.

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L'impression protéique a initialement été utilisée pour reproduire et comprendre l’influence de la matrice extracellulaire sur les cellules et certains de leurs composants. Au cours de la dernière décennie, l'impression subcellulaire s’est développée, permettant d’étudier les interactions protéiques et leur rôle dans les voies de signalisation ainsi que dans la formation de synapses, immunologiques ou neuronales.La connexion synaptique est médiée par les protéines d’adhésion synaptique présentes de chaque côté de la synapse. En raison de la complexité de l’environnement synaptique mais également du manque de modèle in vitro permettant d’étudier la connexion synaptique dans un environnement biomimétique et contrôlé, les rôles exacts de ces protéines dans la synaptogénèse restent encore incertains. L’impression protéique subcellulaire est une solution potentielle pour combler ce manque. Pour cela, nous avons développé deux modèles biomimétiques basés sur l’impression protéique : un premier, utilisant des cellules hétérologues, permettant d’obtenir des informations sur la cinétique d’interaction des couples protéiques et ainsi de lier cela à leur fonction potentielle. Et un deuxième, utilisant des neurones primaires hippocampique, permettant de former des synapses artificielles pour étudier la nano-organisation de la synapse au cours du développement.Le système d’impression protéique PRIMO, commercialisé par Alvéole, qui co-finance cette thèse, est peu utilisé par les neuroscientifiques. En plus des objectifs biologiques, l'objectif industriel de cette thèse est de développer des méthodologies et des preuves de concept afin de démontrer les avantages et la faisabilité de la technologie PRIMO en neuroscience.En couplant notre premier modèle avec des techniques d’imagerie sur cellules vivantes (sptPALM et FRAP), nous avons pu différencier des cinétiques d’interaction entre différents couples de protéines d’adhésion synaptique mais également pour des interactions avec des protéines d’échafaudage. Une interaction labile pour SynCAM1, qui est connue pour son rôle dans la morphologie synaptique. Une forte et stable interaction pour Neuroligine1- Neurexine1β, due à la dimérisation de Neuroligine1, qui est indispensable pour la fonctionnalité de la synapse.Avec le second modèle, nous avons démontré, en présence de LRRTM2, la formation spécifique de synapses artificielles. Ces hémi-synapses présentent des caractéristiques morphologiques et fonctionnelles proches de synapses natives, avec la présence de vésicules et d’une activité calcique spontanée. Cependant, nous n’avons pas réussi à former de postsynapses artificielles avec Neurexine1β. Basés sur nos observations et une analyse bibliographique, nous avons formulé l’hypothèse que la postsynapse pourrait être le compartiment initiateur de la synaptogenèse.En conclusion, cette étude démontre : (1) que l’impression subcellulaire est un excellent modèle pour étudier la connectivité synaptique et l’adhésion de manière générale, aussi bien d’un point de vue fonctionnel qu’organisationnel. (2) Que les modèles d’hémi-synapses utilisant l’impression protéique sont plus spécifiques que les anciens modèles. (3) Que le système PRIMO ouvre de nombreuses perspectives en neurosciences via ses capacités d’impressions quantitatives
Micropatterning was initially employed to replicate and understand the influence of the extracellular matrix on cells and some of their components. Over the past decade, subcellular printing has emerged, enabling the study of protein interactions and their role in signaling pathways as well as in the formation of synaptic, immunological, or neuronal pathways.The synaptic connection is mediated by synaptic adhesion proteins present on each side of the synapse. Due to the complexity of the synaptic environment and the lack of in vitro models to study synaptic connection in a biomimetic and controlled environment, the exact roles of these proteins in synaptogenesis remain uncertain. Subcellular protein printing presents a potential solution to address this gap. For this purpose, we have developed two biomimetic models based on protein printing: a first one using heterologous cells, providing insights into the interaction kinetics of protein pairs and linking them to their potential function. And a second one using primary neurons, allowing the formation of artificial synapses to study synaptic nano-organization during development.The protein printing system PRIMO, commercialized by Alvéole, which is co-funding this thesis, is underutilized by neuroscientists. Besides these biological objectives, the industrial aim of this thesis is to develop methodologies and proofs of concept to demonstrate the advantages and feasibility of the PRIMO technology in neuroscience.By coupling our first model, based on heterologous cells, with live-cell imaging techniques (sptPALM and FRAP), we differentiated interaction kinetics among various synaptic adhesion protein pairs and also for interactions with scaffold proteins. A labile interaction was observed for SynCAM1, known for its role in synaptic morphology. A strong and stable interaction was evident for Neuroligin1/Neurexine1β due to Neuroligin1's dimerization, which is essential for synaptic functionality.With the second model using primary hippocampal neurons, we demonstrated, in the presence of LRRTM2, the specific formation of artificial synapses. These hemi-synapses exhibited morphological and functional characteristics close to native synapses, including the presence of vesicles and spontaneous calcium activity. However, we were unable to form artificial postsynapses with Neurexine1β. Based on our observations and bibliographic analysis, we hypothesize that the postsynapse could be the initiating compartment for synaptogenesis.In conclusion, this study demonstrates: (1) that subcellular printing is an excellent model to study synaptic connectivity and adhesion from both a functional and organizational perspective. (2) That models of hemi-synapses using micropatterning are more specific than previous models. (3) That the PRIMO system opens numerous perspectives in neuroscience through its quantitative printing capabilities

