Characterisation of photoconvertible fluorescent proteins for single-molecule localisation microscopy

Fluorescence microscopy is a powerful tool for the observation of biological specimens and the understanding of molecular processes. The last two decades have seen tremendous advances in the field, notably with the development of “super-resolution” techniques, which allow the observation of structures smaller than the diffraction limit of visible light (~200 nm). One of the most popular of these techniques is Photoactivated Localisation Microscopy (PALM), which uses Phototransformable Fluorescent Proteins (PTFPs) to image single molecules and localise them with 10-20 nm precision. PTFPs are proteins from the Green Fluorescent Protein (GFP) family, which not only produce fluorescence, but can also undergo light-induced reactions such as fluorescence activation or change in emission color. These specific properties are at the base of PALM, since they allow stochastic temporal separation of the fluorescence events and imaging of sparse single-molecules. The fact that PALM deals with single molecules prompted the development of a variety of applications, among which single-particle tracking PALM (sptPALM) and quantitative PALM (qPALM). These advanced applications already provide amazing insights into biological phenomena, but their use remains challenging. One of the reasons for this is the complex photophysical behaviour of PTFPs, beyond the transormations that are useful for PALM imaging.Therefore, this thesis focused on characterising the light-induced reactions occurring in Photoconvertible Fluorescent Proteins (PCFPs, some of the most popular PALM markers) with the aim of improving single-particle tracking and quantitative approaches in PALM. In particular, the work was directed to the understanding of transient losses of fluorescence, known as blinking, that are detrimental to PALM experiments. After a thorough characterisation of light-induced reactions in both the green and the red form of the investigated PCFPs, a strategy was proposed to alleviate blinking and the artefacts it produces. Finally, insights were given into the application of this strategy to improve a qPALM experiment.This work constitutes an further step towards a better understanding of PCFPs photophysics, and improved extraction of quantitative information from PALM datasets.