| 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.identifier.uri | http://dx.doi.org/10.53846/goediss-6934 | |
| dc.language.iso | eng | de |
| dc.rights.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 | |