Intersystem crossing in fluorescent proteins as a novel mechanism for dynamic contrast in confocal fluorescence microscopy – dISCern

Intersystem crossing processes in FPs, and thermal photoswitching steps in RSFPs, will be studied at a fundamental level by an innovative combination of experimental approaches involving either cw or flash excitation to drive the photochemical reactions. We will carry out time-resolved spectroscopy measurements of pump-probe and pump-pump-probe type on ns-µs-ms timescales on selected proteins to characterize their mechanisms in detail (including the fluorescence and photoreactivity of short-lived species). Environment effects will be studied by determining the photodynamic parameters (triplet cross-section and lifetime, characteristic photoswitching times at high intensity, etc.) of series of FPs and RSFPs, thanks to an automated optical setup providing cw illumination of intensity variable up to ~100 kW.cm-2. Next, we will exploit the adjustability of the scanning parameters of a commercial confocal microscope to theoretically design and then implement imaging protocols enabling us to distinguish several FPs or RSFPs thanks to their triplet or photoswitching dynamics.

Will be informed at the end of the project.

Will be informed at the end of the project.

« Dynamic contrast with reversibly photoswitchable fluorescent labels for imaging living cells », R. Chouket, A. Pellissier-Tanon, A. Lemarchand, A. Espagne, T. Le Saux, L. Jullien, Chem. Sci. 2020, 11, 2882-2887, DOI: 10.1039/D0SC00182A
« Dynamic contrast for overcoming spectral interferences in fluorescence imaging », R. Chouket, R. Zhang, A. Pellissier-Tanon, A. Lemarchand, A. Espagne, T. Le Saux, L. Jullien, J. Phys.: Photonics 2020, 2, 032003, DOI: 10.1088/2515-7647/ab9099

Although ubiquitously used as fluorescent probes in biological imaging, fluorescent proteins (FPs) of the GFP family exhibit a variety of photochemical reactions, which mechanisms are still incompletely understood. Some of these reactions led to innovative applications. Thus, dynamic contrast microscopies exploit the transition dynamics of reversibly photoswitchable fluorescent proteins (RSFPs) between their fluorescent and non-fluorescent states to image them selectively in the presence of spectrally similar species, photoswitchable or not.
Our consortium of photochemists, spectroscopists and theoreticians proposes to examine two hitherto poorly known aspects of FP photochemistry of high interest for dynamic contrast microscopies, namely:
- the formation, photoreactivity and dynamics of triplet states;
- the nature and kinetics of µs-ms reaction steps involved in RSFP photoswitching.
Our objectives are more specifically:
1) to characterize at a fundamental level the mechanisms and dynamics of these reactions and their sensitivity to protein environment in different FPs,
2) to develop novel protocols of dynamic contrast, confocal fluorescence imaging of FPs and RSFPs based on these reactions.
The project builds on recent work (to which Partner 2 contributed) indicating that standard FPs, until now considered as non-photoswitchable, can actually be reversibly photoswitched between their singlet and triplet states. These photoswitching properties make standard FPs highly promising probes for dynamic contrast microscopy, as a complement to RSFPs. Due to their low yields (~1%) and short lifetimes (ms), the formation of FP triplets requires high illumination densities, that can be achieved in laser scanning confocal microscopy (10-100 kW.cm-2). Under these illumination conditions, the photoswitching dynamics of RSFPs is limited by the kinetics of thermal reaction steps (µs-ms timescale).
Intersystem crossing processes in FPs, and thermal photoswitching steps in RSFPs, will be studied at a fundamental level by an innovative combination of experimental approaches involving either cw or flash excitation to drive the photochemical reactions. Partner 2 will carry out time-resolved spectroscopy measurements of pump-probe and pump-pump-probe type on ns-µs-ms timescales on selected proteins to characterize their mechanisms in detail (including the fluorescence and photoreactivity of short-lived species). Environment effects will be studied by determining the photodynamic parameters (triplet cross-section and lifetime, characteristic photoswitching times at high intensity, etc.) of series of FPs and RSFPs, thanks to an automated optical setup recently developed by Partner 1 and providing cw illumination of intensity variable up to ~100 kW.cm-2. Next, Partners 1 and 3 will exploit the adjustability of the scanning parameters of a commercial confocal microscope to theoretically design and then implement imaging protocols enabling us to distinguish several FPs or RSFPs thanks to their triplet or photoswitching dynamics.
Our project will advance the fundamental understanding of FP and RSFP photochemistry and it should have a substantial impact on the general field of fluorescence microscopy. In fact, the accumulation of FP triplet states presumably leads to reduced fluorescence signals in many microscopy experiments. The project is moreover expected to considerably expand the scope of dynamic contrast microscopy through the use of novel fluorophores, novel discrimination parameters and novel imaging modalities.