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 remodeling proceeds differently in response to different stresses. Using simulations, it discusses how the small size of bacteria complicates the interpretation of sptPALM data. The work highlights the value of sptPALM for the study of bacteria but also identifies weaknesses in current analysis pipelines that may lead to erroneous data interpretation.​​

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