Relevant bibliographies by topics / FLIM-FRET

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  1. Journal articles
  2. Dissertations / Theses
  3. Books
  4. Book chapters
  5. Conference papers

Academic literature on the topic 'FLIM-FRET'

Author: Grafiati
Published: 4 June 2021
Last updated: 27 April 2022

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Journal articles on the topic "FLIM-FRET"

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Garsha, Karl. "A Comment on using FLIM with FRET." Microscopy Today 14, no. 3 (May 2006): 52–53. http://dx.doi.org/10.1017/s1551929500057709.

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Depending on the nature of the study and what sort of information one is trying to gather through the use of FRET, FLIM has some compelling advantages in certain situations, and can provide a quantitative evaluation of the donor, acceptor and FRET pair stoichiometry. It does require access to specialized equipment and software. Different approaches to FLIM data acquisition have different strengths and weaknesses. For dynamic studies requiring high time resolution, FLIM acquisition times can fall well short of ideal.If a yes/no answer to whether FRET is occurring is all that is required, then the polarization anisotropy of the acceptor can be used to determine FRET between fluorescent proteins (Rizzo and Piston, 2005). This is a relatively simple and robust method for confirming the presence/absence of FRET.
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Lee, Jiung-De, Ping-Chun Huang, Yi-Cheng Lin, Lung-Sen Kao, Chien-Chang Huang, Fu-Jen Kao, Chung-Chih Lin, and De-Ming Yang. "In-Depth Fluorescence Lifetime Imaging Analysis Revealing SNAP25A-Rabphilin 3A Interactions." Microscopy and Microanalysis 14, no. 6 (November 6, 2008): 507–18. http://dx.doi.org/10.1017/s1431927608080628.

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AbstractThe high sensitivity and spatial resolution enabled by two-photon excitation fluorescence lifetime imaging microscopy/fluorescence resonance energy transfer (2PE-FLIM/FRET) provide an effective approach that reveals protein-protein interactions in a single cell during stimulated exocytosis. Enhanced green fluorescence protein (EGFP)–labeled synaptosomal associated protein of 25 kDa (SNAP25A) and red fluorescence protein (mRFP)–labeled Rabphillin 3A (RPH3A) were co-expressed in PC12 cells as the FRET donor and acceptor, respectively. The FLIM images of EGFP-SNAP25A suggested that SNAP25A/RPH3A interaction was increased during exocytosis. In addition, the multidimensional (three-dimensional with time) nature of the 2PE-FLIM image datasets can also resolve the protein interactions in the z direction, and we have compared several image analysis methods to extract more accurate and detailed information from the FLIM images. Fluorescence lifetime was fitted by using one and two component analysis. The lifetime FRET efficiency was calculated by the peak lifetime (τpeak) and the left side of the half-peak width (τ1/2), respectively. The results show that FRET efficiency increased at cell surface, which suggests that SNAP25A/RPH3A interactions take place at cell surface during stimulated exocytosis. In summary, we have demonstrated that the 2PE-FLIM/FRET technique is a powerful tool to reveal dynamic SNAP25A/RPH3A interactions in single neuroendocrine cells.
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Rajoria, Shilpi, Lingling Zhao, Xavier Intes, and Margarida Barroso. "FLIM-FRET for Cancer Applications." Current Molecular Imaging 3, no. 2 (February 4, 2015): 144–61. http://dx.doi.org/10.2174/2211555203666141117221111.

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Bücherl, Christoph A., Arjen Bader, Adrie H. Westphal, Sergey P. Laptenok, and Jan Willem Borst. "FRET-FLIM applications in plant systems." Protoplasma 251, no. 2 (January 4, 2014): 383–94. http://dx.doi.org/10.1007/s00709-013-0595-7.

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Wang, Shiqi, Binglin Shen, Sheng Ren, Yihua Zhao, Silu Zhang, Junle Qu, and Liwei Liu. "Implementation and application of FRET–FLIM technology." Journal of Innovative Optical Health Sciences 12, no. 05 (September 2019): 1930010. http://dx.doi.org/10.1142/s1793545819300106.

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With the development of the new detection methods and the function of fluorescent molecule, researchers hope to further explore the internal mechanisms of organisms, monitor changes in the intracellular microenvironment, and dynamic processes of molecular interactions in cells. Fluorescence resonance energy transfer (FRET) describes the energy transfer process between donor fluorescent molecules and acceptor fluorescent molecules. It is an important means to detect protein–protein interactions and protein conformation changes in cells. Fluorescence lifetime imaging microscopy (FLIM) enables noninvasive measurement of the fluorescence lifetime of fluorescent particles in vivo. The FRET–FLIM technology, which is use FLIM to quantify and analyze FRET, enables real-time monitoring of dynamic changes of proteins in biological cells and analysis of protein interaction mechanisms. The distance between donor and acceptor and their respective fluorescent lifetime, which are of great importance for studying the mechanism of intracellular activity can be obtained by data analysis and algorithm fitting.
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Ellis, Jonathan D., David Llères, Marco Denegri, Angus I. Lamond, and Javier F. Cáceres. "Spatial mapping of splicing factor complexes involved in exon and intron definition." Journal of Cell Biology 181, no. 6 (June 16, 2008): 921–34. http://dx.doi.org/10.1083/jcb.200710051.

