Detection of fast-diffusing molecules in the membrane of living cells [Elektronische Ressource] / put forward by Steffen Joachim Sahl

Dissertationsubmitted to theCombined Faculties for the Natural Sciences and for Mathematicsof the Ruperto-Carola University of Heidelberg, Germanyfor the degree ofDoctor of Natural SciencesPut forward bySte en Joachim Sahl, BA MA MSciborn in Aalen (Wurt t.)Oral examination: November 24th, 2010Detection of fast-di using moleculesin the membrane of living cellsReferees: Prof. Dr. Dr. h.c. Stefan W. HellProf. Dr. Dirk DubbersAbstractWe present a novel optical scheme for the observation of the dynamics of single u-orescent molecules, with a exible choice of high spatial localization accuracy (10{20 nm standard deviation or20{40 nm full-width-at-half-maximum) and temporalresolution (<1 ms, <0.5 ms for much of a molecule trajectory). The uorescencesignal during individual passages of uorescent molecules through a spot of excita-tion light allows the sequential localization and thus spatio-temporal tracking of themolecule if its uorescence is collected on at least three separate point detectors ar-ranged in close proximity. Time-correlated-single-photon-counting is shown to be avital feature within this approach, allowing useful extensions in the analysis and im-proving signal-to-noise ratio in molecular tracking data. We show two-dimensionaltrajectories of individual, small organic dye labeled lipids di using in the plasmamembrane of living cells and directly observe transient events of trapping on<20 nmspatial scales.