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

Nedbal, Jakub, Visitkul, Viput, Ortiz-Zapater, Elena, Weitsman, Gregory, Chana, Prabhjoat, Matthews, Daniel R., Ng, Tony and Ameer-Beg, Simon M. (2015) Time-domain microfluidic fluorescence lifetime flow cytometry for high-throughput Förster resonance energy transfer screening. Cytometry Part A, 87 2: 104-118. doi:10.1002/cyto.a.22616

Author Nedbal, Jakub
Visitkul, Viput
Ortiz-Zapater, Elena
Weitsman, Gregory
Chana, Prabhjoat
Matthews, Daniel R.
Ng, Tony
Ameer-Beg, Simon M.
Title Time-domain microfluidic fluorescence lifetime flow cytometry for high-throughput Förster resonance energy transfer screening
Journal name Cytometry Part A   Check publisher's open access policy
ISSN 1552-4930
Publication date 2015
Year available 2014
Sub-type Article (original research)
DOI 10.1002/cyto.a.22616
Open Access Status
Volume 87
Issue 2
Start page 104
End page 118
Total pages 15
Place of publication Hoboken, NJ, United States
Publisher Wiley-Liss
Collection year 2015
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.
Keyword Flow cytometry
Fluorescence lifetime
Time-correlated single photon counting
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Queensland Brain Institute Publications
Official 2015 Collection
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