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We have analyzed the interaction between serine/arginine-rich (SR) proteins and splicing components that recognize either the 5′ or 3′ splice site. Previously, these interactions have been extensively characterized biochemically and are critical for both intron and exon definition. We use fluorescence resonance energy transfer (FRET) microscopy to identify interactions of individual SR proteins with the U1 small nuclear ribonucleoprotein (snRNP)–associated 70-kD protein (U1 70K) and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) in live-cell nuclei. We find that these interactions occur in the presence of RNA polymerase II inhibitors, demonstrating that they are not exclusively cotranscriptional. Using FRET imaging by means of fluorescence lifetime imaging microscopy (FLIM), we map these interactions to specific sites in the nucleus. The FLIM data also reveal a previously unknown interaction between HCC1, a factor related to U2AF65, with both subunits of U2AF. Spatial mapping using FLIM-FRET reveals differences in splicing factors interactions within complexes located in separate subnuclear domains.
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Llères, David, John James, Sam Swift, David G. Norman, and Angus I. Lamond. "Quantitative analysis of chromatin compaction in living cells using FLIM–FRET." Journal of Cell Biology 187, no. 4 (November 16, 2009): 481–96. http://dx.doi.org/10.1083/jcb.200907029.

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We present a quantitative Förster resonance energy transfer (FRET)–based assay using multiphoton fluorescence lifetime imaging microscopy (FLIM) to measure chromatin compaction at the scale of nucleosomal arrays in live cells. The assay uses a human cell line coexpressing histone H2B tagged to either enhanced green fluorescent protein (FP) or mCherry FPs (HeLaH2B-2FP). FRET occurs between FP-tagged histones on separate nucleosomes and is increased when chromatin compacts. Interphase cells consistently show three populations of chromatin with low, medium, or high FRET efficiency, reflecting spatially distinct regions with different levels of chromatin compaction. Treatment with inhibitors that either increase chromatin compaction (i.e., depletion of adenosine triphosphate) or decrease chromosome compaction (trichostatin A) results in a parallel increase or decrease in the FLIM–FRET signal. In mitosis, the assay showed variation in compaction level, as reflected by different FRET efficiency populations, throughout the length of all chromosomes, increasing to a maximum in late anaphase. These data are consistent with extensive higher order folding of chromatin fibers taking place during anaphase.
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Kelly, Douglas J., Sean C. Warren, Dominic Alibhai, Sunil Kumar, Yuriy Alexandrov, Ian Munro, Anca Margineanu, et al. "Automated multiwell fluorescence lifetime imaging for Förster resonance energy transfer assays and high content analysis." Analytical Methods 7, no. 10 (2015): 4071–89. http://dx.doi.org/10.1039/c5ay00244c.

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Sambrook, Joseph, and David W. Russell. "Probing Protein Interactions Using GFP and FRET Stage 3: FLIM-FRET Measurements." Cold Spring Harbor Protocols 2006, no. 1 (June 2006): pdb.prot3822. http://dx.doi.org/10.1101/pdb.prot3822.

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Kelleher, M. T., F. Festy, P. R. Barber, C. Gillett, E. Ofo, A. Coolen, S. Pinder, et al. "Use of novel optical proteomics to profile breast cancer patients leading to individualised prognosis and tailored treatment." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): e22090-e22090. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.e22090.

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e22090 Background: Optical proteomics quantifies interactions between proteins and post-translational modifications by measuring Förster resonance energy transfer (FRET) quantified by fluorescence lifetime imaging microscopy (FLIM). This project aims to derive multiple high throughput optical proteomic markers, to predict metastatic risk at first diagnosis, and to perturb ‘high risk' protein-protein interactions using targeted therapeutics. This initial step develops robust FRET/FLIM assays, suitable for use in formalin fixed paraffin embedded (FFPE) tissue to be correlated with patient outcome. Methods: Fluorophore-conjugated antibodies to proteins involved in cell migration and survival, were applied to tissue microarrays (TMA), created from archived FFPE invasive ductal breast carcinoma samples. Where fluorophores are located within nanometer proximity, FRET occurs, thus allowing quantification of protein-protein interaction. Ezrin and PKCα phosphorylation, distribution, and interaction were imaged on four TMAs (patients diagnosed with early breast cancer 1984 -1987: 20 years follow-up data). Results: 71 patient samples were optically imaged. Patients were clustered based on the pairwise distances between 18 optical variables ‘input data'. Data are represented on self organising maps and dendrograms and correlated with clinical outcome ‘output data', displaying a heatmap distribution. Conclusions: Ezrin and PKCα phosphorylation, distribution, and interaction imaged optically within FFPE contain prognostic information regarding metastatic outcome in breast cancer, thus stepping ever closer to individualising prognosis. These advanced optics-based parameters informing on metastatic potential will be validated in prospective studies in conjunction with FRET/FLIM assays measuring HER2/HER3 dimerisation, and EGFR and HER2 ubiquitination in order to improve patient selection for targeted therapy. No significant financial relationships to disclose.
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Dissertations / Theses on the topic "FLIM-FRET"

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Yadav, Rahul B. "Studies of the mTOR signalling pathway using advanced FRET-FLIM technique." Thesis, Oxford Brookes University, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.543796.

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West, Lucien. "Illuminating cAMP dynamics at the synapse with multiphoton FLIM-FRET Imaging." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/43387.

