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Updated: Aug 18, 2026

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules
Published on: April 25, 2025
Determination of photophysical parameters from photon arrival time trajectories in single molecule fluorescence
Michal Martyński1, Jolanta Zydlewicz, Noël Boens
1Laboratory for Dynamics of Physicochemical Processes, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznań, Poland.
Abstract:
Triplet blinking is a phenomenon observed commonly in single molecule fluorescence spectroscopy. At high to moderate excitation intensities one can distinguish bright (on) and dark (off) periods in the fluorescence intensity trajectory caused by sojourns into the nonemissive triplet state. In this work, we focus on triplet blinking of an immobilized molecule in the low excitation regime, where a threshold between on and off intensity levels cannot be set, and, therefore, a standard on/off analysis of fluorescence intensity trajectories is not possible. In the low excitation regime triplet blinking parameters can be recovered from photon arrival time trajectories, i.e., records of individual photon arrival time. We use computer-generated data to compare the recovery of the triplet blinking parameters from the intensity correlation function (ICF) and the histogram of interarrival time. We have found that the ICF offers a better statistics for the recovery of the triplet blinking parameters.
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