  By circumventing the optical diffraction limit, super-resolved fluorescence
microscopies enable the study of larger cellular structures and molecular
assemblies. However, fluorescence nanoscopy currently lacks the spatiotemporal
resolution to resolve distances on the size of individual molecules and reveal
the conformational fine structure and dynamics of molecular complexes. Here we
establish FRET nanoscopy by combining colocalization STED microscopy with
multiparameter FRET spectroscopy. We simultaneously localize donor and acceptor
dyes of single FRET pairs with nanometer resolution and quantitatively measure
intramolecular distances with sub-nanometer precision over a large dynamic
range. While FRET provides isotropic 3D distance information, colocalization
measures the projected distance onto the image plane. The combined information
allows us to directly determine its 3D orientation using Pythagoras's theorem.
Studying two DNA model systems and the human guanylate binding protein hGBP1,
we demonstrate that FRET nanoscopy unravels the interplay between their spatial
organization and local molecular conformation in a complex environment.
