Print Email Facebook Twitter Photon efficient orientation estimation using polarization modulation in single-molecule localization microscopy Title Photon efficient orientation estimation using polarization modulation in single-molecule localization microscopy Author Thorsen, R.Ø. (TU Delft ImPhys/Computational Imaging) Hulleman, C.N. (TU Delft ImPhys/Computational Imaging) Rieger, B. (TU Delft ImPhys/Computational Imaging) Stallinga, S. (TU Delft ImPhys/Imaging Physics) Department ImPhys/Imaging Physics Date 2022 Abstract Combining orientation estimation with localization microscopy opens up the possibility to analyze the underlying orientation of biomolecules on the nanometer scale. Inspired by the recent improvement of the localization precision by shifting excitation patterns (MINFLUX, SIMFLUX), we have adapted the idea towards the modulation of excitation polarization to enhance the orientation precision. For this modality two modes are analyzed: i) normally incident excitation with three polarization steps to retrieve the in-plane angle of emitters and ii) obliquely incident excitation with p-polarization with five different azimuthal angles of incidence to retrieve the full orientation. Firstly, we present a theoretical study of the lower precision limit with a Cramér-Rao bound for these modes. For the oblique incidence mode we find a favorable isotropic orientation precision for all molecular orientations if the polar angle of incidence is equal to arccos √2/3 ≈ 35 degrees. Secondly, a simulation study is performed to assess the performance for low signal-to-background ratios and how inaccurate illumination polarization angles affect the outcome. We show that a precision, at the Cramér-Rao bound (CRB) limit, of just 2.4 and 1.6 degrees in the azimuthal and polar angles can be achieved with only 1000 detected signal photons and 10 background photons per pixel (about twice better than reported earlier). Lastly, the alignment and calibration of an optical microscope with polarization control is described in detail. With this microscope a proof-of-principle experiment is carried out, demonstrating an experimental in-plane precision close to the CRB limit for signal photon counts ranging from 400 to 10,000. Subject OA-Fund TU Delft To reference this document use: http://resolver.tudelft.nl/uuid:3654a58f-cb99-40c3-8a8c-1e5da39e8b9c DOI https://doi.org/10.1364/BOE.452159 ISSN 2156-7085 Source Biomedical Optics Express, 13 (5), 2835-2858 Part of collection Institutional Repository Document type journal article Rights © 2022 R.Ø. Thorsen, C.N. Hulleman, B. Rieger, S. Stallinga Files PDF boe_13_5_2835.pdf 25.8 MB Close viewer /islandora/object/uuid:3654a58f-cb99-40c3-8a8c-1e5da39e8b9c/datastream/OBJ/view