Dissertation/ Thesis
The Dark Secrets of Fluorescent Proteins : Manipulating Fluorophore Photophysics to Boost Quantitative SMLM ; Les secrets obscurs des protéines fluorescentes : manipulation de la photophysique des fluorophores pour améliorer la SMLM quantitative
| العنوان: | The Dark Secrets of Fluorescent Proteins : Manipulating Fluorophore Photophysics to Boost Quantitative SMLM ; Les secrets obscurs des protéines fluorescentes : manipulation de la photophysique des fluorophores pour améliorer la SMLM quantitative |
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| المؤلفون: | Wulffele, Jip |
| المساهمون: | 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), Université Grenoble Alpes 2020-., Dominique Bourgeois, Joanna Timmins |
| المصدر: | https://theses.hal.science/tel-04541214 ; Structural Biology [q-bio.BM]. Université Grenoble Alpes [2020-.], 2023. English. ⟨NNT : 2023GRALV094⟩. |
| بيانات النشر: | CCSD |
| سنة النشر: | 2023 |
| المجموعة: | Université Grenoble Alpes: HAL |
| مصطلحات موضوعية: | Fluorophore photophysics, Fluorescent Proteins, Photo activated localization microscopy, Single particle tracking PALM, Photophysique, Protéines fluorescentes, Microscopie de localisation, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] |
| الوصف: | Fluorescence single-molecule localization microscopy (SMLM) has become an indispensable tool in integrated structural and cell biology, providing insights into macromolecular organizations and dynamics at the nanoscale in cellulo. A popular SMLM technique is photoactivated localization microscopy (PALM), which relies on the ‘smart’ photophysical behavior of phototransformable fluorescent proteins (PTFPs). Yet, the complex photophysical behaviors of PTFPs hinder quantitative PALM applications, such as counting (qPALM) and single-particle tracking (sptPALM). Besides suboptimal fluorophore behaviors, imaging artifacts and the necessity for sophisticated data analysis contribute to the still limited usage of quantitative PALM techniques. Aiming to push the application of quantitative PALM, my PhD work consists of two projects, dealing with different aspects of quantitative PALM.The first project is focused on the characterization of PTFPs, aiming to develop strategies to improve their behavior for SMLM. This work starts with a comparison between different protein immobilization platforms for the photophysical characterization of PTFPs. Next, follows an investigation of the effects of different illumination conditions on the behavior of the popular green-to-red photoconvertible FP mEos4b, using a combination of single-molecule and ensemble fluorescence microscopy and simulations. Lastly, my thesis contributes to the development of a new photophysical model describing the behavior of the reversibly photoswitchable FP rsEGFP2 at cryogenic temperature. Altogether, this work contributes to a deeper understanding of the photophysical behavior of PTFPs and provides guidelines for optimized imaging schemes for PALM imaging.The second project involves the application of sptPALM to study stress-induced nucleoid remodeling in Deinococcus radiodurans, one of the most radioresistant bacterium known today. By monitoring the diffusion dynamics of the mEos4b labeled nucleoid associated protein HU, this work reveals that nucleoid ... |
| نوع الوثيقة: | doctoral or postdoctoral thesis |
| اللغة: | English |
| Relation: | NNT: 2023GRALV094 |
| الاتاحة: | https://theses.hal.science/tel-04541214 https://theses.hal.science/tel-04541214v1/document https://theses.hal.science/tel-04541214v1/file/WULFFELE_2023_archivage.pdf |
| Rights: | info:eu-repo/semantics/OpenAccess |
| رقم الانضمام: | edsbas.F46FE74A |
| قاعدة البيانات: | BASE |
| الوصف غير متاح. |