High speed multispectral fluorescence lifetime imaging.

Area of Science:

• Biophotonics
• Fluorescence Spectroscopy
• Microscopy

Background:

• Time-gated fluorescence lifetime imaging (FLIM) systems offer high count rates but often have limitations in gate width and decay curve sampling.
• Accurate characterization of fluorescence lifetimes and spectral properties is crucial for advanced biological imaging.

Purpose of the Study:

• To report a novel spectrally resolved fluorescence lifetime imaging system.
• To characterize the system's performance, including error analysis for lifetime and spectral detection.
• To demonstrate the utility of phasor analysis for complex FLIM data.

Main Methods:

• Development of a spectrally resolved FLIM system utilizing time-gated single photon detection with a 200 ps gate width and 7 spectral channels.
• High-speed transceiver for fluorescence signal sampling.
• Error analysis to evaluate the impact of gate width and spectral channel width on accuracy.
• Phasor analysis for interpreting spectrally resolved FLIM data.

Main Results:

• The system achieves accurate fluorescence lifetimes (error < 2%) at count rates up to 5 MHz, limited by PMT performance.
• Performance evaluation demonstrated the system's capability at count rates up to 12 MHz.
• Phasor analysis was successfully applied to images of fibroblast cells with two labeled components, enabling fast and intuitive data interpretation.

Conclusions:

• The developed spectrally resolved FLIM system provides accurate lifetime measurements at high count rates.
• Phasor analysis is a powerful and efficient tool for analyzing complex spectrally resolved FLIM data, particularly in biological imaging applications.