a-c | Major classes of diffraction-unlimited fluorescence nanoscopy concepts. a | In stimulated emission depletion (STED) and reversible saturable/switchable optical linear fluorescence transitions (RESOLFT) nanoscopy, a specific light pattern (red, doughnut-shaped) is used to switch the fluorescence ability of fluorophores 'off', whereas fluorophores remain 'on' only at the intensity minima (shown by yellow star). This approach can also be parallelized (red, periodic pattern). b | In methods such as photo-activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), ground state depletion with individual molecule return (GSDIM) and points accumulation for imaging in nanoscale topography (PAINT), single on-state fluorophores are established at distances larger than the diffraction limit (λ/2NA, where λ is the wavelength, and NA is the numerical aperture of the objective). c | In MINFLUX (nanoscopy with minimal photon fluxes), single fluorophores can be localized at the nanometre scale with minimal photon numbers because their position is inferred from the positioning of the intensity minimum of the light pattern used for excitation. d | All diffraction-unlimited super-resolution concepts utilize fluorophore state transitions to render adjacent molecules within a common diffraction zone transiently discernible. Transitions between a bright light-emitting on state and a dark off state have proved the most effective and have allowed the diffraction barrier to be broken. e | Comparison of routine levels of 3D resolution obtained using different microscopy techniques for cellular imaging. The ellipsoids indicate the lateral (x, y) and axial (z) resolution levels of the listed methods. Each ellipsoid can be interpreted as an uncertainty range from where detected photons originate. Imaging methods with limited resolution owing to diffraction are shown in red (assumed emission wavelength: ∼650 nm). The conceptually diffraction-unlimited methods (that is, the nanoscopy methods) are shown in green. The methods indicated in yellow have been shown to feature extended resolution over the diffraction limit, but their practical applicability and/or reliability with respect to resolution need to be further explored. The indicated resolutions have been repeatedly demonstrated in cell-imaging applications and do not simply represent ultimate or best values provided in the literature. The resolutions shown for lattice light-sheet microscopy and patterned-activation nonlinear structured illumination microscopy (PA NL-SIM) were taken from Refs 33,38. Confocal RESOLFT resolutions are shown for green fluorescent proteins. The resolution of diffraction-unlimited nanoscopy is limited by the photostabilities and physical sizes (compare with Fig. 6) of the marker fluorophores as well as the attachment strategy. The recently demonstrated MINFLUX concept (see part c) fundamentally addresses the limited photon budget and promises further advances in resolution and recording speed. The dithered mode in the ellipsoid corresponding to lattice light-sheet microscopy involves the rapid scanning of the light sheet along the x-axis, with only one image being recorded per z-plane. iPALM, interferometric photoactivated localization microscopy; isoSTED, isotropic STED; NA, numerical aperture; PSF, point spread function; SIM, structured illumination microscopy; SOFI, super-resolution optical fluctuation imaging.