Caractérisation de protéines fluorescentes photoconvertibles pour la microscopie super-résolution par localisation de molécules uniques

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.La microscopie de fluorescence est une puissante technique pour l'observation d'échantillons biologiques et la compréhension de processus moléculaires. Les deux dernières décennies ont été témoins de grandes avancées dans ce domaine, notamment avec le développement des techniques de "super-résolution", qui permettent l'observation de structures plus fines que la limite de diffraction de la lumière visible (~200 nm). Une de ces techniques les plus populaires est le PALM (Photoactivated Localisation Microscopy, microscopie par localisation photoactivée), qui utilise des protéines fluorescentes phototransformables (PTFPs) pour observer des molécules uniques, et les localiser avec une précision de 10-20 nm. Les PTFPs sont des protéines de la même famille que la GFP (Green Fluorescent Protein, protéine fluorescente verte), qui non-seulement produisent de la fluorescence, mais peuvent aussi subir des réactions photo-induites, comme l'activation de fluorescence ou le changement de couleur d'émission. Ces propriétés sont à la base du PALM, puisqu'elle permettent la séparation temporelle stochastique des émissions de fluorescence, et l'imagerie de molécules uniques. Le fait que le PALM détecte des molécules uniques a conduit au développement d'un grand nombre d'applications, dont le sptPALM (single-particle tracking PALM, suivi de molécules uniques en PALM) et le PALM quantitatif (qPALM). Ces applications avancées permettent déjà d'acquérir des informations précieuses sur le fonctionnement des systèmes biologiques, mais leur utilisation reste difficile. Une des raisons en est le comportement photophysique complexe des PTFPs, qui va au-delà des transformations utiles pour l'imagerie PALM.L'objet de cette thèse était donc de caractériser les réactions photo-induites subies par les protéines fluorescentes photoconvertibles (PCFPs, qui sont parmi les marqueurs les plus populaires en PALM), dans le but d'améliorer les approches de suivi de molécules uniques et quantitatives en PALM. En particulier, ce travail cherchait à comprendre les pertes transitoires de fluorescence, connues sous le terme de scintillement (blinking en anglais), qui sont détrimentales aux expériences PALM. Une caractérisation poussée des réactions photo-induites ayant lieu dans les formes verte et rouge de la PCFP étudiée nous a suggéré une stratégie pour réduire l'influence du scintillement, et des artéfacts qu'il produit. Enfin, cette stratégie a été appliquée à une expérience de qPALM.Ce travail constitue un pas en avant vers une meilleure compréhension de la photophysique des PCFPs, et une meilleure extraction d'informations quantitatives des données PALM

mEos4b Photoconversion Efficiency Depends on Laser Illumination Conditions Used in PALM

International audienceGreen-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 lead 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

Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy

International audienceGreen-to-red photoconvertible fluorescent proteins (PCFPs) such as mEos2 and its derivatives are widely used in PhotoActivated Localization Microscopy (PALM). However, the complex photophysics of these genetically encoded markers complicates the quantitative analysis of PALM data. Here, we show that intense 561 nm light (∼1 kW/cm(2)) typically used to localize single red molecules considerably affects the green-state photophysics of mEos2 by populating at least two reversible dark states. These dark states retard green-to-red photoconversion through a shelving effect, although one of them is rapidly depopulated by 405 nm light illumination. Multiple mEos2 switching and irreversible photobleaching is thus induced by yellow/green and violet photons before green-to-red photoconversion occurs, contributing to explain the apparent limited signaling efficiency of this PCFP. Our data reveals that the photophysics of PCFPs of anthozoan origin is substantially more complex than previously thought, and suggests that intense 561 nm laser light should be used with care, notably for quantitative or fast PALM approaches

Photophysical studies at cryogenic temperature reveal a novel photoswitching mechanism of rsEGFP2

Single-molecule-localization-microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below the glass transition temperature hampers efficient photoswitching at low temperature. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent protein at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at ∼110 K. At this cryogenic temperature, on-off photoswitching involves the formation of 2 dark states with blue shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these dark states can be switched back to the fluorescent state by 405 nm light, while both of them are sensitive to UV light at 355 nm. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins. It suggests that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 or possibly other FPs could improve the achievable effective labeling efficiency in this technique. Table of Contents artwor

Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM

International audienceGreen-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of longer tracks in sptPALM with minimum effort

Mechanistic Investigations of Green mEos4b Reveal a Dynamic Long-Lived Dark State

International audienceGreen-to-red photoconvertible fluorescent proteins (PCFPs) are key players in advanced microscopy schemes such as photoactivated localization microscopy (PALM). Whereas photoconversion and red-state blinking in PCFPs have been studied intensively, their green-state photophysical behavior has received less attention. Yet dark states in green PCFPs can become strongly populated in PALM schemes and exert an indirect but considerable influence on the quality of data recorded in the red channel. Furthermore, green-state photoswitching in PCFPs can be used directly for PALM and has been engineered to design highly efficient reversibly switchable fluorescent proteins (RSFPs) amenable to various nanoscopy schemes. Here, we demonstrate that green mEos4b efficiently switches to a long-lived dark state through cis-trans isomerization of its chromophore, as do most RSFPs. However, by combining kinetic crystallography, molecular dynamics simulations, and Raman spectroscopy, we find that the dark state in green mEos4b is much more dynamic than that seen in switched-off green IrisFP, a biphotochromic PCFP engineered from the common EosFP parent. Our data suggest that H-bonding patterns maintained by the chromophore in green PCFPs and RSFPs in both their on- and off-states collectively control photoswitching quantum yields. The reduced number of H-bonds maintained by the dynamic dark chromophore in green mEos4b thus largely accounts for the observed lower switching contrast as compared to that of IrisFP. We also compare the long-lived dark states reached from green and red mEos4b, on the basis of their X-ray structures and Raman signatures. Altogether, these data provide a unifying picture of the complex photophysics of PCFPs and RSFPs