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- Title
Extra kinetic dimensions for label discrimination.
- Authors
Chouket, Raja; Pellissier-Tanon, Agnès; Lahlou, Aliénor; Zhang, Ruikang; Kim, Diana; Plamont, Marie-Aude; Zhang, Mingshu; Zhang, Xi; Xu, Pingyong; Desprat, Nicolas; Bourgeois, Dominique; Espagne, Agathe; Lemarchand, Annie; Saux, Thomas Le; Jullien, Ludovic
- Abstract
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. Label discrimination is challenging in fluorescence microscopy due to broad spectra and narrow lifetime distribution. Here, the authors introduce extra kinetic dimensions by illuminating reversibly photoswitchable fluorophores with different intensities, and discriminate 20 spectrally similar fluorophores.
- Subjects
FLUORESCENCE microscopy; FLUORESCENT proteins; FLUOROPHORES; LIGHT intensity
- Publication
Nature Communications, 2022, Vol 13, Issue 1, p1
- ISSN
2041-1723
- Publication type
Article
- DOI
10.1038/s41467-022-29172-0