Fluorescence lifetime excitation cytometry by kinetic dithering

Area of Science:

• Biomedical Engineering
• Analytical Chemistry
• Cell Biology

Background:

• Flow cytometers analyze individual cells/particles using spectroscopic data.
• Fluorophore spectral overlap limits simultaneous measurements in traditional flow cytometry.
• Time-resolved fluorescence measurements offer an alternative to intensity-based methods.

Purpose of the Study:

• Introduce Fluorescence Lifetime Excitation Cytometry by Kinetic Dithering (FLECKD) for multi-lifetime analysis.
• Develop a hybrid time-domain flow cytometry method to overcome spectral overlap limitations.
• Enhance the capability of high-throughput cellular analysis.

Main Methods:

• Utilized a hybrid time-domain approach with rapid dithering for pulsed light excitation.
• Implemented FLECKD to measure complex fluorescence decay kinetics in flow cytometry.
• Validated the method through simulations and experiments with fluorescent microspheres and single cells.

Main Results:

• Simulations showed resolvable fluorescence lifetimes as low as 1.8 ns with <20% error.
• FLECKD instrument measured an average fluorescence lifetime of 2.4 ns (±0.3 ns SEM) in microspheres.
• Successfully detected two distinct excited-state lifetimes from fluorophores in single cells.

Conclusions:

• FLECKD enables the resolution of multiple fluorescence lifetimes within a flow cytometry system.
• This technique expands high-throughput analysis capabilities beyond traditional intensity-based assays.
• FLECKD addresses the growing need for multi-fluorophore labeling in single-cell studies.