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The identification of fluorescent dyes in cultural heritage materials is challenging due to analyte fading, as well as sample scarcity and complexity. Here, we demonstrate a blinking-based methodology to identify single dye molecules in ink, relying solely on the dyes’ intrinsic fluorescence intermittency. Using widefield fluorescence microscopy, change point detection, and multinomial logistic regression, we define four quantitative determination factors that provide for positive and exclusive identification among three structurally similar rhodamine dyes. This approach is then applied to wet and dry commercial ballpoint ink samples and demonstrates the presence of rhodamine B, which is validated by bulk surface-enhanced Raman scattering (SERS) measurements. As compared to SERS, blinking-based identification yields exclusive and positive identification of rhodamine dyes with single-molecule sensitivity and without the need for plasmonic substrates. This minimally invasive and ultrasensitive method offers a powerful new tool for characterizing artists’ materials, opening opportunities for conservation and heritage science. 

Abstract Single-molecule fluorescence experiments have transformed our understanding of complex materials and biological systems. Whether single molecules are used to report on their nano-environment or provide for localization, understanding their blinking dynamics (i.e., stochastic fluctuations in emission intensity under continuous illumination) is paramount. We recently demonstrated another use for blinking dynamics called blink-based multiplexing (BBM), where individual emitters are classified using a single excitation laser based on blinking dynamics, rather than color. This study elucidates the structure-activity relationships governing BBM performance in a series of model rhodamine, BODIPY, and anthraquinone fluorophores that undergo different photo-physical and-chemical processes during blinking. Change point detection and multinomial logistic regression analyses show that BBM can leverage spectral fluctuations, electron and proton transfer kinetics, as well as photostability for molecular classification—even within the context of a shared blinking mechanism. In doing so, we demonstrate two- and three-color BBM with ≥ 93% accuracy using spectrally-overlapped fluorophores.