Anwendung von Click-Chemie zur Darstellung neuronaler Proteine in X10-Expansion

Use of Click chemistry for neuronal protein staining in X10 expansion microscopy

by Felix König
Doctoral thesis
Date of Examination:2025-03-18
Date of issue:2025-03-04
Advisor:Prof. Dr. Silvio O. Rizzoli
Referee:Prof. Dr. Tiago Fleming Outeiro
Referee:Prof. Dr. Margarete Schön
Persistent Address: http://dx.doi.org/10.53846/goediss-11123

 

 

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Abstract

English

X10 Expansion Microscopy is a cost-effective method that allows for high-resolution imaging by expanding samples by a factor of ten through water absorption, enabling resolution down to 20 nm with conventional fluorescence microscopes. This technique can be combined with various staining methods for precise protein localization. However, traditional immunostaining methods suffer from a limitation known as the linkage error, where fluorophores are positioned up to 30 µm away from the target due to the size of antibodies. Click chemistry offers a solution to this problem by enabling the direct, covalent binding of a fluorophore to the protein of interest (POI), eliminating the linkage error. This study investigates the use of click chemistry for localizing neuronal proteins in X10 expansion and compares it to traditional immunostaining. Hamster kidney cells (BHK), rat hippocampal neurons (HPC), and PC12 cells were transfected with constructs encoding unnatural amino acids for POI labeling via click chemistry. After expansion using X10, samples were imaged with conventional fluorescence microscopy. The results show that click chemistry enables precise localization of proteins in X10 expansion, with PC12 cells demonstrating more accurate localization compared to traditional immunostaining. This study highlights the potential of combining click chemistry with X10 expansion to overcome the limitations of conventional staining methods, expanding the possibilities of super-resolution imaging.
Keywords: click chemistry; neuronal proteins; expansion microscopy; super resolution microscopy; x10 expansion
 
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