Identification of Nonradiative Decay Pathways in Cy3

Photoexcited fluorescent markers are extensively used in spectroscopy, imaging, and analysis of biological systems. The performance of fluorescent markers depends on high levels of emission, which are limited by competing nonradiative decay pathways. Small-molecule fluorescent dyes have been increasingly used as markers due to their high and stable emission. Despite their prevalence, the nonradiative decay pathways of these dyes have not been determined. Here, we investigate these pathways for a widely used indocarbocyanine dye, Cy3, using transient grating spectroscopy. We identify a nonradiative decay pathway via a previously unknown dark state formed within ∼1 ps of photoexcitation. Our experiments, in combination with electronic structure calculations, suggest that the generation of the dark state is mediated by picosecond vibrational mode coupling, likely via a conical intersection. We further identify the vibrational modes, and thus structural elements, responsible for the formation and dynamics of the dark state, providing insight into suppressing nonradiative decay pathways in fluorescent markers such as Cy3.