Digital analysis and sorting of fluorescence lifetime by flow cytometry

Area of Science:

• Biotechnology
• Analytical Chemistry
• Cell Biology

Background:

• Traditional flow cytometry primarily analyzes fluorescence intensity and light scatter.
• Fluorescence lifetime, a valuable but underutilized parameter, offers additional biological information.
• Existing frequency-domain flow cytometry methods often rely on complex analog systems.

Purpose of the Study:

• To develop and validate a digital signal processing approach for frequency-domain flow cytometry.
• To enable real-time fluorescence decay lifetime analysis and cell sorting.
• To improve the accessibility and application of fluorescence lifetime measurements in flow cytometry.

Main Methods:

• Modified the Open Reconfigurable Cytometric Acquisition System (ORCAS) with enhanced digital signal processing (DSP) capabilities.
• Implemented real-time Fourier analysis on radiofrequency (RF)-modulated detector signals for fluorescence lifetime calculation.
• Performed error analysis using simulated waveforms and validated the system against analog methods and known fluorophores.

Main Results:

• Achieved standard deviations in digitally acquired lifetime values ranging from 0.112 to >2 ns, with low coefficients of variation (<1%) under optimal conditions.
• Demonstrated comparable precision and accuracy to analog phase-sensitive flow cytometry.
• Successfully sorted fluorescent microsphere populations with distinct lifetimes (2 ns and 7 ns) to ~98% purity.

Conclusions:

• The introduced digital signal acquisition and processing methods provide a robust and simple approach for phase-sensitive flow cytometry.
• The system allows for inexpensive implementation on commercial flow sorters, promoting wider adoption of fluorescence lifetime analysis.
• This technology enhances the exploitation of fluorescence lifetime as a key parameter in cytometry.