Microscopy: Overcoming the traditional resolution limit for the fast co-tracking of molecules

The processes within our body are characterized by the interaction of various biomolecules such as proteins and DNA. These processes are often only a few nanometers in scale. Consequently, they cannot be observed with fluorescence microscopy, which has a resolution limit of about 200 nanometers due to diffraction. When the two dye-labeled positions of biomolecules are closer than this optical limit, their fluorescence cannot be detected under a microscope. Since this fluorescence is used to localize them, it becomes impossible to precisely determine their positions.

This resolution limitation has traditionally been overcome in super-resolution microscopy methods by blinking the dyes and turning their fluorescence on and off. This temporally separates their fluorescence, making it distinct and enabling localization below the classical resolution limit. However, for applications studying fast dynamic processes, this trick has a significant drawback: blinking prevents the simultaneous localization of multiple dyes. This significantly reduces the temporal resolution when investigating dynamic processes involving multiple biomolecules.

Led by LMU chemist Professor Philip Tunfeld and supported by Professor Fernando Stefani (Buenos Aires), LMU researchers have now developed pMINFLUX multiplexing, an elegant approach to solving this problem. The team recently published a paper on their method in the journal Nature Photonics. MINFLUX is a super-resolution microscopy method, enabling localization with a precision of only one nanometer. Unlike conventional MINFLUX, pMINFLUX registers the time difference between dye excitation with a laser pulse and subsequent fluorescence with sub-nanosecond resolution. In addition to localizing dyes, this provides insight into another fundamental property of their fluorescence: their fluorescence lifetime. It tells how long, on average, it takes for a dye molecule to fluoresce after being excited.

“The fluorescence lifetime depends on the dye used,” explains Fiona Cole, co-first author of the publication. “We took advantage of the difference in fluorescent lifetime when using different colors to assign fluorescent photons to the dye that emits without blinking and the resulting temporal separation.” To this end, the researchers adapted the localization algorithm and added a multi-exponential fit model to achieve the desired separation. “This allowed us to determine the position of multiple dyes simultaneously and to probe fast dynamic processes between multiple molecules with nanometer precision,” says Jonas Zehringer, also co-first author. By jumping between different positions on the DNA origami nanostructure, the researchers precisely isolated the translational and rotational motions of the DNA origami nanostructure and measured the distance between the antibodies’ antigen-binding sites. demonstrated their method by tracking NA strands. “But this is only the beginning,” says Philip Tunfield. “I believe that pMINFLUX multiplexing, with its high temporal and spatial resolution, will provide new insights into protein interactions and other biological phenomena in the future.”