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The study of signalling pathways within mammalian physiology has long been hindered by the size of the players involved, being far beyond the realms of the conventional light microscope. The advent of advanced fluorescent imaging techniques has revolutionised our capabilities to probe biological processes. The work in this thesis particularly utilised Förster resonance energy transfer (FRET), a fluorescence-based technique that can provide functional readouts of the processes underlying cellular function. Specifically I worked to develop and optimise a fluorescence imaging system for investigating the dynamics and function of cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger. The neuroscientific study of how the brain can learn and recall memories is a rapidly advancing field. The current challenges of tackling dementias, such as Alzheimer's disease, and preventing memory loss can only be addressed through better understanding of how memories can be stored. It is now believed that neurons retain memories within their synapses, the femtolitre structures that determine the strength of these connections. cAMP has been shown to play a distinctive role in orchestrating the retention of long term memory at the synaptic level. However, its spatial and temporal activation profiles are still not fully understood. To address this, my PhD project combined FRET readouts with cutting edge imaging techniques applied to synapses in neuronal cultures that provide reasonably convenient optical access. By examining the structure of these synapses, along with the measurement of cAMP concentration in different neuronal regions, this project uncovered the highly compartmentalised nature of this signalling molecule, seen to act directly at the sites of strengthening synapses. Through the optimisation of a FRET imaging system for studying activity in neuronal tissues, this project establishes a method for the future investigation of a plethora of pathways underlying the healthy functioning of the mammalian brain.
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Doll, Franziska [Verfasser]. "Visualizing Protein-Specific Post-Translational Modifications with FLIM-FRET Microscopy / Franziska Doll." Konstanz : KOPS Universität Konstanz, 2018. http://d-nb.info/1223372219/34.

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Andrews, Natalie Julia. "Spatio-temporal mapping of protein activity in live zebrafish using FRET FLIM OPT." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/59958.

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Optical projection tomography is a “mesoscopic” imaging technique, which fills a size gap in imaging, between techniques that provide subcellular resolution such as confocal laser scanning microscopy and cellular/whole body imaging techniques such as PET/MRI. OPT provides three dimensional (3D) whole body imaging utilising absorption or fluorescence contrast in mesoscopic (1-10 mm), transparent samples. This has the potential to be developed for use in biomedical research, for example in drug screening, developmental biology, and the study of disease mechanisms. To fully exploit the advantages of OPT, we image live zebrafish. These are the optimal size for imaging, and transparent mutants are readily available resulting in less scattering of optical radiation. They are also genetically tractable and many transgenic lines with fluorophore labels have been created. This thesis reports on the use of optical projection tomography (OPT) and fluorescence lifetime imaging (FLIM) to detect enzyme activity in whole live zebrafish. FRET biosensors for Caspase 1 and 3 activity were generated and the Caspase 3 FRET biosensor was validated in mammalian cell culture. Transgenic zebrafish expressing the biosensors ubiquitously under control of the ubiquitin (Ubi), macrophage (mpeg) and neutrophil (LysC) promoters were generated and used to validate the biosensors within live zebrafish. Caspase 3 is activated as part of the apoptotic pathway, in response to gamma irradiation. After exposure to 25 Gy irradiation, Caspase 3 activity is evident in the head region of 24 hpf zebrafish ubiquitously expressing the Caspase 3 biosensor, through both confocal and OPT FLIM imaging. FLIM OPT imaging allows visualisation of activity via production of 3D lifetime maps. Programs were generated to enable qualification and quantification of the data. Preliminary validation of the novel Caspase 1 biosensor indicates the biosensor is functional and it is possible to use FLIM to detect Caspase 1 activity in live zebrafish, in response to immune activation via tail transection. Overall, these findings demonstrate that FLIM OPT is a useful tool in mesoscopic imaging, able to identify enzyme activity in a whole live zebrafish, in 3D.
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Loukil, Abdelhalim. "Etude de la cycline A2 : interactions, dégradation et mise en évidence du rôle de l'autophagie." Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20115.

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Le cycle cellulaire est finement régulé dans le temps et l'espace. Nous avons abordé les aspects dynamiques des interactions que la cycline A2 entretient avec ses partenaires Cdk1, Cdk2 et l'ubiquitine au cours du cycle cellulaire, dans des lignées cellulaires humaines. A cette fin, nous avons eu recours aux approches de FRET (Förster/fluorescence resonance energy transfer) et de FLIM (fluorescence lifetime imaging microscopy). Ceci nous a permis de montrer que les formes ubiquitinylées de la cycline A2 apparaissent principalement sous forme de foyers en prométaphase et se propagent ensuite à l'ensemble de la cellule. En outre, nous avons découvert que l'autophagie participe à la dégradation de cette cycline en mitose. Nous discutons les implications de ces observations quant à un rôle éventuel de la cycline A2 au moment de la formation de l'anneau de constriction, ainsi que de la participation de l'autophagie via cette cycline, dans la réponse aux dommages à l'ADN en mitose
The cell cycle is finely regulated in time and space. We have studied the dynamical aspect of the interactions between cyclin A2 and its partners Cdk1, Cdk2 and ubiquitin during the cell cycle, in human cell lines. To this aim, we have used FRET (Förster/fluorescence resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) techniques. We have thus shown that ubiquitylated forms of cyclin A2 are detected predominantly in foci in prometaphase, before spreading throughout the cell. Moreover, we have shown that autophagy contributes to cyclin A2 degradation in mitosis. We discuss the implications of these observations regarding a possible role of cyclin A2 when the cleavage furrow forms, and the participation of autophagy in DNA damage response in mitosis
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Nobis, Max. "In vivo FLIM-FRET imaging of pharmacodynamics and disease progression in mouse cancer models." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7283/.

