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dc.contributor.authorCoucke, Quinten
dc.contributor.authorParveen, Nagma
dc.contributor.authorSolis-Fernandez, Guillermo 
dc.contributor.authorQian, Chen
dc.contributor.authorHofkens, Johan
dc.contributor.authorDebyser, Zeger
dc.contributor.authorHendrix, Jelle
dc.identifier.citationBiophys Rep (NY). 2023 Sep 13;3(3):100122.es_ES
dc.description.abstractFluorescence lifetime imaging microscopy (FLIM) is a popular modality to create additional contrast in fluorescence images. By carefully analyzing pixel-based nanosecond lifetime patterns, FLIM allows studying complex molecular populations. At the single-molecule or single-particle level, however, image series often suffer from low signal intensities per pixel, rendering it difficult to quantitatively disentangle different lifetime species, such as during Förster resonance energy transfer (FRET) analysis in the presence of a significant donor-only fraction. In this article we investigate whether an object localization strategy and the phasor approach to FLIM have beneficial effects when carrying out FRET analyses of single particles. Using simulations, we first showed that an average of ∼300 photons, spread over the different pixels encompassing single fluorescing particles and without background, is enough to determine a correct phasor signature (SD < 5% for a 4-ns lifetime). For immobilized single- or double-labeled dsDNA molecules, we next validated that particle-based phasor-FLIM-FRET readily allows estimating fluorescence lifetimes and FRET from single molecules. Thirdly, we applied particle-based phasor-FLIM-FRET to investigate protein-protein interactions in subdiffraction HIV-1 viral particles. To do this, we first quantitatively compared the fluorescence brightness, lifetime, and photostability of different popular fluorescent protein-based FRET probes when genetically fused to the HIV-1 integrase enzyme in viral particles, and conclude that eGFP, mTurquoise2, and mScarlet perform best. Finally, for viral particles coexpressing FRET-donor/acceptor-labeled IN, we determined the absolute FRET efficiency of IN oligomers. Available in a convenient open-source graphical user interface, we believe that particle-based phasor-FLIM-FRET is a promising tool to provide detailed insights in samples suffering from low overall signal intensities.es_ES
dc.description.sponsorshipJ. Hofkens acknowledges financial support from the Research Foundation - Flanders (FWO grant nos. G0F2322N and ZW15_09-GOH6316), the Flemish government through long-term structural funding Methusalem (CASAS2, Meth/15/04), and the MPI as MPI fellow. J. Hendrix acknowledges Research Foundation - Flanders (FWO, projects G0B4915, G0B9922N, and G0H3716N) and KU Leuven Special Research Fund (C14/16/053).es_ES
dc.publisherElsevier es_ES
dc.titleParticle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particleses_ES
dc.typeresearch articlees_ES
dc.rights.licenseAttribution-NonCommercial-NoDerivatives 4.0 Internacional*
dc.contributor.funderResearch Foundation - Flanders es_ES
dc.identifier.journalBiophysical reportses_ES
dc.repisalud.centroISCIII::Unidad Funcional de Investigación de Enfermedades Crónicas (UFIEC)es_ES
dc.rights.accessRightsopen accesses_ES

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Attribution-NonCommercial-NoDerivatives 4.0 Internacional
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