Three dimensional modulation-enhanced single-molecule localization techniques, such as ModLoc, offer advancements in axial localization precision across the entire field of view and axial capture range, by applying phase shifting to the illumination pattern. However, this improvement is limited by the pitch of the illumination pattern that can be used and requires registration between separate regions of the camera. To overcome these limitations, we present ZIMFLUX, a method that combines astigmatic point-spread-function (PSF) engineering with a structured illumination pattern in all three spatial dimensions. In order to achieve this we address challenges such as optical aberrations, refractive index mismatch, supercritical angle fluorescence (SAF), and imaging at varying depths within a sample, by implementing a vectorial PSF model. In scenarios involving refractive index mismatch between the sample and immersion medium, the astigmatic PSF loses its ellipticity at greater imaging depths, leading to a deterioration in axial localization precision. In contrast, our simulations demonstrate that ZIMFLUX maintains high axial localization precision even when imaging deeper into the sample. Experimental results show unbiased localization of 3D 80 nm DNA-origami nanostructures in SAF conditions with a 1.5-fold improvement in axial localization precision when comparing ZIMFLUX to conventional SMLM methods that rely solely on astigmatic PSF engineering.

Structured illumination microscopy (SIM) is a widely used imaging technique that doubles the effective resolution of widefield microscopes. Most current implementations rely on diffractive elements, either gratings or programmable devices, to generate structured light patterns in the sample. These can be limited by spectral efficiency, speed, or both. Here we introduce the concept of fiber SIM that allows for camera frame rate limited pattern generation and manipulation over a broad wavelength range. Illumination patterns are generated by coupling laser beams into radially opposite pairs of fibers in a hexagonal single mode fiber array where the exit beams are relayed to the microscope objective’s back focal plane. The phase stepping and rotation of the illumination patterns are controlled by fast electro-optic devices. We achieved a rate of 111 SIM frames per second and imaged with excitation patterns generated by both 488 nm and 532 nm lasers.

MINFLUX offers a breakthrough in single molecule localization precision, but is limited in field of 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 micrometer-sized field of view. We show a near two-fold improvement in precision over standard localization with the same photon count on DNA-origami nanostructures and tubulin in cells, using DNA-PAINT and STORM imaging.