Processes within our bodies are characterized by the interplay of various biomolecules such as proteins and DNA. These processes occur on a scale often within a range of just a few nanometers. Consequently, they cannot be observed with fluorescence microscopy, which has a resolution limit of about 200 nanometers due to diffraction. When two dyes marking positions of biomolecules are closer than this optical limit, their fluorescence cannot be distinguished under the microscope. As this fluorescence is used for localizing them, accurately determining their positions becomes impossible.
This resolution limit has traditionally been overcome in super-resolution microscopy methods by making the dyes blink and turning their fluorescence on and off. This temporally separates their fluorescence, making it distinguishable and enabling localizations below the classical resolution limit. However, for applications involving the study of rapid dynamic processes, this trick has a significant drawback: blinking prevents the simultaneous localization of multiple dyes. This significantly decreases the temporal resolution when investigating dynamic processes involving multiple biomolecules.
