Super-Resolved FRET Imaging by Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy

FRET-based methods are a special tool for detecting interactions between (bio)molecules and their immediate environment. The spatial distribution of molecular interactions and functional states can be seen using FLIM (Fluorescence Lifetime IMaging) and FRET imaging. The spatial information, accuracy, and dynamic range of the observed signals are, however, constrained by the fact that conventional FLIM and FRET imaging only provides average information over an ensemble of molecules within a diffraction-limited volume. On the other hand, conventional Single Molecule Localization Microscopy (SMLM) relies on highly sensitive multi-pixel detectors (e.g. sCMOS or EM-CCD) whose time resolution is not suitable for fluorescence lifetime measurements. Here, we demonstrate a method for obtaining super-resolved FRET imaging using confocal fluorescence-lifetime single-molecule localization microscopy. The proof of concept was carried out using a DNA origami sample for performing DNA-PAINT measurements in combination with fluorogenic probes for reducing background signal. With this method, We show that FRET events separated by sub-diffraction distances can be distinguished based on lifetime modifications.