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

Hulleman, C. N., Thorsen, R. Ø., Kim, E., Dekker, C., Stallinga, S., & Rieger, B. (2021). Simultaneous orientation and 3D localization microscopy with a Vortex point spread function. Nature Communications, 12(1): 5934. doi:10.1038/s41467-021-26228-5.

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 Creators:
Hulleman, Christiaan N.1, Author
Thorsen, Rasmus Ø.1, Author
Kim, Eugene2, 3, Author              
Dekker, Cees3, Author
Stallinga, Sjoerd1, Author
Rieger, Bernd1, Author
Affiliations:
1Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands, ou_persistent22              
2Research Group Structure and Dynamics of Chromosomes, Max Planck Institute of Biophysics, Max Planck Society, ou_3291486              
3Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, Netherlands, ou_persistent22              

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 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.

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Language(s): eng - English
 Dates: 2020-11-012021-09-212021-10-11
 Publication Status: Published online
 Pages: 14
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1038/s41467-021-26228-5
BibTex Citekey: hulleman_simultaneous_2021
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Title: Nature Communications
  Abbreviation : Nat. Commun.
Source Genre: Journal
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Publ. Info: London : Nature Publishing Group
Pages: - Volume / Issue: 12 (1) Sequence Number: 5934 Start / End Page: - Identifier: ISSN: 2041-1723
CoNE: https://pure.mpg.de/cone/journals/resource/2041-1723