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

Louis Obeid Mogridge1, Jakub Nedbal1, Istvan Gyongy2

  • 1Department of Physics, King's College London, Strand, London WC2R 2LS, United Kingdom.

Methods and Applications in Fluorescence
|May 6, 2026
PubMed
Summary

We developed a binomial model to quantify lost photons in time-correlated single photon counting (TCSPC) imaging. This correction is crucial for accurate wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM).

Keywords:
fluorescence lifetime imaging (FLIM)single photon avalanche diode (SPAD)time-correlated single photon counting (TCSPC)

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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.