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

2025 · doi:10.1101/2025.11.21.689649
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This study characterized the two-photon excitation sensitivity of ATTO 490LS at 940 nm with peak emission at 640 nm, enabling successful duplexed imaging with Alexa Fluor 488 using a single laser.

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⚙ AI-generated deep summary by claude@2026-07, 2026-07-14 · read from full text ⓘ

The paper characterizes the two-photon excitation and emission properties of the long-Stokes-shift dye ATTO 490LS, which has a 165-nm Stokes shift under single-photon excitation but had not been previously evaluated for two-photon use. Using excitation and emission spectral analyses in ex vivo Drosophila melanogaster brains, the authors identify two-photon excitation sensitivity at 940 nm with peak emission at 640 nm. They further demonstrate duplexed multicolour imaging by measuring ATTO 490LS alongside Alexa Fluor 488 using a single 920-nm fibre laser and dual photomultiplier tubes. The study’s key limitation is that spectral characterization and validation are performed in an ex vivo insect brain context rather than in human tissue. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.

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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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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. Competing Interest Statement The authors have declared no competing interest. Footnotes Small wording edits in the main text, figure panel labels, and figure legend, in line with Reviewer/Editor comments.

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