Photon efficiency optimization in time-correlated single photon counting technique for fluorescence lifetime imaging...

Area of Science:

• Photon Counting and Spectroscopy
• Biophysical Imaging Techniques

Background:

• Time-correlated single photon counting (TCSPC) systems face signal throughput limitations due to pile-up effects, distorting fluorescence lifetime (FLT) calculations and reducing signal-to-noise ratio (SNR) in high fluorescence intensity (FI) biological applications.
• While high-repetition-rate light sources (50-100 MHz) in fluorescence lifetime imaging (FLIM) mitigate pile-up, they introduce dead-time-related distortions in modern TCSPC configurations.

Purpose of the Study:

• To address dead-time-related distortions in high-repetition-rate TCSPC for FLIM.
• To optimize the F-value, which quantifies the relationship between relative standard deviations in FLT and FI measurements, for minimizing signal distortion and acquisition time.

Main Methods:

• Described signal-to-noise ratio (SNR) loss using the F-value, a metric relating relative standard deviation in estimated FLT to that in FI.
• Applied F-value optimization to fluorescent solutions (Fluorescein, Rhodamine B, Erythrosine) with varying FLT values (4 ns, 1.67 ns, 140 ps).

Main Results:

• Demonstrated that F-value optimization effectively minimizes signal distortions in TCSPC-based FLIM.
• Showcased the method's capability to shorten acquisition times for specific samples.
• Validated the approach across a range of fluorescence lifetimes.

Conclusions:

• F-value optimization is a crucial strategy for improving the accuracy and efficiency of FLIM experiments using high-repetition-rate TCSPC.
• This approach enhances the reliability of fluorescence lifetime measurements in demanding biological applications.