STED microscopy in the visible range [Elektronische Ressource] / presented by Katrin I. Willig

Dissertationsubmitted to theCombined Faculties for the Natural Sciences and for Mathematicsof the Ruperto Carola University of Heidelberg, Germanyfor the degree ofDoctor of Natural Sciencespresented byDiplom Physikerin Katrin I. Willigborn in Kunz¨ elsauOral examination: February 16th, 2006STED microscopy in the visible rangeConfocalSTEDReferees: Prof. Dr. Stefan W. HellProf. Dr. Josef BilleAbstractLight microscopy is a key scientific instrument in the life sciences. However, the resolu tion of far field light microscopy is limited by diffraction. Exploiting a saturated deple tionof themolecularexcited stateby stimulated emission, stimulated emission depletion(STED)breakstheresolutionbarrierintheimportantsubfieldoffluorescencemicroscopy.To this end, STED microscopy utilizes a doughnut shaped beam featuring a central zerowhichiscapableofquenchingthefluorescencesolelyinthefocalperiphery. WhileSTEDmicroscopy was initially restricted to near infrared emitting fluorophores, in this thesisSTEDmicroscopyisshowntobeviablewithvisible(green yellow red)fluorophores. Inparticular,STEDisestablishedwiththegreenandyellowfluorescentproteins(GFP,YFP)which are endogenous cellular markers of outstanding biological importance. The spec tral conditions for STED with these fluorophores are given. The expansion of STED tothevisiblerangehasenabledthefirstapplicationtobiophysicsandtoaddressingunsolvedproblemsofcellbiology.