MINFLUX offers a breakthrough in single molecule localization precision, but is limited in fieldof-view. Here, we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micron sized field-of-view. We show a near twofold improvement in precision over standard localization with the same photon count on DNA-origami nano-structures and tubulin in cells, using DNA-PAINT and STORM imaging. Single-molecule localization microscopy 1,2,3 circumvents the diffraction limit using centroid estimation of sparsely activated, stochastically switching, single-molecule fluorescence images. Improvement over state-of-the-art image resolutions of around 20 nm towards values below 5 nm is desired for truly imaging at the molecular scale. This needs improvements in labelling strategy to reduce linker sizes 4,5,6,7 and methods to overcome low labelling density such as data fusion 8 , but also a step in localization precision. Efforts so far have targeted an increase in the number of detected photons N by chemical engineering of brighter fluorophores 9 , or by avoiding photo-bleaching via e.g. cryogenic techniques 10,11,12. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

MINFLUX offers a breakthrough in single molecule localization precision, but suffers from a tiny fieldof-view and a lack of practical parallelism. Here, we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micron sized field-of-view. We show a near twofold improvement in precision over standard localization with the same photon count on DNA-origami nano-structures. Main textSingle-molecule localization microscopy 1,2,3 circumvents the diffraction limit using centroid estimation of sparsely activated, stochastically switching, single-molecule fluorescence images. Improvement over state-of-the-art image resolutions of around 20 nm towards values below 5 nm is desired for truly imaging at the molecular scale. This needs improvements in labelling strategy to reduce linker sizes 4,5,6,7 and methods to overcome low labelling density such as data fusion 8 , but also a step in localization precision. Efforts so far have targeted an increase in the number of detected photons by chemical engineering of brighter fluorophores 9 , or by avoiding photo-bleaching via e.g. cryogenic techniques 10,11,12 . These improvements scale the localization precision with ( % ) ⁄ , with the fluorescence emission wavelength, and the microscope objective numerical aperture 13 .Recently, a new concept called MINFLUX was proposed 14 , in which a doughnut illumination spot is shifted over an area of size ~50 nm, and the position of a single molecule in the scan range is determined by triangulation based on the detected photon count for the different doughnut positions.

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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.

We have developed an engineered Point Spread Function (PSF) to enable the simultaneous estimation of dipole orientation, 3D position and degree of rotational constraint of single-molecule emitters from a single 2D focal plane. Besides giving access to orientation information, the Vortex PSF along with the vectorial PSF fitter avoids localization bias common in localization microscopy for fixed dipole emitters. We demonstrate this technique on reorienting single-molecules and using binding-activated localization microscopy on DNA intercalators, corroborating perpendicular azimuthal angles to the DNA axis for in-plane emitters but find a non-uniform distribution as a function of the polar angle. The Vortex PSF is realized by an affordable glass phase mask and has a compact footprint that can easily be combined with localization microscopy techniques on rotationally constrained emitters.