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bioRxiv · 10.1101/2025.11.21.689649

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

Cheung, K. Y.·Wu, Y.·Lee, S. Y.·Zhang, X.·Fukuda, M.·Claridge-Chang, A.
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Abstract

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.

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Cheung, K. Y., Wu, Y., Lee, S. Y., Zhang, X., Fukuda, M., Claridge-Chang, A.. 2025-11-22. Two-photon characterisation of long-Stokes-shift dye ATTO 490LS for single-laser multicolour imaging. https://doi.org/10.1101/2025.11.21.689649

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