We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolution microscopy, MINFLUX attained ~1-nm precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30 ribosomal subunits in living As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.

SignificancePopular localization of single molecules through calculating the centroid of the diffraction pattern produced by molecular fluorescence on a camera is typically limited to spatiotemporal resolutions of >10 nm per >10 milliseconds. By requiring at least 10–100 times fewer detected photons and being free of bias due to molecular orientation, the localization concept called MINFLUX propels molecular tracking to the hitherto-unachievable regime of single-digit nanometer precision within substantially less than a millisecond. Our experiments herald the feasibility to detect molecular interactions and conformational changes at microsecond timescales.

In this thesis MINFLUX is presented, a novel optical scheme to determine the position of individual photon emitters in space. By probing the emitter with adapted illumination profiles featuring an intensity zero, the number of emitted photons needed for precise localization can be minimized. Proofof-concept measurements on static emitters reduced the number of photon detections by 22-fold at equal localization precision compared to widespread camera-based methods. Localization precisions of 2.5 nanometer were realized at 400 microsecond time resolution using only 100 photons. With MINFLUX, the temporal resolution in single molecule tracking experiments of mEos2 fused to 30S ribosomal subunits could be increased by 100-fold.Using the available photons more effectively, the number of localizations was enhanced by more than an order of magnitude permitting a 3-fold improvement in the diffusion coefficient estimation precision. Theoretical evaluations showed that it can be expected that future MINFLUX implementations will improve the spatio-temporal resolution beyond the presented experimental results and thus further facilitate the study of fundamental processes in living organisms at their characteristic time and length scales.