  FRET-based approaches are a unique tool for sensing the immediate
surroundings and interactions of (bio)molecules. FRET imaging and FLIM
(Fluorescence Lifetime Imaging Microscopy) enable the visualization of the
spatial distribution of molecular interactions and functional states. However,
conventional FLIM and FRET imaging provide average information over an ensemble
of molecules within a diffraction-limited volume, which limits the spatial
information, accuracy, and dynamic range of the observed signals. Here, we
demonstrate an approach to obtain super-resolved FRET imaging based on
single-molecule localization microscopy using an early prototype of a
commercial time-resolved confocal microscope. DNA Points Accumulation for
Imaging in Nanoscale Topography (DNA-PAINT) with fluorogenic probes provides a
suitable combination of background reduction and blinking kinetics compatible
with the scanning speed of usual confocal microscopes. A single laser is used
to excite the donor, a broad detection band is employed to retrieve both donor
and acceptor emission, and FRET events are detected from lifetime information.
