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

Abstract

Non-invasive separation-free protocols are attractive for analyzing complex mixtures. To increase selectivity, an analysis under kinetic control, through exploitation of the photochemical reactivity of labeling contrast agents, is described. The simple protocol is applied in optical fluorescence microscopy, where autofluorescence, light scattering, as well as spectral crowding presents limitations. Introduced herein is 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, thus opening attractive perspectives for multiplexed observations in biological samples.

DOI: 10.1002/anie.201408985

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Cite this paper

@article{Qurard2015PhotoswitchingKA, title={Photoswitching kinetics and phase-sensitive detection add discriminative dimensions for selective fluorescence imaging.}, author={J{\'e}r{\^o}me Qu{\'e}rard and T L Markus and Marie-Aude Plamont and Carole Gauron and Pengcheng Wang and Agathe Espagne and Michel Volovitch and Sophie Vriz and Vincent Croquette and Arnaud Gautier and Thomas Le Saux and Ludovic Jullien}, journal={Angewandte Chemie}, year={2015}, volume={54 9}, pages={2633-7} }