Compensating for photon counting losses in a TCSPC SPAD array enables quantitative time-resolved fluorescence...

Area of Science:

• Photon counting techniques
• Fluorescence imaging
• Optical instrumentation

Background:

• Accurate wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM) requires precise quantification of lost photons.
• Single photon avalanche diode (SPAD) array cameras, like QuantICAM, are used in TR-FAIM but can lose photon counts during exposure.
• Lost photons can compromise quantitative accuracy in TR-FAIM measurements.

Purpose of the Study:

• To develop and validate a theoretical model for accounting for lost photon counts in time-correlated single photon counting (TCSPC)-based SPAD array cameras.
• To enable quantitatively correct TR-FAIM by addressing photon loss during image acquisition.
• To assess the impact of lost photons on various TR-FAIM parameters.

Main Methods:

• A theoretical model based on a binomial distribution with a single adjustable parameter was devised.
• Experimental validation was performed using a TCSPC-based QuantICAM SPAD array camera.
• The model was fitted to experimental data of recorded versus true photon counts for different exposure times.

Main Results:

• The binomial model accurately describes experimental data for photon counts at various arrival times and fluorescence decays.
• Least-squares fits of the model successfully extracted the correct number of excitation cycles during exposure.
• Compensation for lost photons was shown to be essential for quantitatively correct steady-state anisotropy and G-factor histograms in TR-FAIM.

Conclusions:

• The developed model effectively accounts for lost photons in TCSPC-based TR-FAIM.
• Photon loss compensation is critical for obtaining accurate anisotropy and G-factor measurements.
• Rotational correlation time, initial anisotropy (r0), and hindered rotation parameter (r∞) histograms are minimally affected by photon loss under the studied conditions.