Flow cytometric time-resolved measurements by frequency heterodyning of fluorescence emission signals

A first generation phase-sensitive flow cytometer has been developed that combines flow cytometry (FCM) and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making time-resolved fluorescence measurements in the frequency-domain or particles/cells labeled with fluorescent probes. Cells are analyzed as they flow through a chamber and intersect a high-frequency, intensity-modulated (sine-wave) laser excitation beam. Fluorescence emission signals are currently processed by analog homodyne methods to quantify lifetimes and resolve heterogeneous fluorescence based on differences in lifetimes expressed as phase shifts, while maintaining the capability to make conventional FCM measurements. in this study we report the current status of our phase flow cytometer using homodyne signal processing, including recent applications, along with the description of a flow cytometric method for quantifying fluorescence lifetimes by frequency heterodyning techniques. Lifetimes are determined from homodyned and heterodyned (1 MHz difference frequency) signals using analog signal processing electronics. These signals are then digitized and displayed as frequency distribution histograms in real time.