Single-photon peak event detection (SPEED): a computational method for fast photon counting in fluorescence lifetime...

Area of Science:

• Biophotonics and Imaging Science
• Computational Microscopy
• Cellular Metabolism and Dynamics

Background:

• Fluorescence lifetime imaging microscopy (FLIM) offers valuable contrast by analyzing fluorescence emission's temporal properties.
• Current FLIM methods face limitations in accuracy and acquisition speed, hindering broader applications.

Purpose of the Study:

• To introduce a novel computational single-photon counting method for time-domain FLIM data.
• To enhance accuracy and speed in fluorescence lifetime and intensity measurements.
• To enable high-throughput and high-dynamic-range FLIM imaging.

Main Methods:

• Developed Single-photon PEak Event Detection (SPEED), a computational method for processing directly sampled time-domain FLIM data.
• Achieved over 160 Mega-counts-per-second acquisition rates with sub-nanosecond time resolution.
• Implemented real-time GPU-accelerated processing for SPEED.

Main Results:

• Demonstrated that SPEED provides more accurate fluorescence lifetime and intensity measurements compared to direct pulse sampling.
• Showcased SPEED's superior speed over established photon counting FLIM methods.
• Successfully applied SPEED to image NAD(P)H metabolic dynamics during apoptosis in human breast cancer cells.

Conclusions:

• SPEED significantly improves FLIM accuracy and throughput, overcoming limitations of existing techniques.
• Computational photon counting methods like SPEED open new avenues for advanced FLIM applications.
• This method provides a foundation for future innovations in FLIM technology and biological research.