Single molecule (SM) fluorescence spectroscopy has proven to be a powerful, noninvasive tool in life science, materials science, and photophysics. Here we present an innovative approach to SM fluorescence spectroscopy, able to collect two-dimensional excitation-emission (2D-EEM) maps rapidly and under ambient conditions. If emission occurs from the initially excited state, excitation spectra are equivalent to absorption spectra and are sensitive to couplings of the SM with the local environment or other molecules. The high signal to noise ratio of the measurements presented in this work allow for a characterization of molecular properties on electronic ground and excited states. Among such properties are reorganization energies, the strength of system-bath interaction as well as vibrational anharmonicity constants. As a result, excitation/emission spectra provide unique insight into SMs, beyond effects related to inhomogeneity which are unavoidable in ensemble measurements.
Our approach to SM 2D-EEM is based on Fourier-transform spectroscopy. We employ an innovative, compact, fast, versatile and highly stable common-path interferometer based on birefringent crystals. It generates two phase-locked replicas of the excitation light without the need for active stabilization or auxiliary tracking beams. It provides adjustable excitation wavelength resolution (down to the sub-nm range). We collected sixty SM 2D-EEM maps from terrylene diimide dye with data quality equal to bulk spectra obtained with commercial absorption spectrometers. Based on statistical analysis, we discuss the distribution of spectral shapes of individual molecules due to a combination of intrinsic molecular variety and different interactions of the molecules with their local environment.