Spatially encoded fast single-molecule fluorescence spectroscopy with full field-of-view

We report a simple single-molecule fluorescence imaging method that increases the temporal resolution of any type of array detector by >5-fold with full field-of-view. We spread single-molecule spots to adjacent pixels by rotating a mirror in the detection path during the exposure time of a single frame, which encodes temporal information into the spatial domain. Our approach allowed us to monitor fast blinking of an organic dye, the dissociation kinetics of very short DNA and conformational changes of biomolecules with much improved temporal resolution than the conventional method. Our technique is useful when a large field-of-view is required, for example, in the case of weakly interacting biomolecules or cellular imaging.