Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods.

Area of Science:

• Biophotonics
• Microscopy
• Spectroscopy

Background:

• Fluorescence lifetime imaging (FLIM) is a powerful microscopy technique.
• Two primary methods for FLIM are time-correlated single-photon counting (TCSPC) and frequency-domain (FD) methods.
• Comparing their performance is crucial for optimizing imaging protocols.

Purpose of the Study:

• To implement and compare TCSPC and FD methods within the same laser scanning microscope system.
• To evaluate the signal-to-noise ratio (SNR) and data acquisition speed of both techniques.
• To determine the optimal method based on sample intensity and imaging requirements.

Main Methods:

• Utilized a laser scanning microscope equipped for both TCSPC and FD fluorescence lifetime imaging.
• Acquired fluorescence lifetime images from various samples under both methods.
• Quantitatively analyzed SNR and data acquisition speed for each method.

Main Results:

• TCSPC method demonstrated a superior SNR for low-intensity fluorescence images.
• FD method exhibited faster data acquisition speeds, particularly beneficial for bright samples.
• FD method resulted in less image distortion when imaging bright specimens.

Conclusions:

• Both TCSPC and FD fluorescence lifetime imaging methods have distinct advantages.
• TCSPC is preferred for high-SNR imaging of weakly fluorescent samples.
• FD is the more efficient method for rapidly imaging brightly fluorescent samples with minimal distortion.