%0 Generic
%A Middendorff, Claas von
%D 2008
%F heidok:8736
%K BildtheoriePALM , FCS , STED-FCS
%R 10.11588/heidok.00008736
%T Experimental Stochastics in High-Resolution Fluorescence Microscopy : Imaging Theory of PALMIRA Microscopy; Improved Models for FCS
%U https://archiv.ub.uni-heidelberg.de/volltextserver/8736/
%X This thesis presents a statistical imaging theory for photo-activation localization microscopy with independently running acquisition (PALMIRA). In this type of sub-resolution microscopy the switching of the fluorescence capability of macromolecules reduces imaging to the high-precision localization of individual fluorescent molecules. The point-spread function and the imaging equation of a PALMIRA imaging system are calculated and stochastic expressions for the measurement time and the confidence level of the image as a function of the spatial resolution are provided. Different localization schemes like astigmatic imaging, multi-channel defocus imaging, 4pi imaging and a multi-point setup using photo-diodes are analyzed. The theory for multi-color and polarization-resolved measurements is addressed and estimators for data evaluation procedures are provided. The role of background noise in producing artefacts is studied. Finally, it is assessed whether the quality of images can be augmented by a suitable deconvolution procedure. Furthermore, stochastic methods are applied to solve a couple of persistent problems in fluorescence correlation spectroscopy (FCS). The development of computational methods for the simulation of FCS experiments necessitates the analytical description of the architecture of a multiple-lag-time correlator that is used to estimate autocorrelations from intensity time traces. Recently, FCS has been combined with stimulated emission depletion (STED) focal volumes. The general phenomenology of STED-FCS correlation curves is studied as a function of the STED beam intensity. It is shown that the quality of a measurement is mainly determined by the fraction of signal originating from the focal plane. Then, an improved fit model taking into account the exact spatial dependency of intersystem crossing rates is presented and tested on synthetic data. Finally, the influence of second-order correlations among the points of the FCS curve on the determination of fit parameters is studied. Analytical results are provided wherever possible. Otherwise, Monte-Carlo computations are performed.