Cytometric sorting based on the fluorescence lifetime of spectrally overlapping signals

Abstract

Flow cytometry is a well-established and powerful high- throughput fluorescence measurement tool that also allows for the sorting and enrichment of subpopulations of cells expressing unique fluorescence signatures. Owing to the reliance on intensity-only signals, flow cytometry sorters cannot easily discriminate between fluorophores that spectrally overlap. In this paper we demonstrate a new method of cell sorting using a fluorescence lifetime-dependent methodology. This approach, referred to herein as phase-filtered cell sorting (PFCS), permits sorting based on the average fluorescence lifetime of a fluorophore by separating fluorescence signals from species that emit differing average fluorescence lifetimes. Using lifetime-dependent hardware, cells and microspheres labeled with fluorophores were sorted with purities up to 90%. PFCS is a practical approach for separating populations of cells that are stained with spectrally overlapping fluorophores or that have interfering autofluorescence signals.

À la Fizeau in flow: Pulse shape‐assisted fluorescence lifetime

“…To name a few, these processes are: internal conversion, vibrational relaxation, intersystem crossing, F€ orster resonance energy transfer (FRET), dynamic quenching, solvent relaxation, charge transfer, and photolysis (photobleaching); and most of these processes involve interactions of the excited fluorophore with its local environment (1,3). For this reason fluorescence lifetime can be used to monitor locally the concentrations of ions and molecules, electric field and polarity, temperature, viscosity, and refractive index (1)(2)(3)(6)(7)(8)(9). By spanning a much larger range than the wavelength of the emitted fluorescence, it is more amenable for multiplexing than wavelength (8,10).…”

“…By spanning a much larger range than the wavelength of the emitted fluorescence, it is more amenable for multiplexing than wavelength (8,10). It can be used as a contrast parameter, for example, for discriminating between emitters having overlapping emission spectra (9). It is also a state parameter, meaning that it is independent of conditions of excitation such as wavelength, intensity, and polarization of the exciting light.…”

“…Scattering solution of metal powder, milk, or a light reflector mirror having zero retardance or a fluorophore of known lifetime introduced in the sample position can serve as reference. The unknown lifetime can be determined from the relative phase delay and relative demodulation of the sample as compared to the reference (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)14).…”

“…In spite of this drawback, it has been successfully used recently for discriminating between different states of fluorophores also in flow showing that the high throughput property can be advantageously combined with the phase detection. Combination of multiplexing feature of flow cytometry with the phase method-that is, to measure the lifetimes in several fluorescence channels in parallelmay be hindered by the insufficient speed of data processing electronics and the substantial cost increase (e.g., each excitation beam should be modulated independently) (8,9).…”

“…The final aim would be to obtain the full fluorescence decay curve-impulse response function-per each cell by deconvoluting the fluorescence pulse shape from the exciting one encoded in the light scattering pulse profile. Having the full decay curve for each cell the frequency distribution curves of the pre-exponentials describing the amounts of two forms of a dye, or the amounts of a dye situated in two different environments (polar-apolar, bound-unbound) can be constructed (9).…”