Array detection enables large localization range for simple and robust MINFLUX

Fluorescence microscopy enables visualization of cellular structures with molecular specificity, yet conventional methods remain limited by diffraction to resolutions of ~250 nm. Super-resolution microscopy overcomes this resolution barrier, with minimal fluorescence photon fluxes microscopy (MINFLUX) representing the current state-of-the-art in point scanning microscopy. MINFLUX probes single emitters with an intensity minimum in a targeted coordinate pattern (TCP) and typically achieves 1 nm to 3 nm localization uncertainty. However, this performance is coupled to a small localization range as the localization uncertainty grows rapidly when the emitter lies outside the TCP. Therefore MINFLUX requires a pre-localization step and iterative recentering to keep the emitter inside the TCP, combined with increasing laser power to ensure signal from the emitter. Here we present ISM-FLUX, a technique that combines the MINFLUX concept with image scanning microscopy (ISM). ISM-FLUX combines orbital scanning of a donut-shaped excitation with a 5 × 5 single-photon avalanche diode (SPAD) array detector, extending the localization range to ~600 × 600 nm² while achieving 2 nm to 10 nm localization uncertainty with ~10³ photons. Unlike single-element detection, the SPAD array detector provides spatial information of photons, resulting in camera-like information that prevents Cramér-Rao bound (CRB) divergence across the extended field. The orbital scanning geometry operates on standard galvanometric mirrors, requiring only a vortex phase plate and SPAD array to convert a confocal microscope into an ISM-FLUX system. We validated ISM-FLUX experimentally using DNA-origami nanorulers with 20 nm and 40 nm binding-site separations, resolving these distances even when the structures are positioned outside the TCP orbit. To support long acquisitions, we built a custom 3D active stabilization module that maintains sub-nanometer sample drift over hours in closed loop. The same platform integrates a co-registered widefield single-molecule localization microscopy (SMLM) path for large field-of-view (FoV) context and correlative workflows, achieving σ_NeNA ≈ 7.83 nm. Together, these results show that ISM-FLUX delivers MINFLUX-style localization over an extended range within a point-scanning architecture. By simplifying MINFLUX microscopy and extending its capabilities within existing confocal infrastructure, ISM-FLUX provides an accessible path toward molecular-scale imaging that could accelerate broader adoption of single-molecule localization techniques in biological research.