Two-photon characterisation of long-Stokes-shift dye ATTO 490LS for single-laser multicolour imaging

Long-Stokes-shift fluorophores enable high sensitivity and multiplexed imaging with single-wavelength excitation. Under single-photon illumination ATTO 490LS exhibits a 165-nm Stokes shift, but its two-photon properties remain uncharacterised. Emission and excitation spectral analyses of ATTO 490LS in ex vivo Drosophila melanogaster brains identified two-photon excitation sensitivity at 940 nm, with peak emission at 640 nm. We demonstrate successful duplexed imaging of ATTO 490LS alongside Alexa Fluor 488 using a single 920-nm fibre laser and dual photomultiplier tubes, enabling distinct measurement of red and green fluorescence signals. These findings establish ATTO 490LS as suitable for multicolour two-photon microscopy with single-laser systems.