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Abstract

Optical investigations of nanometer distances between proteins, their subunits, or other biomolecules have been the exclusive prerogative of Forster Resonance Energy Transfer (FRET) microscopy for decades. Here we show that MINFLUX fluorescence nanoscopy measures intra-molecular distances down to 1 nm - and in planar projections down to 1 Angstrom - directly, linearly, and with Angstrom precision. Our method is validated by quantifying well-characterized 1-to-10 nm distances in polypeptides and proteins. Moreover, we visualize the orientations of immunoglobulin subunits and reveal specific configurations of a histidine kinase domain dimer. Our results open the door for examining proximities and interactions of macromolecules under physiological conditions.