MPI Campus Seminar: MINFLUX NANOSCOPY FOR BIOLOGICAL IMAGING WITH MOLECULAR RESOLUTION

The ultimate goal of biological fluorescence microscopy is to provide three-dimensional resolution at the size scale of a fluorescent marker. The resolution of conventional optical microscopes is limited by the diffraction of light, however, implyingthat only features in a distance of about half of the wavelength can be discerned. Over the last decades, several super-resolution techniques, like STED(stimulated emission depletion) microscopyor STORM (stochasticoptical reconstruction microscopy) promisedunlimitedspatialresolutionin theory. Inexperiments, thefinite number of photons that fluorescent molecules emit beforetransitioning into a permanent dark stateconstrainthe resolution to 10-20 nanometers. MINFLUX nanoscopy breaks this limitationby probing the position of switchable fluorophores with a minimum of excitation light, thus renderingthe emitted photons more informative while leaving the photon budget untouched. In proof-of-concept experiments, thisapproachallowed imaging test structureswith molecular precision[1]. Here we show that MINFLUX nanoscopy canprovide resolutions in the range of 1 to 3 nm also for structures in fixed and living cells [2]. This progress has been facilitated by approaching each fluorophore iteratively with the probing-donut minimum, making the resolution essentially uniform and isotropic over scalable fields of view. By exploiting a minimum of excitation light confined in all dimensions, we further demonstrate MINFLUX imaging of the nucleoporin Nup96 in mammalian cells with nanometer-scale resolution in three dimensions and two color channels. We show first 3D two-color MINFLUX acquisitions of dense protein distributions in human mitochondria. The unprecedented 3D resolution of the images paves the way for new quantitative analysis approaches that we exploit for studying the distribution of proteins within the heterooligomeric MICOS protein complex[3].

[1] F. Balzarotti*, Y. Eilers*, K. C. Gwosch*, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell. “Nanometer Resolution Imaging and Tracking of Fluorescent Molecules with Minimal Photon Fluxes”. In: Science (2017). https://doi.org/10.1126/science.aak9913

[2] K. C. Gwosch*, J. K. Pape*, F. Balzarotti*, P. Hoess, J. Ellenberg, J. Ries,and S. W. Hell. “MINFLUX Nanoscopy Delivers 3D Multicolor Nanometer Resolution in Cells”. In: Nature methods (2020). https://doi.org/10.1038/s41592-019-0688-0

[3] J. K. Pape*, T. Stephan*, F. Balzarotti, R. Büchner, F. Lange, D. Riedel, S. Jakobs, & S. W. Hell.“Multicolor 3D MINFLUX nanoscopy of mitochondrial MICOS proteins”. In: Proceedings of the National Academy of Sciences(2020).https://doi.org/10.1073/pnas.2009364117