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

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Resonant out-of-phase fluorescence microscopy and remote imaging overcome spectral limitations, Nature Communications 8, 969 (2017)


In fluorescence microscopy and remote imaging, the discrimination of a fluorophore usually results from optimizing its brightness and its spectral properties1. Despite the widespread use of fluorescence for labeling biological samples, this approach still suffers from limitations. First, extraction of a fluorescent signal is challenging in light-scattering and autofluorescent samples. Second, spectral deconvolution of overlapping absorption and emission bands can only discriminate a few labels, falling short from the several tens needed for advanced bioimaging.



Speed OPIOM distinguishes spectrally similar RSFPs (but other photoswitchable fluorophores would be relevant as well) by leveraging their photochemical and kinetic characteristics. Thanks to its phase-sensitive detection scheme, speed OPIOM is both reference-free and band-pass-selective thereby overcoming some limitations encountered with SAFIRe and OLID. Despite intrinsic limitations associated to noise and precision on phase retrieval, speed OPIOM imaging can typically enhance contrast of photoswitchable fluorophores against non-photoactive spectrally interfering fluorophores or ambient light by a 102–103 factor. Eventually, speed OPIOM application will be facilitated by the ever-expanded number of RSFPs developed for super-resolution microscopy; by requiring one order of magnitude departure of the photochemical properties for efficient discrimination, we estimate that speed OPIOM should soon give access to multiplexed imaging of five to ten distinct RSFPs. Hence, speed OPIOM should considerably expand the scope of fluorescence microscopy and remote imaging at video rate.


Corresponding Authors : Thomas LE SAUX ( - Ludovic JULLIEN (


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Nature Communications 8, 969 (2017)


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.


Chimie Biophysique
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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 Le Saux & Ludovic Jullien


Nature Communications 8, 969 (2017)


DOI: 10.1038/s41467-017-00847-3