title: 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.
authors.affiliation: École Normale Supérieure-PSL Research University, Department of Chemistry, 24 rue Lhomond, 75005 Paris (France); Sorbonne Universités, UPMC Univ Paris 06, PASTEUR, 75005 Paris (France); CNRS, UMR 8640 PASTEUR, 75005 Paris (France).
authors.forename: Jérôme
authors.initials: J
authors.lastname: Querard
authors.forename: Tal-Zvi
authors.initials: TZ
authors.lastname: Markus
authors.forename: Marie-Aude
authors.initials: MA
authors.lastname: Plamont
authors.forename: Carole
authors.initials: C
authors.lastname: Gauron
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Wang
authors.forename: Agathe
authors.initials: A
authors.lastname: Espagne
authors.forename: Michel
authors.initials: M
authors.lastname: Volovitch
authors.forename: Sophie
authors.initials: S
authors.lastname: Vriz
authors.forename: Vincent
authors.initials: V
authors.lastname: Croquette
authors.forename: Arnaud
authors.initials: A
authors.lastname: Gautier
authors.forename: Thomas
authors.initials: T
authors.lastname: Le Saux
authors.forename: Ludovic
authors.initials: L
authors.lastname: Jullien
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D061848:Optical Imaging; D055668:Photochemical Processes; D015027:Zebrafish
chemical_list: D005456:Fluorescent Dyes
keywords: analytical methods; fluorescence; fluorescent probes; imaging agents; photochemistry