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http://hdl.handle.net/1942/42050
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DC Field | Value | Language |
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dc.contributor.author | Coucke, Quinten | - |
dc.contributor.author | Parveen, Nagma | - |
dc.contributor.author | Fernandez, Guillermo Solis | - |
dc.contributor.author | Qian, Chen | - |
dc.contributor.author | Hofkens, Johan | - |
dc.contributor.author | Debyser, Zeger | - |
dc.contributor.author | HENDRIX, Jelle | - |
dc.date.accessioned | 2024-01-08T08:56:25Z | - |
dc.date.available | 2024-01-08T08:56:25Z | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2024-01-05T14:05:16Z | - |
dc.identifier.citation | biophysical report, 3 (3) (Art N° 100122) | - |
dc.identifier.uri | http://hdl.handle.net/1942/42050 | - |
dc.description.abstract | Fluorescence 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 Forster 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 similar to 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. | - |
dc.description.sponsorship | Jens de Wachter is thanked for assistance with the DNA experiments. Dr. Waldemar Schrimpf is thanked for implementing phasor analysis in PAM and evaluating the manuscript. Namjoo Vanderveken is acknowledged for assistance in producing the viral particle preparations. Rik Nuyts, Carine Jackers, and Debora Linhares are gratefully acknowledged for assistance in the lab and essential logistics. Dr. Kris Janssen is thanked for writing the software for galvanometric mirror scanning. Dr. Irena Zurnic Bönisch is acknowledged for critically reading the manuscript. J. 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). | - |
dc.language.iso | en | - |
dc.publisher | ELSEVIER | - |
dc.rights | 2023 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) | - |
dc.title | Particle-based phasor-FLIM-FRET resolves protein- protein interactions inside single viral particles | - |
dc.type | Journal Contribution | - |
dc.identifier.issue | 3 | - |
dc.identifier.volume | 3 | - |
local.format.pages | 16 | - |
local.bibliographicCitation.jcat | A1 | - |
dc.description.notes | Hendrix, J (corresponding author), Katholieke Univ Leuven, Dept Chem, Mol Imaging & Photon Div, Leuven, Belgium.; Hendrix, J (corresponding author), Hasselt Univ, Adv Opt Microscopy Ctr, Dynam Bioimaging Lab, Hasselt, Belgium.; Hendrix, J (corresponding author), Hasselt Univ, Biomed Res Inst, Hasselt, Belgium. | - |
dc.description.notes | jelle.hendrix@uhasselt.be | - |
local.publisher.place | RADARWEG 29, 1043 NX AMSTERDAM, NETHERLANDS | - |
local.type.refereed | Refereed | - |
local.type.specified | Article | - |
local.bibliographicCitation.artnr | 100122 | - |
dc.identifier.doi | 10.1016/j.bpr.2023.100122 | - |
dc.identifier.pmid | 37649577 | - |
dc.identifier.isi | 001124456500001 | - |
dc.contributor.orcid | Hofkens, Johan/0000-0002-9101-0567; Qian, Chen/0000-0002-3005-5761; | - |
dc.contributor.orcid | Solis Fernandez, Guillermo/0000-0002-4785-0040; Coucke, | - |
dc.contributor.orcid | Quinten/0000-0002-2493-2668 | - |
dc.identifier.eissn | - | |
local.provider.type | wosris | - |
local.description.affiliation | [Coucke, Quinten; Parveen, Nagma; Fernandez, Guillermo Solis; Hofkens, Johan; Hendrix, Jelle] Katholieke Univ Leuven, Dept Chem, Mol Imaging & Photon Div, Leuven, Belgium. | - |
local.description.affiliation | [Parveen, Nagma] Indian Inst Technol Kanpur, Dept Chem, Kanpur, India. | - |
local.description.affiliation | [Fernandez, Guillermo Solis] Natl Inst Hlth Carlos III, UFIEC, Madrid, Spain. | - |
local.description.affiliation | [Qian, Chen] Ludwig Maximilians Univ Munchen, Ctr Nano Sci CENS, Ctr Integrated Prot Sci Munich CIPSM & Nanosyst In, Dept Chem, Munich, Germany. | - |
local.description.affiliation | [Hofkens, Johan] Max Planck Inst Polymer Res, Mainz, Germany. | - |
local.description.affiliation | [Debyser, Zeger] Katholieke Univ Leuven, Dept Pharmaceut & Pharmacol Sci, Lab Mol Virol & Gene Therapy, Leuven, Belgium. | - |
local.description.affiliation | [Hendrix, Jelle] Hasselt Univ, Adv Opt Microscopy Ctr, Dynam Bioimaging Lab, Hasselt, Belgium. | - |
local.description.affiliation | [Hendrix, Jelle] Hasselt Univ, Biomed Res Inst, Hasselt, Belgium. | - |
local.uhasselt.international | yes | - |
item.fullcitation | Coucke, Quinten; Parveen, Nagma; Fernandez, Guillermo Solis; Qian, Chen; Hofkens, Johan; Debyser, Zeger & HENDRIX, Jelle (2023) Particle-based phasor-FLIM-FRET resolves protein- protein interactions inside single viral particles. In: biophysical report, 3 (3) (Art N° 100122). | - |
item.accessRights | Open Access | - |
item.fulltext | With Fulltext | - |
item.contributor | Coucke, Quinten | - |
item.contributor | Parveen, Nagma | - |
item.contributor | Fernandez, Guillermo Solis | - |
item.contributor | Qian, Chen | - |
item.contributor | Hofkens, Johan | - |
item.contributor | Debyser, Zeger | - |
item.contributor | HENDRIX, Jelle | - |
crisitem.journal.issn | 2667-0747 | - |
Appears in Collections: | Research publications |
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Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles.pdf | Published version | 1.37 MB | Adobe PDF | View/Open |
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