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Etude photophysique des protéines fluorescentes photoconvertibles utilisées en microscopie de super-résolution

Abstract : Super-resolution PALM microscopy (photoactivated localization microscopy) is a powerful tool to investigate the cells with nanoscopic accuracy. Advanced PALM microscopy allows to quantitatively and dynamically study biological objects and events. These applications are nevertheless limited by the complex photophysical behavior of the green-to-red photoconvertible fluorescent proteins (PCFPs) used as markers. In particular, PCFPs red forms repeated and stochastic transitions between a fluorescent and a dark state (blinking) as well as photoconversion uncompleteness complicate the extraction of quantitative information.Our study, by combining X-ray crystallography and localization microscopy, evidences that a single aminoacid well conserved among PCFPs, the arginine 66, controls the blinking and photobleaching behavior of two popular PCFPs: mEos2 and Dendra2.Preliminary results suggest that in their green forms and under PALM classical illumination conditions, PCFPs switch to a long-lived dark state resulting in a photoconversion slowing down.Our results open the door to future rational engineering of enhanced PCFPs for quantitative and dynamic PALM.
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Submitted on : Thursday, March 30, 2017 - 6:43:12 PM
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Romain Berardozzi. Etude photophysique des protéines fluorescentes photoconvertibles utilisées en microscopie de super-résolution. Biochimie [q-bio.BM]. Université Grenoble Alpes, 2016. Français. ⟨NNT : 2016GREAV015⟩. ⟨tel-01498997⟩

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