Pulsed dipolar hyperfine spectroscopy for molecular distance measurements in the angstrom to nanometer scale

Hyperfine spectroscopy is a fundamental method in biophysical and material sciences to detect nuclear spins in vicinity of paramagnetic centers, leading to molecular structural information. Among variants of this experiment, only electron-nuclear double resonance (ENDOR) has been established to detect nuclei at interspin distances up to about 1.7 nanometers using ¹⁹F labels. This limit is dictated by the ENDOR linewidth of 10 to 30 kilohertz, which appeared insurmountable given dipolar broadening of the detected nucleus with the nuclear spin bath. Herein, we present ENDOR experiments based on nuclear sublevel coherence spectroscopy that push the boundaries of ENDOR sensitivity and resolution by one order of magnitude. In particular, we introduce an experiment, in which the electron-nuclear dipolar interaction can be exquisitely extracted from other nuclear broadening mechanisms, thus enabling to access distance distributions. This methodology paves the way for structural studies using ¹⁹F ENDOR in biomolecular systems. Moreover, it offers opportunities to access spin dynamics in electron-nuclear coupled spin systems.