Blinking characteristics of organic fluorophores for blink-based multiplexing.

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.

Rapid, Accurate Classification of Single Emitters in Various Conditions and Environments for Blinking-Based Multiplexing.

Although single-molecule imaging is widely applied in biology and materials science, most studies are limited by their reliance on spectrally distinct fluorescent probes. We recently introduced blinking-based multiplexing (BBM), a simple approach to differentiate spectrally overlapped single emitters based solely on their intrinsic blinking dynamics. The original proof-of-concept study implemented two methods for emitter classification: an empirically derived metric and a deep learning algorithm, both of which have significant drawbacks.

Blinking-Based Multiplexing: A New Approach for Differentiating Spectrally Overlapped Emitters.

Multicolor single-molecule imaging is widely applied to answer questions in biology and materials science. However, most studies rely on spectrally distinct fluorescent probes or time-intensive sequential imaging strategies to multiplex. Here, we introduce blinking-based multiplexing (BBM), a simple approach to differentiate spectrally overlapped emitters based solely on their intrinsic blinking dynamics.