Autofluorescence lifetime flow cytometry with time-correlated single photon counting

Area of Science:

• Cellular metabolism and function analysis
• Advanced microscopy and flow cytometry techniques
• Biophotonics and single-cell analysis

Background:

• Autofluorescence lifetime imaging microscopy (FLIM) detects cellular metabolic changes via NAD(P)H co-enzymes.
• Traditional FLIM using laser-scanning microscopes is costly, slow, and requires extensive post-processing.
• There is a need for higher-throughput, real-time methods for analyzing single-cell metabolic states.

Purpose of the Study:

• To develop a fluorescence lifetime-sensitive flow cytometer with high temporal resolution.
• To enable real-time, label-free analysis of single-cell metabolism and function.
• To overcome the limitations of throughput and cost associated with traditional FLIM.

Main Methods:

• Utilized a 375 nm picosecond-pulsed diode laser and time-correlated single-photon counting (TCSPC) detection.
• Integrated an FPGA-based time tagger for real-time phasor-based classification of flowing cells.
• Employed a microfluidic system with simultaneous brightfield imaging and two-color analysis, achieving high throughput and low light dose.

Main Results:

• Demonstrated real-time, phasor-based gating of flowing cells with TCSPC temporal resolution.
• Confirmed cellular viability post-measurement and sensitivity to metabolic perturbations in Jurkat T cells.
• Successfully differentiated quiescent versus activated states in primary human T cells and mouse neural stem cells, consistent with FLIM studies.

Conclusions:

• The developed TCSPC-based autofluorescence lifetime flow cytometer offers a high-throughput, label-free alternative to laser-scanning FLIM.
• This system provides valuable real-time insights into single-cell function and metabolism.
• The technology has broad applications in cell biology, immunology, and neuroscience research.