Time‐domain microfluidic fluorescence lifetime flow cytometry for high‐throughput Förster resonance energy transfer screening

Abstract

Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry

Real-time fluorescence lifetime actuation for cell sorting using a CMOS SPAD silicon photomultiplier

“…The limiting factor of sorting techniques in flow cytometry is the complexity and the large volume of fluorescence data to be processed to detect the passage of each individual cell and identify its chemical or physical characteristics to perform a reliable sorting decision. For this reason, existing time-resolved fluorescence lifetime flow cytometers such as reported in [5] and [6] do not perform cell sorting. Processing of the fluorescence signal is performed offline on a PC causing large delays from slow data transfer rates.…”

New high-speed centre of mass method incorporating background subtraction for accurate determination of fluorescence lifetime

“…In practice, although data can be acquired quite rapidly with no delay between acquisitions, sorting and analysis is usually performed offline which means that one cannot readily achieve real-time lifetime readouts [17]. Whilst this immediate lifetime analysis is not required in most cases, it could be extremely important in some instances such as medical diagnostics [18], in flow cytometry [19] and high content screening applications [20,21].…”

Phasor plotting with frequency-domain flow cytometry

“…We and others have demonstrated different versions of time-resolved flow cytometry [3,[7][8][9][10][11][12][13][14], and the main objective for augmenting flow cytometers to detect fluorescence lifetimes is to enhance cytometric data with a quantitative parameter that is independent of the measured fluorescence intensity [11,[15][16][17]. As a parameter, fluorescence lifetime can be used to discriminate among spectrally overlapping fluorescence signals as well as to validate the fluorescence intensity changes that arise from quenched fluorophores such as during Förster resonance energy transfer (FRET) events like those arising from changes in association state of tagged cytoplasmic proteins.…”