Photoswitching kinetics and phase-sensitive detection add discriminative dimensions for selective fluorescence imaging. Querard, J., Markus, T., Plamont, M., Gauron, C., Wang, P., Espagne, A., Volovitch, M., Vriz, S., Croquette, V., Gautier, A., Le Saux, T., & Jullien, L. Angewandte Chemie (International Ed. in English), 54(9):2633--2637, February, 2015. doi abstract bibtex 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.
@article{querard_photoswitching_2015,
title = {Photoswitching kinetics and phase-sensitive detection add discriminative dimensions for selective fluorescence imaging},
volume = {54},
issn = {1521-3773},
doi = {10.1002/anie.201408985},
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.},
language = {eng},
number = {9},
journal = {Angewandte Chemie (International Ed. in English)},
author = {Querard, Jérôme and Markus, Tal-Zvi and Plamont, Marie-Aude and Gauron, Carole and Wang, Pengcheng and Espagne, Agathe and Volovitch, Michel and Vriz, Sophie and Croquette, Vincent and Gautier, Arnaud and Le Saux, Thomas and Jullien, Ludovic},
month = feb,
year = {2015},
pages = {2633--2637}
}
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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. 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