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Photoswitching Kinetics and Phase‐Sensitive Detection Add Discriminative Dimensions for Selective Fluorescence Imaging
Author(s) -
Querard Jérôme,
Markus TalZvi,
Plamont MarieAude,
Gauron Carole,
Wang Pengcheng,
Espagne Agathe,
Volovitch Michel,
Vriz Sophie,
Croquette Vincent,
Gautier Arnaud,
Le Saux Thomas,
Jullien Ludovic
Publication year - 2015
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201408985
Subject(s) - autofluorescence , fluorescence , monochromatic color , fluorescence lifetime imaging microscopy , microscopy , chemistry , materials science , optics , physics
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.