Lifetime-based analysis of binary fluorophores mixtures in the low photon count limit

Single biomolecule sensing often requires the quantification of multiple fluorescent species. Here, we theoretically and experimentally use time-resolved fluorescence via Time Correlated Single Photon Counting (TCSPC) to accurately quantify fluorescent species with similar chromatic signatures. A modified maximum likelihood estimator is introduced to include two fluorophore species, with compensation of the instrument response function. We apply this algorithm to simulated data of a simplified two-fluorescent species model, as well as to experimental data of fluorophores' mixtures and to a model protein, doubly labeled with different fluorophores' ratio. We show that 100 to 200 photons per fluorophore, in a 10-ms timescale, are sufficient to provide an accurate estimation of the dyes' ratio on the model protein. Our results provide estimation for the desired photon integration time toward implementation of TCSPC in systems with fast occurring events, such as translocation of biomolecules through nanopores or single-molecule burst analyses experiments.