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Agathe Espagne
Agnès Pellissier-Tanon
Aliénor Lahlou
Annie Lemarchand
Diana Kim
Dominique Bourgeois
Ludovic Jullien
Marie-Aude Plamont
Mingshu Zhang
Nicolas Desprat
Pingyong Xu
Raja Chouket
Ruikang Zhang
Thomas Le Saux
Xi Zhang
Extra kinetic dimensions for label discrimination


Due to its sensitivity and versatility, fluorescence is widely used to detect specifically labeled biomolecules. However, fluorescence is currently limited by label discrimination, which suffers from the broad full width of the absorption/emission bands and the narrow lifetime distribution of the bright fluorophores. We overcome this limitation by introducing extra kinetic dimensions through illuminations of reversibly photoswitchable fluorophores (RSFs) at different light intensities. In this expanded space, each RSF is characterized by a chromatic aberration-free kinetic fingerprint of photochemical reactivity, which can be recovered with limited hardware, excellent photon budget, and minimal data processing. This fingerprint was used to identify and discriminate up to 20 among 22 spectrally similar reversibly photoswitchable fluorescent proteins (RSFPs) in less than 1s. This strategy opens promising perspectives for expanding the multiplexing capabilities of fluorescence imaging.

Github repository doi

Journal: Nature Communications
Page: 1482