Optimization of tools for multiplexed super resolution imaging of the synapse

by Shama Sograte Idrissi
Cumulative thesis
Date of Examination:2019-10-16
Date of issue:2020-07-09
Advisor:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Blanche Schwappach
Persistent Address: http://dx.doi.org/10.53846/goediss-8086

 

 

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Abstract

English

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.
Keywords: Nanobodies; Nanobody; Single domain antibody; Single domain antibodies; Super resolution microscopy; STED; STORM; DNA PAINT; synapse
 
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