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

• Published in: Nature communications Vol. 12; no. 1; p. 5934
• Main Authors: Hulleman, Christiaan N., Thorsen, Rasmus Ø., Kim, Eugene, Dekker, Cees, Stallinga, Sjoerd, Rieger, Bernd
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 11.10.2021; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/245/2225; 631/57/2265; 639/624/1107/328/1652; 639/624/1107/328/2238; 639/624/1107/328/2239; Binding Sites; Constraints; Deoxyribonucleic acid; Dipoles; DNA; DNA - metabolism; DNA - ultrastructure; DNA, Superhelical - metabolism; DNA, Superhelical - ultrastructure; Emitters; Field of view; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Heterocyclic Compounds, 4 or More Rings - chemistry; Heterocyclic Compounds, 4 or More Rings - metabolism; Humanities and Social Sciences; Imaging, Three-Dimensional - instrumentation; Imaging, Three-Dimensional - methods; Intercalating Agents - chemistry; Intercalating Agents - metabolism; Localization; Microscopy; Microscopy - instrumentation; Microscopy - methods; multidisciplinary; Orientation; Periodicity; Phase plates; Point spread functions; Polarization; Science; Science (multidisciplinary); Splitting; Vortices
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-26228-5

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. Molecular orientation is often ignored during single-molecule localisation microscopy. Here, the authors use a Vortex point spread function in order to simultaneously estimate the 3D position, dipole orientation and degree of rotational constraint, within 30% of the Cramér-Rao bound limit.