Nonstop nanometric resolution of randomly moving point scatterers with focused light

Abstract In established super-resolution fluorescence microscopy, resolving multiple fluorescent molecules at sub-diffraction distances requires the molecules to emit sequentially so that they become discernible from their neighbors one after another. Simultaneous tracking of multiple fluorophores that are only a few nanometers apart is thus conceptually and practically impossible. We have recently shown that probing a sub-diffraction region with an excitation beam featuring an intensity zero, i.e., MINFLUX, super-resolves and tracks closely packed fluorophores without interruption. Here, we provide a conceptual framework for resolving and tracking constantly emitting fluorophores – more generally, point scatterers – that undergo random changes in position. In particular, we show that the detection rates available in fluorescence microscopy are sufficient to track sub-10 nm distance changes within micro-to milliseconds. By using a DNA origami construct with a fixed and a movable fluorophore as a proxy, we prove the concept that thermally driven conformational changes of biomolecules are continuously detectable with visible light. Conformational changes of the DNA nanostructure leading to random jumps in distance of about 10 nm between two labels are registered within about a millisecond. Our work paves the way towards super-resolving complex conformational transitions of individual biomolecules with focused light.