Investigating Single-Molecule Fluorescence Spectral Heterogeneity of Rhodamines Using High-Throughput Single-Molecule Spectroscopy.

We experimentally investigated several intramolecular coordinate and environmental changes as potential causes of single-molecule fluorescence spectral heterogeneities (smFSH). We developed a high-throughput single-molecule spectroscopy method to analyze more than 5000 single-molecule emission spectra from each of 9 commonly used fluorophores with different structural rigidities and deposited on substrates with different polarities. We observed an unexpectedly high smFSH from structurally rigid Rhodamine B compared with a structurally flexible Cyanine dye-Alexa Fluor 647. Based on experimentally measured smFSH, we ruled out the system's noise uncertainty, single-molecule spectral diffusion, and environmental polarity as the primary causes of the high smFSH. We found that the rotational flexibility of N,N-dialkylated groups contributed to the smFSH. With the high smFSH observed in structurally more rigid model fluorophores, we speculated that other intramolecular coordinate and environmental changes might also contribute to the high smFSH in Rhodamines.