Simultaneous orientation and 3D localization microscopy with a Vortex point spread function

Christiaan N. Hulleman, Rasmus Ø. Thorsen, Eugene Kim, Cees Dekker, Sjoerd Stallinga () and Bernd Rieger ()
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Christiaan N. Hulleman: Delft University of Technology
Rasmus Ø. Thorsen: Delft University of Technology
Eugene Kim: Delft University of Technology
Cees Dekker: Delft University of Technology
Sjoerd Stallinga: Delft University of Technology
Bernd Rieger: Delft University of Technology

Nature Communications, 2021, vol. 12, issue 1, 1-14

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-021-26228-5

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