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dc.contributor.advisor Hell, Stefan Prof. Dr.
dc.contributor.author Masch, Jennifer-Magdalena
dc.date.accessioned 2018-06-20T08:21:01Z
dc.date.available 2018-06-20T08:21:01Z
dc.date.issued 2018-06-20
dc.identifier.uri http://hdl.handle.net/11858/00-1735-0000-002E-E427-D
dc.language.iso eng de
dc.relation.uri http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc 571.4 de
dc.title STED nanoscopy of synaptic substructures in living mice de
dc.type doctoralThesis de
dc.contributor.referee Schild, Detlev Prof. Dr.
dc.date.examination 2017-10-19
dc.description.abstracteng Optical nanoscopy has revolutionized far-field microscopy, enabling the observation of subcellular structures and dynamics from completely new perspectives. Among other fields, neuroscience benefits greatly from the remarkable advances in super-resolution microscopy, which provide unprecedented insights into the molecular organization and function of synapses. STED nanoscopy has been one of the most successful methods for live-cell applications and is the only superresolution technique that has been demonstrated for imaging in living mice so far. However, in vivo nanoscale imaging of synaptic proteins has remained challenging due to the extraordinary complexity of the experiments and the lack of adequate labeling tools. The goal of this thesis was to overcome these challenges and image for the first time the distribution and substructure of a synaptic protein in vivo by STED nanoscopy with markers in the far-red spectral range. In order to achieve this aim, this work focused on the optimization and integration of three essential elements of in vivo super-resolution imaging: (i) the development of a robust STED nanoscope, (ii) the establishment of a protocol for stable animal preparation, and (iii) the identification of an effective fluorescent labeling approach. First, a compact in vivo two-color STED nanoscope for the far-red spectrum was constructed, which best fulfilled the specific requirements for imaging the brains of living mice. A new design concept with minimized optical beam path lengths was developed, which resulted in maximum optical stability and reduced the need for frequent alignment procedures. In addition, a protocol for surgical preparation of the mice was established with particular emphasis on the mechanical stability of the mouse head fixation. This new procedure of animal preparation considerably reduced imaging artifacts due to cardiac and respiration induced motion. Lastly, highly specific labeling strategies based on far-red fluorophores for live-cell imaging were developed. The performance of various far-red fluorescent proteins and organic dyes was evaluated for in vivo applicability and the dyes 610CP and silicon-rhodamine (SiR) were identified as the most suitable fluorophores for in vivo far-red STED nanoscopy at 775 nm depletion wavelength. The synergy of these three essential elements then enabled the first super-resolution study of a synaptic protein in the brains of living mice. The postsynaptic density 95 (PSD-95) protein was examined in a transgenic knock-in mouse line expressing endogenous PSD-95 in fusion with the selflabeling enzyme HaloTag. The results of this study uncovered the native nanoscopic structural organization of PSD-95 in dendritic spines of the visual cortex and revealed complex morphologies which were unresolved by confocal microscopy and not yet reported in such detail in in vitro model systems. The present work underscores the importance of in vivo super-resolution microscopy and introduces a novel approach for future investigations of the molecular organization and function of the brain and other tissues in living animals. de
dc.contributor.coReferee Brose, Nils Prof. Dr.
dc.subject.eng super-resolution microscopy de
dc.subject.eng in vivo STED nanoscopy de
dc.subject.eng PSD-95 de
dc.subject.eng far-red tissue imaging de
dc.subject.eng protein labeling de
dc.identifier.urn urn:nbn:de:gbv:7-11858/00-1735-0000-002E-E427-D-4
dc.affiliation.institute Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) de
dc.subject.gokfull Biologie (PPN619462639) de
dc.identifier.ppn 1024810968

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