Measuring single molecule 3D orientational behavior is a challenge that, if solved in addition to 3D localization, would provide key elements for super resolution structural imaging. Orientation contains indeed information on local conformational properties of proteins, while orientational fluctuations are signatures of local steric, charges or viscosity constraints. Both these properties are not perceptible in pure super resolution imaging, which relies on position localization measurements. Imaging 3D orientation together with 3D localization is however not easily accessible due to the intrinsic coupling between spatial deformation of the single molecules’ point spread function (PSF) and their off-plane orientations, as well as the requirement to measure six parameters which are not directly distinguishable (two angles of orientation, aperture of angular fluctuations, and three spatial position coordinates). In this work, we report a method that is capable of resolving these six parameters in a modality that is compatible with super resolution imaging. The method is based on the use of a stress-engineered spatially-variant birefringent phase plate placed in the Fourier plane of the microscope detection path. This modifies the PSF of single emitters in a way that can be non-ambiguously decomposed onto the nine 3D-analogs of the Stokes parameters. Moreover, the use of two complementary co/counter circular polarizations projections provides a non-ambiguous determination of the 3D spatial position of single emitters with tens of nanometers precision. This method, which opens to nanoscale structural imaging of proteins organization, is presented on model nano-beads emitters and applied to single fluorophores used for cytoskeleton labelling.
Sophie Brasselet, Valentina Curcio, Thomas Brown, and Miguel Alonso, "Simultaneous detection of 3D orientation and 3D spatial localization of single emitters for nanoscale structural imaging (Conference Presentation)," Proc. SPIE 10883, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVI, 1088307 (Presented at SPIE BiOS: February 05, 2019; Published: 13 March 2019); https://doi.org/10.1117/12.2509891.6013172979001.
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