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

Author

Listed:
  • Christiaan N. Hulleman

    (Delft University of Technology)

  • Rasmus Ø. Thorsen

    (Delft University of Technology)

  • Eugene Kim

    (Delft University of Technology
    Max Planck Institute of Biophysics)

  • Cees Dekker

    (Delft University of Technology)

  • Sjoerd Stallinga

    (Delft University of Technology)

  • Bernd Rieger

    (Delft University of Technology)

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.

Suggested Citation

  • Christiaan N. Hulleman & Rasmus Ø. Thorsen & Eugene Kim & Cees Dekker & Sjoerd Stallinga & Bernd Rieger, 2021. "Simultaneous orientation and 3D localization microscopy with a Vortex point spread function," Nature Communications, Nature, vol. 12(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-26228-5
    DOI: 10.1038/s41467-021-26228-5
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    References listed on IDEAS

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    1. Valentina Curcio & Luis A. Alemán-Castañeda & Thomas G. Brown & Sophie Brasselet & Miguel A. Alonso, 2020. "Birefringent Fourier filtering for single molecule coordinate and height super-resolution imaging with dithering and orientation," Nature Communications, Nature, vol. 11(1), pages 1-13, December.
    2. Kim I. Mortensen & Jongmin Sung & Henrik Flyvbjerg & James A. Spudich, 2015. "Optimized measurements of separations and angles between intra-molecular fluorescent markers," Nature Communications, Nature, vol. 6(1), pages 1-9, December.
    3. Katrin I. Willig & Silvio O. Rizzoli & Volker Westphal & Reinhard Jahn & Stefan W. Hell, 2006. "STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis," Nature, Nature, vol. 440(7086), pages 935-939, April.
    4. Andreas S. Biebricher & Iddo Heller & Roel F. H. Roijmans & Tjalle P. Hoekstra & Erwin J. G. Peterman & Gijs J. L. Wuite, 2015. "The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics," Nature Communications, Nature, vol. 6(1), pages 1-12, November.
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    Cited by:

    1. Fabian Hinterer & Magdalena C Schneider & Simon Hubmer & Montserrat López-Martinez & Philipp Zelger & Alexander Jesacher & Ronny Ramlau & Gerhard J Schütz, 2022. "Robust and bias-free localization of individual fixed dipole emitters achieving the Cramér Rao bound for applications in cryo-single molecule localization microscopy," PLOS ONE, Public Library of Science, vol. 17(2), pages 1-15, February.

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