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Perrin, Aurélien. "Caenorhabditis elegans un modèle d’étude des différents compartiments du noyau : de l’étude d’un stress du nucléole par inhibition de la voie de neddylation à la mesure de la compaction de la chromatine in vivo." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT049/document.

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NEDD8, molécule de la famille de l’ubiquitine est essentielle au développement, à la croissance et à la viabilité d’un organisme, de plus c’est une cible prometteuse en thérapeutique. Nous avons découvert que l’inhibiteur spécifique de la NEDDylation, MLN4924 altère la morphologie sans fragmentation et augmente la surface du nucléole de cellules humaines et de noyaux de la lignée germinale de Caenorhabditis elegans. Une approche de protéomique quantitative (SILAC) combiné à l’analyse de la production des ARNr et des ribosomes montrent que MLN4924 change la composition protéique du nucléole sans affecter l’activité transcriptionnelle de l’ARN pol I. Notre analyse montre que MLN4924 active p53 par la voie RPL11/RPL5-Mdm2 caractéristique d’un stress du nucléole. Cette étude identifie le nucléole comme une cible intéressante dans l’utilisation d’inhibiteurs de la NEDDylation et apporte un nouveau mécanisme d’activation de p53 par inhibition de la voie NEDD8.Dans une seconde étude nous avons adapté la méthode de FLIM-FRET (« Fluorescence Lifetime Imaging Microscopy – Förster Resonance Energy Transfer ») à l’étude de la compaction de la chromatine à l’échelle du nanomètre dans un organisme vivant. Le nématode Caenorhabditis elegans s’est révélé être un modèle de choix. Au sein des chromosomes méiotiques, nous avons identifié différentes régions de compaction, de niveau variable par mesure du FRET entre histones fusionnées à des protéines fluorescentes. Par une approche originale d’ARN interférence et injection d’un « extra-chromosome » nous avons défini l’architecture à une nano-échelle de différents états de l’hétérochromatine et montré que cette organisation est contrôlée par les protéines HP1 « Heterochromatin Protein 1 » et SETDB1, une protéine « H3-Lysine 9 methyl transferase ». Nous avons également montré que la compaction de l’hétérochromatine est dépendante des condensines I et II et plus particulièrement la condensine I contrôle l’état faiblement compacté de la chromatine.Nos travaux ont confirmé que C. elegans est un modèle d’intérêt majeur pour l’étude des compartiments nucléaires et parfaitement adapté pour des études pré-clinique
The ubiquitin-like molecule NEDD8 is conserved and essential for viability, growth and development; its activation pathway is a promising target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human cells and Caenorhabditis elegans germ cells in the absence of nucleolar fragmentation. Through SILAC proteomic analysis and rRNA production, processing and ribosome profiling, we show that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of the NEDDylation pathway and provides a mechanism for p53 activation upon NEDD8 inhibition.Then we adapted a quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay the nano-scale chromatin compaction in a living organism, the nematode Caenorhabditis elegans. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1) and SETDB1 H3-lysine-9 methyl-transferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.We confirm that C. elegans is an interesting model to study nuclear signalling and perfectly adapt to be a platform for pre-clinical studies
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Engel, Stephanie Vanessa. "Assembly von Influenzaviren." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15918.

