%0 Generic
%A Held, Marcus Oliver
%C Heidelberg
%D 2021
%F heidok:30257
%K nanoscopy
%R 10.11588/heidok.00030257
%T 4Pi-MINFLUX localizations of fluorescent molecules
%U https://archiv.ub.uni-heidelberg.de/volltextserver/30257/
%X MINFLUX has led to remarkable improvements in localization precision compared to centroid-fitting localization schemes. Yet, molecular localization precision with far fewer than 100 photons remained to be demonstrated. Previously, the localization precision and the number of detected photons were linked by an inverse square root relation. Decoupling these parameters allows for ultrafast tracking and measurements of photo-sensitive samples. An illumination scheme with two opposing objective lenses promises further improvements of the efficient use of detected photons in a  MINFLUX localization. In this thesis, the MINFLUX concept was combined with a 4Pi-optics illumination scheme to explore highest axial localization precision with low  photon counts. Simulations suggested that, for a given signal-background-ratio, a 4Pi-MINFLUX microscope would outperform single objective lens MINFLUX microscopes  by 40 % in terms of detected photons required for molecular localization precision. Our implementation surpassed this expectation and achieved a localization precision  of 1 nm with only several tens of detected photons in single molecule measurements. Detecting more than 100 photons led to an axial localization precision of one one-  thousandth of the excitation wavelength, limited only by vibrations and thermal drift. The axial resolving power was demonstrated with DNA origami-based nanorulers  featuring single fluorophore distances of 10 nm. These results highlight that molecular distances and dynamics on a spatial scale of 1 nm, and below, become accessible  with an extremely low number of detected photons.