Single-molecule excitation–emission spectroscopy

Significance The most popular methods of single-molecule detection are based on fixed-wavelength excitation of individual molecules combined with dispersed detection of emission. While these methods are well established, they mainly provide information on the excited-state environment of the fluorophore. Here, we introduce an approach capable of simultaneously acquiring the full information, involving both ground- and excited states, by recording a 2D excitation versus emission map for single molecules by use of a birefringent interferometer. We interpret the results in terms of optical lineshape theory. The presented approach is easily implementable and applicable to a wide range of scientific problems, ranging from spectroscopy of supramolecular biological complexes to materials science. Single-molecule spectroscopy (SMS) provides a detailed view of individual emitter properties and local environments without having to resort to ensemble averaging. While the last several decades have seen substantial refinement of SMS techniques, recording excitation spectra of single emitters still poses a significant challenge. Here we address this problem by demonstrating simultaneous collection of fluorescence emission and excitation spectra using a compact common-path interferometer and broadband excitation, which is implemented as an extension of a standard SMS microscope. We demonstrate the technique by simultaneously collecting room-temperature excitation and emission spectra of individual terrylene diimide molecules and donor–acceptor dyads embedded in polystyrene. We analyze the resulting spectral parameters in terms of optical lineshape theory to obtain detailed information on the interactions of the emitters with their nanoscopic environment. This analysis finally reveals that environmental fluctuations between the donor and acceptor in the dyads are not correlated.