Blinking-Based Identification of Single Dye Molecules in Ink

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.