Point spread function engineering in fluorescence spectroscopy [Elektronische Ressource] / presented by Andreas Schönle

DissertationsubmittedtotheCombinedFacultiesfortheNaturalSciencesandforMathematicsoftheRuperto-CarolaUniversityofHeidelberg,GermanyforthedegreeofDoctorofNaturalSciencespresentedbyDiplom-PhysikerAndreasSchonle¨borninMunichOralexamination12.2.2003Point Spread Function EngineeringinFluorescence SpectroscopyReferees: Priv. Doz. Dr. Stefan W. HellProf. Dr. Josef BillePoint Spread Function Engineering in Fluorescence Spectroscopy: Thecombination of high resolution imaging with fluorescence spy has ren-dered the microscope into a powerful tool for functional analysis of biologicalspecimens. This thesis explores the potential of techniques, that are usuallyutilized for PSF-engineering, for the development of new spectroscopical appli-cations. The derivation of a simple integral solution for the Fourier transformof the vectorial PSF lays the foundation for numerical modelling of dynamical,intensity dependent processes in the focal region. Subsequently a theory de-scribing the combination of fluorescence correlation spectroscopy with diffractionlimited, periodically modulated detection volumes is derived. This idea leads tothe proposal of a ’diffusion and flow microscope’ with high spatial resolution. Itis readily implemented in a multifocal 4Pi microscope and its potential to ex-tract the parameters of anisotropic diffusion as well as speed and direction offlow inside a fluid is demonstrated in simulations.