Abstract
AbstractSingle-molecule localization microscopy provides insights into the nanometer-scale spatial organization of proteins in cells, however it does not provide information on their conformation and orientation, which are key functional signatures. Detecting single molecules’ orientation in addition to their localization in cells is still a challenging task, in particular in dense cell samples. Here, we present a polarization-splitting scheme which combines Stochastic Optical Reconstruction Microscopy (STORM) with single molecule 2D orientation and wobbling measurements, without requiring a strong deformation of the imaged point spread function. This method called 4polar-STORM allows, thanks to a control of its detection numerical aperture, to determine both single molecules’ localization and orientation in 2D and to infer their 3D orientation. 4polar-STORM is compatible with relatively high densities of diffraction-limited spots in an image, and is thus ideally placed for the investigation of dense protein assemblies in cells. We demonstrate the potential of this method in dense actin filament organizations driving cell adhesion and motility.
Publisher
Springer Science and Business Media LLC
Subject
General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry,Multidisciplinary
Cited by
30 articles.
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