# Blinking-Based Identification of Single Dye Molecules in Ink

## TL;DR

A new method uses blinking patterns of single dye molecules to identify them in ink samples with high sensitivity and without damaging the material.

## Contribution

A novel blinking-based methodology enables exclusive identification of single dye molecules without plasmonic substrates.

## Key findings

- Four quantitative determination factors were defined for identifying three structurally similar rhodamine dyes.
- Rhodamine B was identified in ballpoint ink samples using blinking-based analysis.
- The method outperforms SERS by offering single-molecule sensitivity without plasmonic substrates.

## Abstract

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.