Abstract
Conventional flow cytometry is a valuable quantitative tool. Flow cytometers reveal physical and biochemical information from cells at a high throughput, which is quite valuable for many biomedical, biological, and diagnostic research fields. Flow cytometers range in complexity and typically provide multiparametric data for the user at rates of up to 50,000 cells measured per second. Cytometry systems are configured such that fluorescence or scattered light signals are collected per-cell, and the integrated optical signal at a given wavelength range indicates a particular cellular feature such as phenotype or morphology. When the timing of the optical signal is measured, the cytometry system becomes “time-resolved.” Time-resolved flow cytometry (TRFC) instruments can detect fluorescence decay kinetics, and such measurements are consequential for Förster Resonance Energy Transfer (FRET) studies, multiplexing, and metabolic mapping, to name a few. TRFC systems capture fluorescence lifetimes at rates of thousands of cells per-second, however the approach is challenged at this throughput by terminal cellular velocities. High flow rates limit the total number of photons integrated per-cell, reducing the reliability of the average lifetime as a cytometric parameter. In this contribution, we examine an innovative approach to address this signal-to-noise issue. The technology merges time-resolved hardware with microfluidics and acoustics. We present an “acoustofluidic” time-resolved flow cytometer so that cellular velocities can be adjusted on the fly with a standing acoustic wave (SAW). Our work shows that acoustic control can be combined with time-resolved features to appropriately balance the throughput with the optical signals necessary for lifetime data.
Highlights
Flow cytometry is a robust statistical apparatus common to many research fields as well as the medical industry
time-resolved flow cytometry (TRFC) was recently employed in an application in which the endogenous fluorophore, NAD(P)H, was measured to identify metabolic changes during apoptosis since it is well-known that this metabolite undergoes fluorescence lifetime shifts during enzymatic reactions important for energy generation [6]
The preliminary tests performed on the time-resolved acoustofluidic flow cytometer (TRAFFC) system included fluorescence measurements detected from both fluorescence microspheres and the propidium iodide (PI)-labeled cells
Summary
Flow cytometry is a robust statistical apparatus common to many research fields as well as the medical industry. This high-throughput technique is used to separate subpopulations of a heterogeneous population of cells based on the fluorescence signal measured. To obtain the fluorescence lifetime of a fluorescent compound, a modulated laser excites the fluorophore on or within the cell. This modulated excitation source leads to a fluorescence signal modulated at the same frequency and a modulated scattered light signal [2, 7, 8]. The fluorescence lifetime is calculated from the difference in these phases
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