Optimization of tools for multiplexed super resolution imaging of the synapse

Sograte Idrissi, Shama

dc.contributor.advisor	Rizzoli, Silvio Prof. Dr.
dc.contributor.author	Sograte Idrissi, Shama
dc.date.accessioned	2020-07-09T13:44:56Z
dc.date.available	2020-07-09T13:44:56Z
dc.date.issued	2020-07-09
dc.identifier.uri	http://hdl.handle.net/21.11130/00-1735-0000-0005-140A-A
dc.identifier.uri	http://dx.doi.org/10.53846/goediss-8086
dc.language.iso	eng	de
dc.relation.uri	http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc	572	de
dc.title	Optimization of tools for multiplexed super resolution imaging of the synapse	de
dc.type	cumulativeThesis	de
dc.contributor.referee	Rehling, Peter Prof. Dr.
dc.date.examination	2019-10-16
dc.description.abstracteng	The synapse is the major site of neurotransmission in the brain. Even though the synapse has been extensively studied, artefact-free imaging tools are still necessary for its correct investigation. With the resolution of modern techniques approaching the molecular size, the main current limitations are the few available affinity probes targeting synaptic proteins and the limited multiplexing abilities of most microscopy techniques. Camelid single-domain antibodies (also called nanobodies) are a superior alternative to conventional antibodies for super resolution microscopy applications. Nanobodies have a significantly smaller size than conventional antibodies; they are monovalent binders, they can reach buried epitopes and can be expressed recombinantly in prokaryotic systems. However, nanobodies against just a few targets are available and their selection is laborious. In this thesis, I first established a pipeline that allows selection, production, and validation of nanobodies against various synaptic proteins. Next, I characterized nanobodies binding selectively to primary antibodies (secondary nanobodies) and compared them in immunofluorescences performed with conventional secondary antibodies. Finally, I also established a protocol for coupling a single-stranded DNA to nanobodies using click chemistry. As a proof of principle, I used this procedure to implement a triple color super resolution Exchange PAINT with an automated microfluidic setup. Altogether, this thesis gives rise to a set of tools that allows the characterization of the neuronal synapses with fewer technical constrains at super resolution scales and minimal artefacts.	de
dc.contributor.coReferee	Schwappach, Blanche Prof. Dr.
dc.subject.eng	Nanobodies	de
dc.subject.eng	Nanobody	de
dc.subject.eng	Single domain antibody	de
dc.subject.eng	Single domain antibodies	de
dc.subject.eng	Super resolution microscopy	de
dc.subject.eng	STED	de
dc.subject.eng	STORM	de
dc.subject.eng	DNA PAINT	de
dc.subject.eng	synapse	de
dc.identifier.urn	urn:nbn:de:gbv:7-21.11130/00-1735-0000-0005-140A-A-8
dc.affiliation.institute	Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)	de
dc.subject.gokfull	Biologie (PPN619462639)	de
dc.identifier.ppn	1724163299

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GGNB - Göttinger Graduiertenzentrum für Neurowissenschaften, Biophysik und molekulare Biowissenschaften [1016]
GGNB - Göttingen Graduate Center for Neurosciences, Biophysics and Molecular Biosciences

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