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: Plots in Fig.3 are simulation results for the Gaussian and ex-Gaussian function sets (S1–S4) described previously. Figure3A, B, and C is of three different excitation pulse widths using simulation set S1, emphasizing how the light–cell interaction time dramatically impacts the ability to observe fluorescence decay in an ex-Gaussian curve. Figure3A shows that the ex-Gaussian curve (Fig.3A, ‘o’) synthesized by the convolution of a 1.5-μs Gaussian curve with a 10-ns exponential decay is visually indistinguishable from the Gaussian curve itself (Fig.3A, ‘—’). Therefore with added system noise, which is expected with real data, a 10-ns decay would be very difficult to deconvolve from a 1.5-μs pulse. Instruments performing TCSPC techniques typically use excitation pulses in the picosecond range; therefore compared to a 10-ns decay curve, a Gaussian excitation function with a 15-ps width resembles a “delta function” (Fig.3C, ‘—’), making the convolution decay curve stand out very clearly (Fig.3C, ‘o’). However, TCSPC is designed for thousands of repeated excitations of stationary samples, which are unsuitable for the high-throughput single-pass-measurement requirements of flow cytometry. Therefore results that mimic the FLECKD system are provided in Fig.3B, which plots a 15-ns Gaussian excitation curve (Fig.3B, ‘—’) and the simulated emission, or ex-Gaussian plot, resulting from convolution with a 10-ns decay function (Fig.3B, ‘o’). The simulation results in Fig.3A, B, and C illustrate the point that excitation pulse widths of typical flow cytometers are not sufficient to directly time-resolve fluorescence decays and conversely picosecond excitation widths, albeit optimum for high resolution, are too narrow for flow cytometry. Therefore excitation pulse widths that are on the same order of magnitude as the sought-after fluorescence lifetime decay values can be adequate to produce the required time resolution.
Affiliation: Department of Chemical Engineering, College of Engineering, New Mexico State University, Las Cruces, NM, USA.