  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.
