Dipole orientation reveals single-molecule interactions and dynamics on 2D crystals

Direct observation of single-molecule interactions and dynamic configurations in situ is a demanding challenge but crucial for both chemical and biological systems. However, optical microscopy that relies on bulk measurements cannot meet these requirements due to rapid molecular diffusion in solutions and the complexity of reaction systems. In this work, we leveraged the fluorescence activation of pristine hexagonal boron nitride (h-BN) in organic solvents as a molecular sensing platform, confining the molecules to a two-dimensional (2D) interface and slowing down their motion. Conformational recognition and dynamic tracking were achieved simultaneously by measuring the 3D orientation of fluorescent emitters through polarized single-molecule localization microscopy (SMLM). We found that the orientation of in-plane emitters aligns with the symmetry of the h-BN lattice, and their conformation is influenced by both the local conditions of h-BN and the regulation of the electrochemical environment. Additionally, lateral diffusion of fluorescent emitters at the solid-liquid interface displays more abundant dynamics compared to solid-state emitters. This study opens the door for the simultaneous molecular conformation and photophysics measurement, contributing to the understanding of interactions at the single-molecule level and real-time sensing through 2D materials.