Phasor-based widefield FLIM using a gated 512×512 single-photon SPAD imager.

Area of Science:

• Biophotonics
• Advanced Imaging Technologies
• Fluorescence Microscopy

Background:

• Single-photon avalanche diode (SPAD) imagers offer compact, fast time-resolved imaging.
• Recent SPAD advancements enable widefield microscopy with megapixel arrays and competitive sensitivity.

Purpose of the Study:

• To demonstrate phasor-based fluorescence lifetime imaging microscopy (FLIM) using a gated binary SPAD imager.
• To validate the accuracy and speed of this FLIM approach for measuring short fluorescence lifetimes.

Main Methods:

• Utilized a 512×512 gated binary SPAD imager with short gate lengths (5.75 ns) and small gate steps (17.9 ps).
• Acquired data by shifting a 13.1 ns gate window across a 50 ns laser period for phasor analysis.
• Measured lifetimes of ATTO 550 and Rhodamine 6G solutions and compared results with TCSPC and Levenberg-Marquardt fitting.

Main Results:

• Achieved >90% accuracy for Rhodamine 6G and ATTO 550 lifetimes, comparable to established methods.
• Demonstrated accurate measurement of short lifetimes without minimum gate length constraints.
• Showcased a system capable of up to 98 kfps readout rate (1-bit frames) for FLIM.

Conclusions:

• Phasor-based FLIM with a compact SPAD imager is accurate for short lifetime measurements.
• The system's high frame rate and FPGA parallel computation pave the way for near real-time widefield FLIM.
• This technology advances compact, high-speed, time-resolved imaging for microscopy applications.