Fluorescence lifetime excitation cytometry by kinetic dithering.
Bottom Line: Using the FLECKD instrument, we measured the shortest average fluorescence lifetime value of 2.4 ns and found the system measurement error to be ±0.3 ns (SEM), from hundreds of monodisperse and chemically stable fluorescent microspheres.This approach presents a new ability to resolve multiple fluorescence lifetimes while retaining the fluidic throughput of a cytometry system.The ability to discriminate more than one average fluorescence lifetime expands the current capabilities of high-throughput and intensity-based cytometry assays as the need to tag one single cell with multiple fluorophores is now widespread.
Affiliation: Department of Chemical Engineering, College of Engineering, New Mexico State University, Las Cruces, NM, USA.Show MeSH
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Mentions: In this work, we present a paradigm shift in the instrumentation architecture of a flow cytometer capable of measuring fluorescence lifetime. In lieu of standard frequency-domain techniques, a new type of time-resolved instrument was conceived for the purpose of expanding the ability to measure multiple fluorescence decays from single, transient events. The new approach takes advantage of the time delay between the fluorescence signal and the side scatter signal, similar to the phase delay between emission and excitation in multi-frequency systems. As a cell interacts with the laser beam, a prototypical Gaussian peak results from the interaction between the cell and the interrogating light beam, which normally has a Gaussian profile in the direction of flow (see Fig.1). When shifts in the fluorescence lifetime between the scattering and fluorescence signals are to be detected on the order of nanoseconds, the width of the signal pulses becomes a limiting factor. Therefore reducing the width of the Gaussian interaction peak may improve the measurement of fluorescence-lifetime-induced time delays and achieve the resolution necessary for accurate fluorescence lifetime measurements (Cao R. et al., submitted).
Affiliation: Department of Chemical Engineering, College of Engineering, New Mexico State University, Las Cruces, NM, USA.