UMR 8640 : Chimie Biophysique

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Long-term in vivo single-cell lineage tracing of deep structures using three-photon activation

Light: Science & Applications (2016) 5, e16084

 

Genetic labeling techniques allow for noninvasive lineage tracing of cells in vivo. Two-photon inducible activators provide spatial resolution for superficial cells, but labeling cells located deep within tissues is precluded by scattering of the far-red illumination required for two-photon photolysis. Three-photon illumination has been shown to overcome the limitations of two-photon microscopy for in vivo imaging of deep structures, but whether it can be used for photoactivation remains to be tested. Here we show, both theoretically and experimentally, that three-photon illumination overcomes scattering problems by combining longer wavelength excitation with high uncaging three-photon cross-section molecules. We prospectively labeled heart muscle cells in zebrafish embryos and found permanent labeling in their progeny in adult animals with negligible tissue damage. This technique allows for a noninvasive genetic manipulation in vivo with spatial, temporal and cell-type specificity, and may have wide applicability in experimental biology.

 

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

Proceedings of the National Academy of Sciences, Volume 113 n°.3, January 2016, Pages 497-502

 

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a socalled fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Expanding discriminative dimensions for analysis and imaging

OPTIMAL can discriminate – without any separation or washing step – a targeted photoswitchable probe used as labelling or titration contrast agent among various interfering compounds, photoswitchable or not.

Photoswitching kinetics and phase sensitive detection add discriminative dimensions for selective fluorescence imaging

Non-invasive separation-free protocols are attractive to analyze complex mixtures. To increase selectivity, we propose to perform analysis under kinetic control upon exploiting the photochemical reactivity of labeling contrast agents. Our simple protocol is applied in optical fluorescence microscopy, where autofluorescence, light scattering as well as spectral crowding presently bring limitations. We introduce OPIOM (Out-of-Phase Imaging after Optical Modulation), which exploits the rich kinetic signature of a photoswitching fluorescent probe to increase selectively and quantitatively its contrast. Filtering the specific contribution of the probe only requires phase-sensitive detection upon matching the photoswitching dynamics of the probe and the intensity and frequency of a modulated monochromatic light excitation. After in vitro validation, we applied OPIOM for selective imaging in mammalian cells and zebrafish, opening attractive perspectives for multiplexed observations in biological samples.

 

Photochemical properties of Spinach and its use in selective imaging

We propose a dynamic model that accounts for the photochemical behavior of the Spinach system, a recently described fluorescent probe for RNA imaging. We exploit the dynamic fluorogen exchange and the unprecedented photoconversion properties in a non-covalent fluorescence turn-on system to significantly improve signal-to-background ratio during long-term microscopy imaging.