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Es wird angenommen, dass das Influenzavirus-Glykoprotein Hämagglutinin (HA) für seine Funktion sowohl bei der Virusfreisetzung als auch bei der Fusion von viraler und zellulärer Membran mit Cholesterin- und Sphingolipidreichen Domänen, sogenannten Membran-Rafts, assoziiert sein muss. Aus diesem Grund sollte in dieser Arbeit die Membran-Raft-Affinität von HA in lebenden Zellen mittels FLIM-FRET gemessen werden. Dabei wurde mit Hilfe der Fluroreszenz-Lebenszeit-Messung (FLIM) der Förster-Resonanz-Energie-Transfer (FRET) von fluoreszenzmarkiertem HA auf einen etablierten Raft-Marker bestimmt. Diese Messungen zeigten, dass beide Proteine in gemeinsamen Klustern in der Plasmamembran vorkommen. Durch Cholesterinentzug und durch den Einsatz von Cytochalasin D, welches die Mikrofilamente zerstört, konnte diese Klusterbildung reduziert werden. Demnach tragen sowohl die Membran-Rafts als auch das Aktinnetzwerk zu dieser Klusterbildung bei. Mittels FLIM-FRET konnte zusätzlich bestätigt werden, dass die Signale für die Detergenslöslichkeit von HA in Triton-Extraktionsexperimenten, die Palmitylierung und die stark hydrophoben Aminosäuren zu Beginn der Transmembrandomäne (TMD), auch im lebenden System eine wichtige Rolle spielen. Zusätzlich konnten biochemische Experimente zeigen, dass die hydrophoben Aminosäuren zu Beginn der HA-TMD den intrazellulären Transport, nach der Trimerbildung, entscheidend verzögern. Diese Verzögerung ist vermutlich auf einer erschwerten Integration dieser Proteine in die Membran-Rafts begründet. Die virale Fusion mit der Wirtszellmembran wird durch eine pH5-Behandlung vermittelte Konformationsänderung von HA ausgelöst. FLIM-FRET-Messungen zeigten für die pH5-Konformation von HA eine verglichen mit der pH7-Konformation verringerte Klusterbildung mit dem Raft-Marker. Somit ist offensichtlich, dass die Membranfusion-vermittelnde HA-Konformation eine verringerte Raft-Affinität besitzt. Diese verringerte Raft-Affinität könnte eine wichtige Rolle bei der Störung der Lipide an der Fusionsstelle spielen und somit die Bildung und/oder Vergrößerung der Fusionspore erleichtern.
It has been supposed that the hemagglutinin (HA) of influenza virus is recruited to cholesterol- and sphingolipid-enriched domains, also named membrane-rafts, to accomplish its function in virus budding and membrane fusion. This study aimed at verifying the affinity of HA for membrane-rafts in living cells using fluorescence-lifetime imaging microscopy to measure Förster’s resonance energy transfer (FLIM-FRET). FLIM-FRET revealed strong clustering between a fluorescence-tagged HA-protein and a well-established raft-marker in CHO cells. Clustering was significantly reduced when rafts were disintegrated by cholesterol depletion and when microfilaments were disrupted with cytochalasin D. Thus, membrane-rafts as well as the actin meshwork contribute synergistically to clustering. Clustering was also reduced by the removal of the known signals for the association of HA with detergent-resistant-membranes, the palmitoylation and the first amino acids in the transmembrane region (TMR). Since these mutations are obviously important for the raft-association of HA their function during the transport through the ER and the Golgi-complex was studied. These investigations showed that the exchange of the first three amino acids of the HA-TMR led to a decelerated transport after trimer-formation of the protein, probably due to retarded integration of these proteins into membrane-raft domains. Mediating viral fusion with the host cell membrane requires an irreversible conformational change of HA. FLIM-FRET studies of this low pH conformation unveiled that the clustering with the raft-marker is decisively reduced compared to the pre-fusion conformation of the protein. It might be assumed that the fusion-mediating conformation of HA reduces the proteins affinity for membrane-rafts. Therefore it is likely that this reduced affinity for rafts after the conformational change is relevant to cause perturbation of lipids at the fusion site and thereby facilitating the formation and/or enlargement of the fusion pore.
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Sizaire, Florian. "Développement d’un criblage automatisé de l’activité kinase d’un biosenseur Aurora A par FLIM." Thesis, Rennes 1, 2019. http://www.theses.fr/2019REN1B033.

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La surexpression d’Aurora A est un marquer majeur de certains cancers épithéliaux. Ce gène code pour la kinase multifonctionnelle Aurora A et son activation est requise pour l’entrée et la progression vers la mitose. Jusqu'à présent, aucun inhibiteur de cet oncogène n'a été approuvé par la FDA et il est donc primordial d'identifier de nouvelles molécules. Notre équipe a développé un biosenseur FRET (Forster Resonance Energy Transfer) pour l’activité kinase d’Aurora A, constitué de la kinase entière flanquée de deux fluorophores, une GFP et une mCherry. Le changement de conformation d’Aurora A lorsqu’elle est activée rapproche les fluorophores et augmente l’efficacité du FRET. Il est ainsi possible de suivre l’activation d’Aurora A dans les cellules vivantes exprimant le biosenseur à des niveaux endogènes. Nous pouvons mesurer le FRET en utilisant la technique de FLIM (Fluorescence Lifetime Imaging Microscopy) grâce à un microscope développé dans l’équipe et appelé fastFLIM. Mes travaux de thèse ont consisté à développer une stratégie de criblage robuste et automatisée en combinant les capacités du fastFLIM et le biosenseur d’activité d’Aurora A. Cette stratégie basée sur une automatisation des acquisitions et de l’analyse de données a permis de cribler une banque de molécules en plaque 96 puits afin de trouver de potentielles inhibiteurs de l’activité kinase d’Aurora A. De plus, j’ai participé à la validation du biosenseur pour un suivi de l’activité kinase dans des cellules vivantes en montrant que les variations de FRET mesurées correspondent bien à l’état de phosphorylation d’Aurora A sur le résidu Thréonine 288, marqueur de son activation. Enfin, j’ai participé à l’élaboration de nouvelles techniques de microscopie pour suivre l’activité du biosenseur. Pour cela, j’ai utilisé un biosenseur de type homoFRET avec l’enjeu de pouvoir utiliser plusieurs biosenseurs dans un contexte multiplex. J’ai aussi utilisé la technique de 2c-FCCS (2-colors Fluorescence Cross Correlation Spectroscopy) sur le biosenseur Aurora A afin de pouvoir mesurer le FRET dans des régions où celui-ci est faiblement exprimant et dont la mesure de durée de vie de fluorescence n’est pas possible par le FLIM. Ainsi, mes travaux de thèse s’inscrivent dans la tendance à développer une microscopie quantitative et autonome avec comme enjeu d’apporter un grande nombre de données phénotypiques
Overexpression of Aurora A is a major marker of some epithelial cancers. This gene encodes the multifunctional Aurora A kinase and its activation is required for entry and progression to mitosis. So far, no inhibitor of this oncogene has been approved by the FDA and it is therefore essential to identify new molecules. Our team developed a Forster Resonance Energy Transfer (FRET) biosensor for Aurora A kinase activity, consisting of the entire kinase flanked by two fluorophores, a GFP and a mCherry. The conformational change of Aurora A when it is activated brings the fluorophores closer and increases FRET efficiency. It is thus possible to follow the activation of Aurora A in living cells expressing the biosensor at endogenous levels. We can measure FRET using FLIM (Fluorescence Lifetime Imaging Microscopy) technique using a microscope developed in the team called fastFLIM. My thesis work consisted of developing a robust and automated screening strategy by combining the capabilities of fastFLIM and the Aurora A activity biosensor. This strategy based on automation of acquisitions and data analysis allowed to screen a library of 96-well plate molecules for potential inhibitors of Aurora A kinase activity. In addition, I participated in the validation of the biosensor for kinase activity monitoring in living cells, showing that the FRET variations measured correspond to the phosphorylation state of Aurora A on the Threonine 288 residue, a marker of its activation. Finally, I participated in the development of new microscopy techniques to monitor the activity of the biosensor. For that, I used a homoFRET biosensor with the challenge of being able to use several biosensors in a multiplex context. I also used the 2c-FCCS (2-color Fluorescence Cross Correlation Spectroscopy) technique on the Aurora A biosensor to measure FRET in regions where it is weakly expressing and whose lifetime measurement of Fluorescence is not possible by FLIM. Thus, my thesis work is part of the trend to develop a quantitative and autonomous microscopy with the challenge of providing a large number of phenotypic data
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Ziegler, Cornelia. "Imagerie quantitative de l'assemblage de la NADPH oxydase des phagocytes en cellules vivantes par des approches FRET-FLIM." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS048/document.

