Emission dipole orientation reveals dynamic single-molecule interactions with 2D crystals at solvent interfaces

Direct observation of individual fluorescent emitters is essential for studying quantum materials, chemical reactions, and biological systems. However, current single-molecule tracking methods only focuses on the localizations of molecules, overlooking molecular configuration and orientation. In this work, we introduce a high-throughput polarized single-molecule localization microscopy that simultaneously resolves the locations and emission dipole orientations of single fluorescent emitters with nanometer precision. Using the interface between pristine hexagonal boron nitride (h-BN) and an organic solvent as a challenging platform, we capture over 10⁵ fluorescent events and reveal distinct molecular interaction dynamics at room temperature. The measured dipole orientations align with the three-fold (C₃) rotational symmetry of the h-BN lattice, and molecular dynamics in the liquid environment can be modulated electrochemically, suggesting a route for on-demand control of quantum emitters. We also find that lateral diffusion at the solid–liquid interface is far more dynamic than that of solid-state emitters. This simultaneous tracking of molecular conformation and photophysics advances the understanding of single-molecule interactions and enables real-time sensing through two-dimensional materials. Hexagonal boron nitride substrates affect the dynamics of single molecules at solid-liquid interfaces. Here, the authors use polarized super-resolution microscopy to map the 3D orientation of emitters at interfaces, thereby demonstrating how lattice structure and electricity regulate molecular behavior.