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.
Highlights
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)
The g2 parameter is a weight parameter quantifying the contribution of the fixed versus free dipole component to the overall PSF, which is sufficient to quantify the impact of orientational constraint, irrespective of the form of the constraint, e.g., "wobblein-cone" or harmonic orientational potential well[36]
The slight asymmetry in the PSF arising from the azimuthal orientation (φ = 45∘), is difficult to identify by the eye
Summary
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). 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. Single-Molecule Localization Microscopy (SMLM), with flavors like (F)PALM1,2, (d)STORM3,4, and (DNA)-PAINT5,6, have made nanoscale structural information beyond the diffraction limit more accessible to biologists These superresolution techniques commonly focus on localizing single emitters in the two dimensions of the focal plane, and sometimes in the third dimension along the optical axis. For a limited set of orientations the angles can even be extracted from in focus single-molecule images in case of sufficiently high Signal-to-Noise Ratio (SNR)[11,25,26]
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