Single-Molecule, Non-Chromatic Discrimination in Binary Fluorophores Mixtures
24 Pages Posted: 26 Jul 2021 Publication Status: Under ReviewMore...
SummarySingle biomolecule sensing often involves quantification of multiple fluorescent species, coupled for example to different DNAs or proteins resides. While chromatic separation of the fluorescent species is well-established and broadly used it involves several limitations, particularly when the total photon budget is limited. Here we theoretically and experimentally explore the use of time-resolved fluorescence via Time Correlated Single Photon Counting (TCSPC) to accurately quantify multiple fluorescent species. A modified maximum likelihood estimator (MLE) is introduced to include two fluorophore species, with the compensation of the instrument response. We apply this algorithm to simulated data of a simplified two fluorescent species model, as well as to experimental data of free fluorophores mixtures and doubly labelled proteins in order to estimate the errors in the two species ratios as a function of photon budget available. Moreover, focusing on the case of labeled proteins in which the ratios of the two species per molecule is a discrete variable subject to the chemical labeling numbers of the two dyes, we obtain that between 1,000 to 2,000 photons can already provide an accurate estimation of the dyes’ ratio in labelled Ovalbumin proteins. These values correspond to roughly 100 to 200 photons per fluorophore suggesting a realistic path for dyes ratio determination from a single passage of a protein in the detection volume in roughly 10 ms timescale. Our results highlight the way in which temporal limitations of the TCSPC technique could hinder its implementation in systems with fast occurring events such as translocation of biomolecules through nanopores, or fast diffusion in single-molecule burst analyses experiments and provide estimation for the desired photon integration time.
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