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Частини книг з теми "Fluorescent recovery after photobleaching (FRAP)"

Saito, Takumi, Daiki Matsunaga, and Shinji Deguchi. "Long-Term Fluorescence Recovery After Photobleaching (FRAP)." In Methods in Molecular Biology, 311–22. New York, NY: Springer US, 2023. http://dx.doi.org/10.1007/978-1-0716-2851-5_21.

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Carnell, Michael, Alex Macmillan, and Renee Whan. "Fluorescence Recovery After Photobleaching (FRAP): Acquisition, Analysis, and Applications." In Methods in Molecular Biology, 255–71. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-1752-5_18.

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Giakoumakis, Nickolaos Nikiforos, Maria Anna Rapsomaniki, and Zoi Lygerou. "Analysis of Protein Kinetics Using Fluorescence Recovery After Photobleaching (FRAP)." In Methods in Molecular Biology, 243–67. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-6810-7_16.

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van Royen, Martin E., Pascal Farla, Karin A. Mattern, Bart Geverts, Jan Trapman, and Adriaan B. Houtsmuller. "Fluorescence Recovery After Photobleaching (FRAP) to Study Nuclear Protein Dynamics in Living Cells." In The Nucleus, 363–85. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-60327-461-6_20.

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Takeshi, Shimi, Chan-Gi Pack, and Robert D. Goldman. "Analyses of the Dynamic Properties of Nuclear Lamins by Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS)." In Methods in Molecular Biology, 99–111. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3530-7_5.

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Warrington, Samantha J., Helen Strutt, and David Strutt. "Use of Fluorescence Recovery After Photobleaching (FRAP) to Measure In Vivo Dynamics of Cell Junction–Associated Polarity Proteins." In Methods in Molecular Biology, 1–30. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2035-9_1.

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Badrinarayanan, Anjana, and Mark C. Leake. "Using Fluorescence Recovery After Photobleaching (FRAP) to Study Dynamics of the Structural Maintenance of Chromosome (SMC) Complex In Vivo." In Methods in Molecular Biology, 37–46. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3631-1_4.

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Badrinarayanan, Anjana, and Mark C. Leake. "Fluorescence Recovery After Photobleaching (FRAP) to Study Dynamics of the Structural Maintenance of Chromosome (SMC) Complex in Live Escherichia coli Bacteria." In Methods in Molecular Biology, 31–41. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2221-6_4.

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Caydasi, Ayse Koca, and Gislene Pereira. "Evaluation of the Dynamicity of Mitotic Exit Network and Spindle Position Checkpoint Components on Spindle Pole Bodies by Fluorescence Recovery After Photobleaching (FRAP)." In Methods in Molecular Biology, 167–82. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-6502-1_13.