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La NADPH oxydase des phagocytes (NOX2) est responsable de la production d’anions superoxydes qui sont les précurseurs des autres formes réactives de l’oxygène. NOX2 est une enzyme majeure de la réponse immunitaire. Les dysfonctionnements de NOX2 sont associés à de nombreuses pathologies et donc il convient d’en comprendre les détails de la régulation. Cette oxydase est composée de cinq sous-unités : deux protéines membranaires, gp91phox et p22phox et 3 protéines cytosoliques p47phox, p67phox et p40phox. D’après les études in vitro avec des protéines purifiées, les protéines cytosoliques sont supposées former un complexe ternaire qui se déplace à la membrane avec une petite protéine G, Rac, au moment l’activation.L’objectif de ce projet est de caractériser les interactions spécifiques entre les sous-unités cytosoliques de NOX2 en cellules vivantes en utilisant le phénomène de transfert résonant d’énergie de type Förster (FRET) entre deux fluorophores, un donneur et un accepteur. Ici les fluorophores seront des protéines fluorescentes de la famille de la GFP. Elles sont fusionnées à deux sous-unités. L’efficacité du FRET dépend de la distance entre les fluorophores et permet ainsi de caractériser les interactions entre les protéines d’intérêt. Une méthode rapide d’identification des situations où le FRET est positif a été mise au point par cytométrie en flux. Des études détaillées et quantitatives ont ensuite été réalisées en utilisant l’imagerie de durée de fluorescence (FLIM) du donneur. Le FLIM, combiné à l’utilisation de donneurs présentant une durée de vie mono-exponentielle, permet de déterminer directement des efficacités de FRET apparentes et moléculaires, qui contiennent, toutes les deux, des informations qualitatives et quantitatives sur l’interaction et la structure des protéines impliquées. De ces données, il est possible d’extraire la fraction des donneurs interagissant avec un accepteur. Les informations obtenues à partir des données de FRET-FLIM permettent une meilleure compréhension de l’organisation et de la régulation de NOX2 tout en permettant une estimation des constantes de dissociation (Kd). Afin de confirmer ces résultats, des expériences de spectroscopie de corrélation de fluorescence à deux couleurs (FCCS) ont été réalisées. Cette méthode complétement indépendante n’est pas basée sur la distance entre fluorophores comme le FRET mais sur leur co-diffusion à travers un petit volume d’observation dans le cytoplasme cellulaire.L’approche FRET-FLIM nous a tout d’abord permis d’observer les interactions entre hétéro-dimères formés de deux sous-unites différentes en cellules vivantes et d’exclure la formation d’homo-dimères entre deux sous-unités identiques. Etant donné la bonne précision des mesures de FLIM, nous avons pu comparer les informations structurales obtenues en cellules avec les données structurales issues d’études sur les protéines purifiées in vitro et nous avons constaté qu’elles sont en bon accord. Nous avons ensuite aligné les structures disponibles pour proposer un premier modèle 3D du complexe cytosolique de la NADPH oxydase au repos dans le cytosol cellulaire.Les fractions de protéines en interaction sont pour tous les hétéro-dimères autour de 20% ce qui n’est pas en accord avec l’hypothèse courante qui propose que toutes les sous-unités cytosoliques soient sous forme de complexe. Toutefois nos premiers résultats de FCCS confirment ce résultat extrait des données de FRET-FLIM. Nous proposons donc que la complexation des sous-unités cytosolique pourrait être impliquée dans la régulation de la NADPH oxydase. Des études complémentaires seront nécessaires pour valider cette nouvelle hypothèse. Les constantes de dissociation Kd estimées à partir de nos résultats sont micromolaires et donc un ordre de grandeur plus élevé que les valeurs nanomolaires déterminées in vitro. Des mesures plus détaillées de FCCS pourront compléter et valider ces résultats
The phagocyte NADPH oxidase (NOX2) is a key enzyme of the immune system generating superoxide anions, which are precursors for other reactive oxygen species. Any dysfunctions of NOX2 are associated with a plethora of diseases and thus detailed knowledge about its regulation is needed. This oxidase is composed of five subunits, the membrane-bound gp91phox and p22phox and the cytosolic p47phox, p67phox, and p40phox. The latter are assumed to be in a ternary complex that translocates together with the small GTPase Rac to the membranous subunits during activation.Our aim was to discover and to characterize specific interactions of the cytosolic subunits of NOX2 in live cells using a Förster Resonance Energy Transfer (FRET) based approach: Since FRET depends on the distance between two fluorophores, it can be used to reveal protein-protein interactions non-invasively by studying fluorescent protein tagged subunits. To have a rapid method on hand to reveal specific interactions, a flow cytometer based FRET approach was developed. For more detailed studies, FRET was measured by fluorescence lifetime imaging microscopy (FLIM), because it allows a direct determination of the apparent and molecular FRET efficiency, which contains both qualitative and quantitative information about the interaction and the structure of the interacting proteins. Furthermore, the FRET-FLIM approach enables an estimation of the fraction of bound donor. This information itself is important for a better understanding of the organisation and regulation of the NOX2, but it is also necessary for the calculation of the dissociation constant Kd from the FRET-FLIM data. To confirm the findings obtained by FRET-FLIM fluorescence cross correlation spectroscopy (FCCS) experiments were performed. This completely independent method is not based on distances like FRET but on the observation of the co diffusion of the fluorescently labelled samples when they move across a small observation volume inside the cells.The FRET-FLIM approach allowed us in a first step to discover heterodimeric interactions between all cytosolic subunits in live cells. Due to the good precision of the results, we were able to extract structural information about the interactions and to compare them with available structural data obtained from in vitro studies. The information from FRET-FLIM was coherent with in vitro data. We then aligned the available structures leading to the first 3D model of the cytosolic complex of the NADPH oxidase in the resting state in live cells.Additionally, the bound fraction for all heterodimeric interactions derived by FRET-FLIM is around 20 %, which is in contrast to the general belief that all cytosolic subunits are bound in complex. The first FCCS results support our findings. Therefore, we believe that the complexation of the cytosolic subunits could be involved in the regulation of the NADPH oxidase and should be investigated further. The estimated Kd derived from the FRET-FLIM approach is in the low micomolar range, which is an order of a magnitude higher than the nanomolar range of in vitro studies.In conclusion, we showed that our quantitative FRET-FLIM approach is not only able to distinguish between specific and unspecific protein-protein interactions, but gives also information about the structural organisation of the interacting proteins. The high precision of the FRET-FLIM data allow the determination of the bound fraction and an estimation of the Kd in live cells. FCCS is a complementary method, which can verify these quantitative findings. However, it cannot replace FRET-FLIM completely as it does not give any structural information.With respect to the biological outcome of this project, we can propose for the first time a 3D-model of the cytosolic complex of the NADPH oxidase covering the in vitro as well as the live cell situation. Additionally, the small bound fraction found here may raise new ideas on the regulation of this vital enzyme
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Books on the topic "FLIM-FRET"

