University of Twente Student Theses


dSTORM for alpha-synuclein clustering on supported lipid bilayers

Berg, Pim Jasper van den (2012) dSTORM for alpha-synuclein clustering on supported lipid bilayers.

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Abstract:α-Synuclein (αS) has been found to be involved in Parkinson’s Disease (PD), where excessive neuronal cell death occurs. Little is known about its precise function and toxicity, but the protein is thought to be involved in regulation of synaptic vesicles that carry neurotransmitters. Interaction of αS with lipid membranes is therefore thought to be an important process in neuronal cell death. α-Synuclein induced domain formation visible on Supported Lipid Bilayers creates areas with a high protein density. A high local protein density may be a starting point of PD pathology. Normal fluorescence microscopy is insufficient to image the initiation of the domain formation or the ordering of the protein clustering. These features are smaller than the diffraction limit, so super- resolution imaging of the domains and their formation is required. A dSTORM setup has been improved and tested. An objective-based TIRF setup was created using an optical fibre to change the TIRF angle. Dual colour imaging was also implemented to perform dSTORM imaging of both the bilayer and the protein in future research. It is found that α-synuclein fibrils can be imaged with a FWHM of 44.2 nm ± 2.4 nm and a FWHM of 26.8 nm ± 2.4 nm when discarding low intensity localisations. The shortest measurement time required for reconstruction of a continuous fibril was found to be two and a half minute, using 5,000 frames at 33 Hz. Analysis of the power- law distributed localisation brightness indicated that there is no effect of an increase in DTT concentration on the localisation brightness. Other photo-physical processes may be more important to the switching of the fluorophores than the thiol concentration. dSTORM imaging showed αS induced domains on SLB’s. The average size and shape of the domains differed for different sample preparation. Differences in coverslip surface treatment and bilayer stability have been argued as possible explanations.
Item Type:Essay (Master)
Faculty:TNW: Science and Technology
Subject:33 physics
Programme:Nanotechnology MSc (60028)
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