High-Throughput Measurement of the Long Excited-state Lifetime of Quantum Dots in Flow Cytometry

The long fluorescence lifetime of quantum dots (QDs) is not often utilized in high-throughput bioassays, despite of the potential for the lifetime to be an optimum parameter for multiplexing with spectrally overlapping excitable species that have short fluorescence lifetimes. The limitation of currently available instruments that can rapidly resolve complex decay kinetics of QDs contributes to this dearth. Therefore work in our laboratory is focused on developing unique and reliable frequency-domain flow cytometry (FDFC) systems as well as QDs applications where fluorescence dynamics are exploited. In this paper we demonstrate both by simulation and experimental validation, the viability of rapidly capturing the fluorescence lifetime of QDs from single QDs-labeled cells and microspheres by employing a home-built FDFC system. With FDFC theory we simulated measurements of long-lived QDs decays and evaluated the potential to discriminate multi-exponential decay profiles of QDs from typical cellular autofluorescence lifetimes. Our FDFC simulation work included calculations of fluorescence phase-shifts at multiple modulation frequencies extracted from square wave modulation signals (i.e. similar to heterodyning frequency-domain spectroscopy). Experimental work to support the result from our simulations involved acquiring measurements from real samples and processing them for multi-frequency phase shifts. Additionally the average excited-state lifetimes of QDs (streptavidin conjugated CdSe/Zns and oleic acid coated CdSxSe1-x/ZnS) measured were found to be greater than 15 ns. The average lifetime results were consistent with published literature values as well as verified with independent time domain measurements. This work opens the possibility of developing powerful bioassays using FDFC based on the long fluorescence lifetime of QDs.