Localizing and tracking of fluorescent molecules with minimal photon fluxes

Abstract

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.