Print Email Facebook Twitter Simultaneous orientation and 3D localization microscopy with a Vortex point spread function Title Simultaneous orientation and 3D localization microscopy with a Vortex point spread function Author Hulleman, C.N. (TU Delft ImPhys/Computational Imaging) Thorsen, R.Ø. (TU Delft ImPhys/Computational Imaging) Kim, E. (TU Delft BN/Cees Dekker Lab; Kavli institute of nanoscience Delft; Max Planck Insitute of Biophysics, Frankfurt) Dekker, C. (TU Delft BN/Cees Dekker Lab; Kavli institute of nanoscience Delft) Stallinga, S. (TU Delft ImPhys/Imaging Physics) Rieger, B. (TU Delft ImPhys/Computational Imaging) Department ImPhys/Imaging Physics Date 2021 Abstract Estimating the orientation and 3D position of rotationally constrained emitters with localization microscopy typically requires polarization splitting or a large engineered Point Spread Function (PSF). Here we utilize a compact modified PSF for single molecule emitter imaging to estimate simultaneously the 3D position, dipole orientation, and degree of rotational constraint from a single 2D image. We use an affordable and commonly available phase plate, normally used for STED microscopy in the excitation light path, to alter the PSF in the emission light path. This resulting Vortex PSF does not require polarization splitting and has a compact PSF size, making it easy to implement and combine with localization microscopy techniques. In addition to a vectorial PSF fitting routine we calibrate for field-dependent aberrations which enables orientation and position estimation within 30% of the Cramér-Rao bound limit over a 66 μm field of view. We demonstrate this technique on reorienting single molecules adhered to the cover slip, λ-DNA with DNA intercalators using binding-activated localization microscopy, and we reveal periodicity on intertwined structures on supercoiled DNA. Subject OA-Fund TU Delft To reference this document use: http://resolver.tudelft.nl/uuid:a5d9b2ca-4a1d-47ef-82eb-4dfb9e81a8b0 DOI https://doi.org/10.1038/s41467-021-26228-5 ISSN 2041-1723 Source Nature Communications, 12 (1) Part of collection Institutional Repository Document type journal article Rights © 2021 C.N. Hulleman, R.Ø. Thorsen, E. Kim, C. Dekker, S. Stallinga, B. Rieger Files PDF s41467_021_26228_5.pdf 4.94 MB Close viewer /islandora/object/uuid:a5d9b2ca-4a1d-47ef-82eb-4dfb9e81a8b0/datastream/OBJ/view