Spectroscopic analysis beyond the diffraction limit.

The recent surge in spectroscopic Single-Molecule Localization Microscopy (sSMLM) offers exciting new capabilities for combining single molecule imaging and spectroscopic analysis. Through the synergistic integration of super-resolution optical microscopy and single-molecule spectroscopy, sSMLM offers combined strengths from both fields. By capturing the full spectra of single molecule fluorescent emissions, sSMLM can distinguish minute spectroscopic variations from individual fluorescent molecules while preserving nanoscopic spatial localization precision. It can significantly extend the coding space for multi-molecule super-resolution imaging. Furthermore, it has the potential to detect spectroscopic variations in fluorescence emission associated with molecular interactions, which further enables probing local chemical and biochemical inhomogeneities of the nano-environments. In this review, we seek to explain the working principle of sSMLM technologies and the status of sSMLM techniques towards new super-resolution imaging applications.