Nasser Maisa, Meller Amit
Department of Biomedical Engineering, Technion - IIT, Haifa 32000, Israel.
iScience. 2021 Dec 2;25(1):103554. doi: 10.1016/j.isci.2021.103554. eCollection 2022 Jan 21.
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.
单生物分子传感通常需要对多种荧光物质进行定量分析。在此，我们通过时间相关单光子计数（TCSPC）在理论和实验上利用时间分辨荧光来准确量化具有相似色度特征的荧光物质。引入了一种改进的最大似然估计器，以包含两种荧光团物质，并对仪器响应函数进行补偿。我们将此算法应用于简化的双荧光物质模型的模拟数据，以及荧光团混合物的实验数据和用不同荧光团比例双标记的模型蛋白。我们表明，在10毫秒的时间尺度内，每个荧光团100至200个光子足以准确估计模型蛋白上染料的比例。我们的结果为在具有快速发生事件的系统（如生物分子通过纳米孔的转运或单分子爆发分析实验）中实施TCSPC所需的光子积分时间提供了估计。