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Brown, Edward, Ania Majewska,, and Rakesh K. Jain. "Photobleaching and Recovery with Nonlinear Microscopy." In Handbook of Biomedical Nonlinear Optical Microscopy, 673–88. Oxford University PressNew York, NY, 1998. http://dx.doi.org/10.1093/oso/9780195162608.003.0026.

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Анотація:

Abstract Fluorescence recovery after photobleaching (FRAP), or fluorescence photobleaching recovery (FPR), describes a family of related techniques that measure transport properties of fluorescently labeled molecules. This is done by monitoring the evolution of a fluorescence signal after a spatially localized population of fluorophores is bleached by light. The classical FRAP experiment with one-photon excitation uses a focused laser beam to bleach a disk-shaped region of fluorescently labeled molecules in a thin sample such as a cell membrane or a lamellipodium (Fig. 26.1) (Axelrod et al., 1976). The same laser beam, greatly attenuated, generates fluorescence signal from that region as unbleached fluorophores diffuse in. The recovery in fluorescence signal is recorded by a photomultiplier tube (PMT) or similar detector, producing a fluorescence-versus-time curve (Fig. 26.2). Simple analytical formulas can often fit the fluorescence recovery curve, with the recovery time revealing the two-dimensional diffusion coefficient of the fluorescent molecule, and the extent of fluorescence recovery revealing the fraction of fluorophores that are mobile. More complex analysis with multiple diffusion coefficients can be used if the dynamics of the system warrant (Feder et al., 1996; Periasamy & Verkman, 1998). The three-dimensional hourglass profile of a focused laser beam (Born & Wolf, 1980) renders analytical solutions difficult unless the sample is relatively thin along the optical axis of the laser beam. Therefore, conventional one-photon FRAP is often limited to analysis of diffusion in thin two-dimensional systems such as cell membranes, where the intersection of the membrane with the laser beam produces a simple disk of photobleaching. In spite of this limitation, conventional one-photon FRAP has provided extensive insight into myriad cellular processes involving lateral membrane diffusion (Brown et al., 2005; Reits & Neefjes, 2001).

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Тези доповідей конференцій з теми "Fluorescent recovery after photobleaching (FRAP)"

Cao, Ziyi, Dustin Harmon, Jiayue Rong, Andreas Geiger, and Garth J. Simpson. "Fourier-transform Fluorescence Recovery after Photobleaching (FT-FRAP) diffusion imaging analysis." In Advanced Chemical Microscopy for Life Science and Translational Medicine 2022, edited by Garth J. Simpson, Ji-Xin Cheng, and Wei Min. SPIE, 2022. http://dx.doi.org/10.1117/12.2607631.

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BIRMINGHAM, J. J. "PHASE-FRAP: A NEW FREQUENCY-DOMAIN VARIANT OF FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING." In Proceedings of the Fifth Royal Society–Unilever Indo-UK Forum in Materials Science and Engineering. A CO-PUBLICATION OF IMPERIAL COLLEGE PRESS AND THE ROYAL SOCIETY, 2000. http://dx.doi.org/10.1142/9781848160163_0007.

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Docimo, Jennifer E., and John E. Novotny. "Measuring Diffusion in Non-Sectioned Articular Cartilage: A FRAP Sensitivity Study." In ASME 2008 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2008. http://dx.doi.org/10.1115/sbc2008-192375.

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Measuring the diffusion of molecules within articular cartilage is essential in characterizing its behavior. Information about this important mechanism may be useful to understanding changes in cartilage during degeneration or osteoarthritis. One method used in quantifying diffusion is fluorescence recovery after photobleaching (FRAP). The FRAP technique has been used in previous studies for cartilage [1] and various tissues [2]. In FRAP, a small region of interest (ROI) is selected within the tissue and fluorescent molecules are bleached using a higher laser power than would be used for imaging. Immediately following the ROI bleaching, bleached molecules diffuse out of the ROI as unbleached molecules diffuse into it. The average intensity data within the ROI is collected as a function of time. This data is then fit to a diffusion model usually resulting in a calculation of the diffusion constant, D (μm2/second) [3].