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FRET and FLIM techniques. Amsterdam: Elsevier, 2009.

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Gadella, Theodorus W. J. FRET and FLIM Techniques. Elsevier Science & Technology Books, 2011.

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Fret and Flim Techniques. Elsevier, 2009. http://dx.doi.org/10.1016/s0075-7535(08)x0001-4.

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Book chapters on the topic "FLIM-FRET"

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Kukk, Olga, Jeffrey Klarenbeek, and Kees Jalink. "Time-Domain Fluorescence Lifetime Imaging of cAMP Levels with EPAC-Based FRET Sensors." In cAMP Signaling, 105–16. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2245-2_7.

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AbstractSecond messenger molecules in eukaryotic cells relay the signals from activated cell surface receptors to intracellular effector proteins. FRET-based sensors are ideal to visualize and measure the often rapid changes of second messenger concentrations in time and place. Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative technique for measuring FRET. Given the recent development of commercially available, sensitive and photon-efficient FLIM instrumentation, it is becoming the method of choice for FRET detection in signaling studies. Here, we describe a detailed protocol for time domain FLIM, using the EPAC-based FRET sensor to measure changes in cellular cAMP levels with high spatiotemporal resolution as an example.
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Morton, Penny E., and Maddy Parsons. "Measuring FRET Using Time-Resolved FLIM." In Methods in Molecular Biology, 403–13. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-207-6_27.

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Periasamy, Ammasi, Nirmal Mazumder, Yuansheng Sun, Kathryn G. Christopher, and Richard N. Day. "FRET Microscopy: Basics, Issues and Advantages of FLIM-FRET Imaging." In Springer Series in Chemical Physics, 249–76. Cham: Springer International Publishing, 2015. http://dx.doi.org/10.1007/978-3-319-14929-5_7.

