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

Jérôme Quérard,Xiaojiang Xie,Pierre Neveu,Samantha Albright,Marie-Aude Plamont,Hanna L Sladitschek,Zsolt Kelemen,Yulia Korepina,Michel Volovitch,Vincent Croquette,Eliane Ipendey,Arnaud Gautier,Ruikang Zhang,Raja Chouket,Lionel Gissot,Jean-Denis Faure,Insa Roemgens,Ludovic Jullien,Thomas Le Saux

doi:10.1038/s41467-017-00847-3

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.

Highlights

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 retrieve the label signal
To drive reversibly photoswitchable fluorescent proteins (RSFPs) photoswitching at optimal rate and extent, speed OPIOM does not use one modulated light source like the original OPIOM,[10] but two modulated light sources synchronized in antiphase at two wavelengths driving RSFP photoswitching between its bright and dark states (Fig. 1a)
In this paper, we used RSFPs to show that speed OPIOM is a powerful approach for multiplexed fluorescence imaging against a background of spectral interferences

Summary

Introduction

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 retrieve the label signal. 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. As SAFIRe, OPIOM relies on a periodically modulated illumination but it exploits phase-sensitive detection and predictable resonance conditions involving the illumination control parameters and the RSFP photoswitching dynamics to selectively retrieve the contribution of a fluorescent probe of interest from the amplitude of the overall out-of-phase fluorescence response. Speed OPIOM correspondingly enables the fast and remote imaging of an RSFP expressed in a strongly autofluorescent biological sample under ambient light and the fast, quantitative and simultaneous microscopy imaging of several spectrally overlapping RSFPs in both fixed and live cells

Methods

Results

Conclusion