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Geiger, Andreas C., Casey J. Smith, and Garth J. Simpson. "Multi-photon excited Fourier-transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination." In Multiphoton Microscopy in the Biomedical Sciences XX, edited by Ammasi Periasamy, Peter T. So, and Karsten König. SPIE, 2020. http://dx.doi.org/10.1117/12.2545908.

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Oubekka, S. Daddi, R. Briandet, F. Waharte, M. P. Fontaine-Aupart, and K. Steenkeste. "Image-based Fluorescence Recovery After Photobleaching (FRAP) to dissect vancomycin diffusion-reaction processes in Staphylococcus aureus biofilms." In European Conference on Biomedical Optics. Washington, D.C.: OSA, 2011. http://dx.doi.org/10.1364/ecbo.2011.80871i.

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Daddi Oubekka, S., R. Briandet, F. Waharte, M. P. Fontaine-Aupart, and K. Steenkeste. "Image-based fluorescence recovery after photobleaching (FRAP) to dissect vancomycin diffusion-reaction processes in Staphylococcus aureus biofilms." In European Conferences on Biomedical Optics, edited by Nirmala Ramanujam and Jürgen Popp. SPIE, 2011. http://dx.doi.org/10.1117/12.889461.

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Simpson, Garth J. "Imaging of molecular mobility by spatial Fourier transform fluorescence recovery after photobleaching (FT-FRAP) with structured illumination." In Advanced Chemical Microscopy for Life Science and Translational Medicine 2024, edited by Garth J. Simpson, Ji-Xin Cheng, and Wei Min. SPIE, 2024. http://dx.doi.org/10.1117/12.3005854.

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Teijeiro Gonzalez, Yurema, Klaus Suhling, Andrew Beavil, Rebecca Beavil, James Levitt, Maddy Parsons, Elena Ortiz-Zapater, et al. "Fluorescence Recovery After Photobleaching (FRAP) with simultaneous Fluorescence Lifetime and time-resolved Fluorescence Anisotropy Imaging (FLIM and tr-FAIM)." In Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVI, edited by Thomas G. Brown and Tony Wilson. SPIE, 2019. http://dx.doi.org/10.1117/12.2508692.

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Cao, Ziyi, Dustin M. Harmon, Ruochen Yang, Aleksandr Razumtcev, Minghe Li, Mark S. Carlsen, Andreas C. Geiger, et al. "Diffusion mapping by Fourier-transform fluorescence recovery after photobleaching (FT-FRAP) for phase separation in drug formulations (Conference Presentation)." In Advanced Chemical Microscopy for Life Science and Translational Medicine 2023, edited by Garth J. Simpson, Ji-Xin Cheng, and Wei Min. SPIE, 2023. http://dx.doi.org/10.1117/12.2648473.

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Albro, Michael B., Vikram Rajan, Clark T. Hung, and Gerard A. Ateshian. "Fickian Behavior and Concentration-Dependence of the Diffusion of Dextran in Agarose." In ASME 2007 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2007. http://dx.doi.org/10.1115/sbc2007-176646.

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Various studies have attempted to quantify the effects of loading on nutrient transport in cartilage and other soft tissues. The application of a dynamic mechanical stimulus has been shown to significantly enhance the mechanical properties of chondrocyte-seeded agarose [1]. While the mechanism for this enhancement is still not completely understood, dynamic loading has been shown theoretically [2] as well as experimentally [3] to increase the uptake of large molecules. Since dextran is available in a wide range of molecular weights and can be conjugated with fluorphores, it has become a popular model system for studying solute transport in statically loaded and free swelling gels and tissues [4, 5]. To better characterize this model system, this study uses fluorescence recovery after photobleaching (FRAP) to investigate the Fickian behavior of linear dextran macromolecules as well as the dependence of its diffusivity on concentration.

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