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Bücherl, Christoph, José Aker, Sacco de Vries, and Jan Willem Borst. "Probing Protein–Protein Interactions with FRET–FLIM." In Plant Developmental Biology, 389–99. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-60761-765-5_26.

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Yoo, Tae Yeon, and Daniel J. Needleman. "Studying Kinetochores In Vivo Using FLIM-FRET." In Methods in Molecular Biology, 169–86. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3542-0_11.

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Kao, Fu-Jen, Gitanjal Deka, and Nirmal Mazumder. "Cellular Autofluorescence Detection Through FLIM/FRET Microscopy." In Topics in Applied Physics, 471–82. Dordrecht: Springer Netherlands, 2014. http://dx.doi.org/10.1007/978-94-017-9392-6_26.

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Day, Richard N. "Chapter 3 Visible Fluorescent Proteins for FRET-FLIM." In Flim Microscopy in Biology and Medicine, 65–92. 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742: CRC Press, 2009. http://dx.doi.org/10.1201/9781420078916-4.

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Evans, Paul R., Long Yan, and Ryohei Yasuda. "Imaging Neuronal Signal Transduction Using Multiphoton FRET-FLIM." In Neuromethods, 111–30. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9702-2_6.

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Richert, Ludovic, Pascal Didier, Hugues de Rocquigny, and Yves Mély. "Monitoring HIV-1 Protein Oligomerization by FLIM FRET Microscopy." In Springer Series in Chemical Physics, 277–307. Cham: Springer International Publishing, 2015. http://dx.doi.org/10.1007/978-3-319-14929-5_8.

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Laptenok, Sergey P., Joris J. Snellenburg, Christoph A. Bücherl, Kai R. Konrad, and Jan Willem Borst. "Global Analysis of FRET–FLIM Data in Live Plant Cells." In Methods in Molecular Biology, 481–502. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-649-8_21.

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Conference papers on the topic "FLIM-FRET"

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Elangovan, Masilamani, Richard N. Day, and Ammasi Periasamy. "FRET-FLIM microscopy." In International Symposium on Biomedical Optics, edited by Ammasi Periasamy and Peter T. C. So. SPIE, 2002. http://dx.doi.org/10.1117/12.470682.

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Garcia, Edwin, Wenjun Guo, Sunil Kumar, Frederik Görlitz, Hugh Sparks, Yuriy Alexandrov, Ian Munro, et al. "FLIM, FRET and high content analysis." In Multiphoton Microscopy in the Biomedical Sciences XX, edited by Ammasi Periasamy, Peter T. So, and Karsten König. SPIE, 2020. http://dx.doi.org/10.1117/12.2547517.

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Warren, Sean, Christopher Kimberley, Anca Margineanu, Romain Laine, Christopher Dunsby, Matilda Katan, and Paul M. French. "FLIM-FRET of Cell Signalling in Chemotaxis." In Optical Molecular Probes, Imaging and Drug Delivery. Washington, D.C.: OSA, 2013. http://dx.doi.org/10.1364/omp.2013.mth1c.5.

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Wallrabe, Horst, Yuangsheng Sun, Zdenek Svindrych, and Ammasi Periasamy. "Comprehensive quantitative evaluation of FLIM-FRET microscopy." In SPIE BiOS, edited by Ammasi Periasamy, Peter T. C. So, and Karsten König. SPIE, 2015. http://dx.doi.org/10.1117/12.2180162.

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Chen, Ye, and Ammasi Periasamy. "Two-photon FLIM-FRET microscopy for protein localization." In Biomedical Optics 2004, edited by Ammasi Periasamy and Peter T. C. So. SPIE, 2004. http://dx.doi.org/10.1117/12.538312.

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Zhang, Yinan, Yu Chen, Jun Yu, and David J. S. Birch. "Endosytosis Study of Gold Nanoparticles through FRET-FLIM Approach." In Biomedical Engineering. Calgary,AB,Canada: ACTAPRESS, 2017. http://dx.doi.org/10.2316/p.2017.852-032.

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Poland, Simon P., Simao Coelho, Nikola Krstajić, David Tyndall, Richard Walker, James Monypenny, David D. Li, Robert Henderson, and Simon Ameer-Beg. "Development of a fast TCSPC FLIM-FRET imaging system." In SPIE BiOS, edited by Ammasi Periasamy, Karsten König, and Peter T. C. So. SPIE, 2013. http://dx.doi.org/10.1117/12.2004199.

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Gabrielaitis, Dovydas. "Development of a novel FLIM-FRET based synaptic sensor." In European Microscopy Congress 2020. Royal Microscopical Society, 2021. http://dx.doi.org/10.22443/rms.emc2020.1135.

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Periasamy, Ammasi, Shagufta R. Alam, Zdenek Svindrych, and Horst Wallrabe. "FLIM-FRET image analysis of tryptophan in prostate cancer cells." In European Conferences on Biomedical Optics, edited by Emmanuel Beaurepaire, Francesco S. Pavone, and Peter T. C. So. SPIE, 2017. http://dx.doi.org/10.1117/12.2283037.

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Krishnan, Ramanujan V., Eva Biener, Victoria E. Centonze, Arieh Gertler, and Brian A. Herman. "Multiphoton FLIM: a reliable FRET detection tool in cell biological applications." In Biomedical Optics 2004, edited by Ammasi Periasamy and Peter T. C. So. SPIE, 2004. http://dx.doi.org/10.1117/12.528050.

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