Dendritic spines are neuronal structures which play a key role in memory formation by strengthening synaptic pathways through actin cytoskeleton reorganization. The protein CaMKII is hypothesized to perform major structural functions in this rearrangement.
Here, we use single-particle tracking and super-resolution imaging to extract information on the bindings dynamic and nanoscale architecture of reconstituted CaMKII-actin assemblies adhered to a coverslip.
To improve resolution over the entire field of view, we use a homogeneously illuminated total internal reflection fluorescence scheme. Our method closes an experimental gap present in the field and provides relevant knowledge of actin/CaMKII interactions at the nanoscale.
KEYWORDS: Super resolution, Microscopes, 3D image processing, Luminescence, Imaging systems, Systems engineering, Super resolution microscopy, Stereoscopy, Point spread functions, Molecules
The appropriate sample illumination method varies depending on the nature of the sample and the goals of the study, especially for single-molecule localization microscopy. Here, we demonstrate a flexible microscopy system which combines the whole-cell sectioning capability of light sheet with conventional epi-illumination and total internal reflection fluorescence (TIRF), both with a homogeneous flat-field profile to significantly improve the resolution across the entire field of view. In conjunction with point spread function engineering capabilities and fast switching of illumination modes, our system enables 3D single-molecule super-resolution imaging optimized for the sample and region of interest.
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