Extended Data Fig. 3Resolution enhancement with triple-view confocal imaging.

a) Lateral maximum intensity projection image of fixed U2OS cells immunolabelled with mouse-anti-alpha tubulin, anti-mouse-biotin, streptavidin Alexa Fluor 488, marking microtubules (fused result after registration and deconvolution of all three raw views). Height: colour bar. b) Raw and fused axial views along the dashed line in a). Fourier transforms of axial view are shown in last row, showing resolution enhancement after fusion. Orange oval: 1/260 nm⁻¹ in half width and 1/405 nm⁻¹ in half height. c) Triple-view reconstruction of whole fixed L1 stage larval worm, nuclei labelled with NucSpot 488. Two maximum intensity projections are shown, taken after rotating volume 220 degrees (top) and 300 degrees (bottom) about the x axis. d) Higher magnification lateral view 10 μm from the beginning of the volume, corresponding to the yellow dashed region in c). e) Higher magnification axial views (single planes) corresponding to the red rectangular region in c), comparing single-view deconvolved result (left) to triple-view result (right). Magenta arrows highlight subnuclear structure better resolved in triple-view result. f) Line profiles corresponding to e1, e2 in e). Scale bars: a, c) 10 µm; b, e) 3 µm.