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Article Dans Une Revue Scientific Reports Année : 2020

tempo-spectral multiplexing in flow cytometry with lifetime detection using QD-encoded polymer beads

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Semiconductor quantum dots (QDs) embedded into polymer microbeads are known to be very attractive emitters for spectral multiplexing and colour encoding. Their luminescence lifetimes or decay kinetics have been, however, rarely exploited as encoding parameter, although they cover time ranges which are not easily accessible with other luminophores. We demonstrate here the potential of QDs made from II/VI semiconductors with luminescence lifetimes of several 10 ns to expand the lifetime range of organic encoding luminophores in multiplexing applications using time-resolved flow cytometry (LT-FCM). For this purpose, two different types of QD-loaded beads were prepared and characterized by photoluminescence measurements on the ensemble level and by single-particle confocal laser scanning microscopy. Subsequently, these lifetime-encoded microbeads were combined with dye-encoded microparticles in systematic studies to demonstrate the potential of these QDs to increase the number of lifetime codes for lifetime multiplexing and combined multiplexing in the time and colour domain (tempo-spectral multiplexing). These studies were done with a recently developed novel luminescence lifetime flow cytometer (LT-FCM setup) operating in the time-domain, that presents an alternative to reports on phase-sensitive lifetime detection in flow cytometry. Flow cytometry (FCM), where either single cells or particles are optically detected in a flow, is an established and widespread multiparametric fluorescence technique used for many routine and research applications in biology, medical diagnostics, and food analysis 1-5. FCM typically relies on organic luminophores for optical encoding and multiplexing as well as analyte quantification 6-10. State-of-the-art FCM instruments are designed to detect spectral and/or intensity codes and can distinguish a large number of different colour and intensity codes, e.g. in the case of bead-based assays 5,11-13. Commonly applied spectral multiplexing approaches utilizing organic lumi-nophores are, however, limited by the spectral overlap of the emission bands of the different encoding dyes which limits the number of distinguishable codes and can require signal compensation and cross talk correction 6,14,15. The exploitation of the luminophore-characteristic luminescence lifetime (LT) as additional encoding parameter could in principle increase the number of accessible codes in flow cytometry analysis, thereby increasing the degree of multiplexing 6,7,14,16-21. Time-resolved fluorescence measurements in flow cytometry and lifetime encoding have been discussed for decades 8,22-28. The concept of lifetime multiplexing in a flow, however, has not been adopted in routine applications up to now due to the limited measurement time per object resulting in reduced photon count numbers 27,29. Moreover, the vast majority of reports on lifetime detection in flow cytometry relies on frequency-domain techniques (phase fluorometry), which used to be less expensive as fast detectors required for photon counting-based detection in the time domain, and present specific applications 8,9,14,18,28,30-32. This has been highlighted e.g., in a recently published comprehensive review article from J.P. Houston et al. 8 , revealing only a very small number of research articles related to cytometry studies in the time domain, mostly associated
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hal-02933848 , version 1 (08-09-2020)

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Daniel Kage, Katrin Hoffmann, Galina Nifontova, Victor Krivenkov, Alyona Sukhanova, et al.. tempo-spectral multiplexing in flow cytometry with lifetime detection using QD-encoded polymer beads. Scientific Reports, 2020, 10 (1), ⟨10.1038/s41598-019-56938-2⟩. ⟨hal-02933848⟩

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