Fiona Cole and Jonas Zähringer, joint lead authors of the paper, calibrate a fluorescence microscope. (LMU)

Researchers at LMU have developed an innovative method to simultaneously track rapid dynamic processes of multiple molecules at the molecular scale.

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.

Under the leadership of LMU chemist Professor Philip Tinnefeld and in cooperation with Professor Fernando Stefani (Buenos Aires), researchers at LMU have now developed pMINFLUX multiplexing, an elegant approach to address this problem. The team recently published a paper on their method in the journal Nature Photonics. MINFLUX is a super-resolution microscopy method, enabling localizations with precisions of just one nanometer. In contrast to conventional MINFLUX, pMINFLUX registers the time difference between the excitation of dyes with a laser pulse and the subsequent fluorescence with sub-nanosecond resolution. In addition to localizing the dyes, this provides insights into another fundamental property of their fluorescence: their fluorescence lifetimes. This describes how long, on average, it takes for a dye molecule to fluoresce after it is excited.

“The fluorescence lifetime depends on the dye used,” explains Fiona Cole, co-first author of the publication. “We exploited differences in fluorescence lifetimes when using different dyes to assign the fluorescent photons to the dye that emitted without the need for blinking and the resulting temporal separation.” For this purpose, the researchers adapted the localization algorithm and included a multiexponential fit model to achieve the required separation. “This allowed us to determine the position of multiple dyes simultaneously and investigate rapid dynamic processes between multiple molecules with nanometer precision,” adds Jonas Zähringer, also co-first author. The researchers demonstrated their method by accurately tracking two DNA strands as they jumped between different positions on a DNA origami nanostructure, as well as by separating translational and rotational movements of a DNA origami nanostructure and by measuring the distance between antigen-binding sites of antibodies. “But this is just the beginning,” says Philip Tinnefeld. “I am certain that pMINFLUX multiplexing, with its high temporal and spatial resolution, will provide new insights into protein interactions and other biological phenomena in the future.”


Philip Tinnefeld is grateful for support from the German Research Foundation (DFG) for Project A13 within Collaborative Research Centre 1032 (Project ID 201269156) and for funding by the Bavarian Ministry of Science and the Arts through the ONE MUNICH project “Munich Multiscale Biofabrication.” Furthermore, he is indebted to the DFG for funding via grant nos. 470075523 and 459594986 and through Germany’s Excellence Strategy (grant no. EXC 2089/1-390776260).


Fiona Cole, Jonas Zähringer, Johann Bohlen, Tim Schröder, Florian Steiner, Martina Pfeiffer, Patrick Schüler, Fernando D. Stefani, Philip Tinnefeld, Super-resolved FRET and co-tracking in pMINFLUX, Nature Photonics, X, YZ (2024).