Far‐field fluorescence microscopy with three‐dimensional resolution in the 100‐nm range

We report three‐dimensional (3D) microscopy with nearly isotropic resolution in the λ/5 − λ/10 range. Our approach combines 4Pi‐confocal two‐photon fluorescence microscopy with image restoration. The 3D resolution is demonstrated with densely clustered beads as well as with F‐actin fibers in mouse fibroblast cells. A comparison with unrestored two‐photon confocal images reveals a total reduction of the uncertainty volume up to a factor of 15.