mEos4b photoconversion efficiency depends on laser illumination conditions used in PALM
| Autor: | Jip Wulffele, Daniel Thédié, Oleksandr Glushonkov, Dominique Bourgeois |
|---|---|
| Přispěvatelé: | Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), M4D, ANR-17-CE11-0047,Cryo-PALM,Microscopie super-résolution par localisation de molécules uniques à température cryogénique.(2017), ANR-20-CE11-0013,STABLE-FP,Nouvelle génération de protéines fluorescentes ultrastables pour la microscopie quantitative super-résolution(2020), ANR-16-CE11-0016,DIVinHD,Imagerie super-résolue de la division bactérienne(2016) |
| Rok vydání: | 2022 |
| Předmět: |
Microscopy
MESH: Microscopy Light [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry Molecular Biology/Structural Biology [q-bio.BM] Lasers Green Fluorescent Proteins MESH: Lighting MESH: Light Luminescent Proteins MESH: Green Fluorescent Proteins MESH: Lasers General Materials Science MESH: Luminescent Proteins Physical and Theoretical Chemistry Lighting |
| Zdroj: | Journal of Physical Chemistry Letters Journal of Physical Chemistry Letters, 2022, 13 (22), pp.5075-5080. ⟨10.1021/acs.jpclett.2c00933⟩ |
| ISSN: | 1948-7185 |
| DOI: | 10.1101/2022.03.31.486573 |
| Popis: | Green-to-red photoconvertible fluorescent proteins (PCFPs) are widely employed as markers in photoactivated localization microscopy (PALM). However, their highly complex photophysical behavior complicates their usage. The fact that only a limited fraction of a PCFP ensemble can form the photoconverted state upon near-UV light illumination, termed photoconversion efficiency (PCE), lowers the achievable spatial resolution in PALM and creates undercounting errors in quantitative counting applications. Here, we show that the PCE of mEos4b is not a fixed property of this PCFP, but strongly depends on illumination conditions. Attempts to reduce long-lived blinking in red mEos4b by application of 488 nm light leads to a reduction of the PCE. Furthermore, the PCE of mEos4b strongly depends on the applied 405-nm power density. A refined photophysical model of mEos4b accounts for the observed effects, involving nonlinear green-state photobleaching upon violet light illumination favored by photon absorption by a putative radical dark state.TOC GRAPHICS |
| Databáze: | OpenAIRE |
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