SummaryWe demonstrate a novel design of two-colour two-photon fluorescence microscope in which isotropic three-dimensional imaging resolution and high scanning speed can be achieved simultaneously. In our scheme, a three-dimensional optical lattice constructed by multi-beam interference is used for twocolour two-photon fluorescence excitation. Our simulation results show that a resolution of 113.5 nm can be achieved in both transverse and axial directions with two pump pulses at the wavelengths of 400 and 800 nm, respectively; meanwhile, imaging speed can be greatly improved compared with that of traditional two-photon scanning fluorescence microscopes.