One of our central themes concerns photophysical studies of phototransformable fluorescent proteins (PTFPs). PTFPs are fundamental players in super-resolution microscopy as well as other advanced fluorescence methods such as pulse-chase imaging, modulated-illumination imaging, photochromic Förster Resonance Energy Transfer (FRET) or biological data storage.
These genetically encoded fluorescent markers are fascinating because their fluorescent state can be altered by illumination with appropriate laser light.
For example, the emission color of certain PTFPs can change from green to red by illumination with violet light (these are called photoconvertible FPs, PCFPs).
Other PTFPs can reversibly switch from a fluorescent to a non-fluorescent state (these are called reversibly photoswitchable FPs, RSFPs), for example by alternating cyan and violet light for RSFPs that fluoresce in green.
Phototransformation properties such as photoactivation, photoconversion and photoswitching need to be understood and optimized for each application. Additionally, blinking (stochastic and transient loss of fluorescence) and photobleaching (irreversible loss of fluorescence) are crucial photophysical properties observed in all fluorescent proteins, that we study in detail or although they still hold many mysteries.
Many groups around the world study and develop fluorescent proteins to create ever more efficient markers or sensors. We contribute to this research field by studying PTFPs using a wide range of tools : we generally use a combination of kinetic crystallography (including XFEL in collaboration with the Dynamop group), spectroscopy in cristallo, single-molecule imaging in vitro and in cellulo, and NMR in collaboration with the NMR group).
In recent years, we have focused our attention on the photophysics of popular PCFPs such as mEos4b. This photophysics seems to present endless complexity, but we understand it a little better every day. For example, in collaboration with the team of P. Dedecker (KUL, Belgium), we deciphered in 2019 a major blinking mechanism in red mEos4b, which usually causes serious problems in the quality and interpretation of quantitative PALM (qPALM) or sptPALM data, and we showed that this mechanism is very closely related to the reversible photoswitching mechanisms at play in RSFPs. This allowed us to propose a trick to reduce blinking in sptPALM, thereby enabling the reconstruction of longer tracks. We have also been interested in the photophysics of the green state in PCFPs. Indeed, although PCFPs are generally detected only once photoconverted to the red state, what happens before photoconversion significantly affects their performance as SMLM markers. For example, the actual labeling efficiency of mEos4b depends non-linearly on the 405 nm laser power used for photoconversion. In collaboration with the teams of J.B Sibarita and M. Sainlos (IINS, Bordeaux), we started in 2020 an ANR-funded project aimed at further improving the photostability of PCFPs, by combining structural studies, including NMR, with high-throughput single-molecule imaging approaches to enable effective semi-rational engineering. An important object of study is the link between the conformational dynamics of PTFPs and their photophysics, which can be addressed by NMR, today a central tool for our research. For example, we discovered that mEos proteins exist in multiple conformational states, both in their green and red forms. Furthermore, we discovered that the maturation times of mEos2,3,4 variants were extremely long, and through a rational approach, we engineered the proteins mEos4Fast.
We also study the photoswitching mechanism of green RSFPs like rsEGFP2, or red ones in collaboration with Dorus Gadella (University of Amsterdam). In particular, we are interested in the photoswitching mechanisms of fluorescent proteins at cryogenic temperature ( 100 K), and we recently discovered that the photoswitching mechanisms of rsEGFP2 at room temperature and cryogenic temperature were completely different, involving cis-trans isomerization at room temperature and probably the generation of stable non-fluorescent radical states at cryogenic temperature.