Resonant out-of-phase fluorescence microscopy and remote imaging overcome spectral limitations

Jérôme Quérard, Ruikang Zhang, Zsolt Kelemen, Marie-Aude Plamont, Xiaojiang Xie, Raja Chouket, Insa Roemgens, Yulia Korepina, Samantha Albright, Eliane Ipendey, Michel Volovitch, Hanna L. Sladitschek, Pierre Neveu, Lionel Gissot, Arnaud Gautier, Jean-Denis Faure, Vincent Croquette, Thomas Saux () and Ludovic Jullien ()
Additional contact information
Jérôme Quérard: UPMC Univ Paris 06, CNRS, PSL Research University
Ruikang Zhang: UPMC Univ Paris 06, CNRS, PSL Research University
Zsolt Kelemen: Universite Paris-Saclay
Marie-Aude Plamont: UPMC Univ Paris 06, CNRS, PSL Research University
Xiaojiang Xie: UPMC Univ Paris 06, CNRS, PSL Research University
Raja Chouket: UPMC Univ Paris 06, CNRS, PSL Research University
Insa Roemgens: UPMC Univ Paris 06, CNRS, PSL Research University
Yulia Korepina: UPMC Univ Paris 06, CNRS, PSL Research University
Samantha Albright: UPMC Univ Paris 06, CNRS, PSL Research University
Eliane Ipendey: UPMC Univ Paris 06, CNRS, PSL Research University
Michel Volovitch: PSL Research University, Collège de France
Hanna L. Sladitschek: European Molecular Biology Laboratory
Pierre Neveu: European Molecular Biology Laboratory
Lionel Gissot: Universite Paris-Saclay
Arnaud Gautier: UPMC Univ Paris 06, CNRS, PSL Research University
Jean-Denis Faure: Universite Paris-Saclay
Vincent Croquette: Laboratoire de Physique Statistique, CNRS, ENS
Thomas Saux: UPMC Univ Paris 06, CNRS, PSL Research University
Ludovic Jullien: UPMC Univ Paris 06, CNRS, PSL Research University

Nature Communications, 2017, vol. 8, issue 1, 1-8

Abstract: Abstract We present speed out-of-phase imaging after optical modulation (OPIOM), which exploits reversible photoswitchable fluorophores as fluorescent labels and combines optimized periodic illumination with phase-sensitive detection to specifically retrieve the label signal. Speed OPIOM can extract the fluorescence emission from a targeted label in the presence of spectrally interfering fluorophores and autofluorescence. Up to four fluorescent proteins exhibiting a similar green fluorescence have been distinguished in cells either sequentially or in parallel. Speed OPIOM is compatible with imaging biological processes in real time in live cells. Finally speed OPIOM is not limited to microscopy but is relevant for remote imaging as well, in particular, under ambient light. Thus, speed OPIOM has proved to enable fast and quantitative live microscopic and remote-multiplexed fluorescence imaging of biological samples while filtering out noise, interfering fluorophores, as well as ambient light.

Date: 2017
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DOI: 10.1038/s41467-017-00847-3

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