title: 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.
authors.affiliation: Division of Cancer Studies, King's College London, United Kingdom; Randall Division of Cell and Molecular Biophysics, King's College London, United Kingdom.
authors.forename: Jakub
authors.initials: J
authors.lastname: Nedbal
authors.forename: Viput
authors.initials: V
authors.lastname: Visitkul
authors.forename: Elena
authors.initials: E
authors.lastname: Ortiz-Zapater
authors.forename: Gregory
authors.initials: G
authors.lastname: Weitsman
authors.forename: Prabhjoat
authors.initials: P
authors.lastname: Chana
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Matthews
authors.forename: Tony
authors.initials: T
authors.lastname: Ng
authors.forename: Simon M
authors.initials: SM
authors.lastname: Ameer-Beg
mesh_terms: D045744:Cell Line, Tumor; D019281:Dimerization; D004815:Epidermal Growth Factor / Q000032:analysis; D066246:ErbB Receptors / Q000032:analysis* / Q000378:metabolism; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D061986:MCF-7 Cells; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D010766:Phosphorylation; D011726:Pyridinium Compounds / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; C541550:di-4-ANEPPDHQ; D004815:Epidermal Growth Factor; D066246:ErbB Receptors
keywords: FRET; flow cytometry; fluorescence lifetime; microfluidics; time-correlated single photon counting