%0 Generic
%A Sahl, Steffen Joachim
%D 2010
%F heidok:11425
%K Lokalisierung , Einzelmolekültracking , Dynamiksingle molecule tracking , fluorescence , plasma membrane dynamics , lipid rafts , nanoscale diffusion
%R 10.11588/heidok.00011425
%T Detection of fast-diffusing molecules in the membrane of living cells
%U https://archiv.ub.uni-heidelberg.de/volltextserver/11425/
%X We present a novel optical scheme for the observation of the dynamics of single fluorescent molecules, with a flexible choice of high spatial localization accuracy (~10-20 nm standard deviation or ~20-40 nm full-width-at-half-maximum) and temporal resolution (<1 ms, <0.5 ms for much of a molecule trajectory). The fluorescence signal during individual passages of fluorescent molecules through a spot of excitation light allows the sequential localization and thus spatio-temporal tracking of the molecule if its fluorescence is collected on at least three separate point detectors arranged in close proximity. Time-correlated-single-photon-counting is shown to be a vital feature within this approach, allowing useful extensions in the analysis and improving signal-to-noise ratio in molecular tracking data. We show two-dimensional trajectories of individual, small organic dye labeled lipids diff using in the plasma membrane of living cells and directly observe transient events of trapping on <20 nm spatial scales. The trapping is cholesterol-assisted and much more pronounced for a sphingo- than for a phosphoglycero-lipid, with average trapping times of ~15 ms and <4 ms, respectively. The results support previous STED nanoscopy measurements and suggest that, at least for nontreated cells, the transient interaction of a single lipid is con fined to macromolecular dimensions. Our experimental approach demonstrates that molecular movements up to diffusion coefficients D~0.1-1 square microns / s can be tracked with minimal invasion, which can reveal new important details of cellular nano-organization. This is further exemplifi ed by uncovering subtle di fferences in the lateral diffusion of several membrane constituents of diff erent chemical structure. The potential of single photon detection and counting for the rapidly developing  field of far- field optical nanoscopy is discussed and proof-of-principle experiments are presented in which nanoscale images are reconstructed from molecular registrations based on their complete photon statistics.