Single-molecule fluorescent probes can be used for nanoreporting, localization, and now multiplexing, but understanding their stochastic fluctuations in emission intensity is crucial for accurate signal interpretation. Here, the authors elucidate the blinking dynamics of rhodamine, BODIPY, and antraquinone dyes, demonstrate that multiplexing performance improves with photophysical differences, and suggest guidelines for the selection and design of organic fluorophores for single-molecule multiplexing.
- Amelia G. Seabury
- Alisha J. Khodabocus
- Kristin L. Wustholz