Artificial Intelligence-Empowered Spectroscopic Single Molecule Localization Microscopy.

Spectroscopic single-molecule localization microscopy (SMLM) has revolutionized the visualization and analysis of molecular structures and dynamics at the nanoscale level. The technique of combining high spatial resolution of SMLM with spectral information, enables multicolor super-resolution imaging and provides insights into the local chemical environment of individual molecules. However, spectroscopic SMLM faces significant challenges, including limited spectral resolution and compromised localization precision because of signal splitting and the difficulties in analyzing complex, multidimensional datasets, that limit its application in studying intricate biological systems and materials. The recent integration of artificial intelligence (AI) with spectroscopic SMLM has emerged as a powerful approach for addressing these challenges. Here, it is reviewed how AI-based methods applied to spectroscopic SMLM enhance and expand the capabilities of these applications. Recent advancements in AI-driven data analysis for spectroscopic SMLM, including improved spectral classification, localization precision, and extraction of rich spectral information from unmodified point-spread functions are discussed, further examining their applications in biological studies, materials science, and single-molecule reaction analysis, which highlight how AI provides new insights into molecular behavior and interactions. The AI-empowered approach adds new dimensions of information and provides new opportunities and insights into the nanoscale world of rapidly evolving field of spectroscopic SMLM.