article · 29/01/2026
Thermal Lensing Effects in Two-Photon Light-Sheet Microscopy
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Résumé
In light-sheet fluorescence microscopy (LSFM), the axial resolution is governed by the illumination beam profile, motivating the development of advanced beam-shaping techniques to enhance imaging performance. Two-photon LSFM (2P-LSFM), in particular, improves the signal-to-background ratio by reducing laser scattering and distortion in biological specimens. However, we report a potentially detrimental thermal effect in 2P-LSFM: the high laser powers required for two-photon excitation induce localized heating, which alters the refractive index of the medium and effectively forms a divergent thermal lens in water. At 500 mW the light-sheet waist broadens by 25% and shifts by 300 μm before stabilizing several seconds after the laser shutter is opened. Both experiments and simulations reveal that this thermal lensing effect scales with laser power and the path length the beam travels through water. The resulting degradation in resolution and signal-to-noise ratio may compromise imaging applications that require high laser powers for rapid volumetric imaging of large specimens or functional brain imaging. This limitation is particularly critical in dynamic sample environments, such as during stepwise repositioning or flow-based delivery of chemical or hydrodynamic sensory stimuli, where changes occur on timescales comparable to the thermal settling time.
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Hubert, A., Trentesaux, H., Pujol, T., Debrégeas, G., & Bormuth, V. (2026). Thermal Lensing Effects in Two-Photon Light-Sheet Microscopy. Biomedical Optics express, 17(2), 1064-1073. https://doi.org/https://doi.org/10.1364/BOE.564339@article{Hubert2026_476,
author = {Hubert, Antoine and Trentesaux, Hugo and Pujol, Thomas and Debrégeas, Georges and Bormuth, Volker},
year = {2026},
month = {1},
title = {Thermal Lensing Effects in Two-Photon Light-Sheet Microscopy},
journal = {Biomedical Optics express},
volume = {17},
number = {2},
pages = {1064-1073},
abstract = {In light-sheet fluorescence microscopy (LSFM), the axial resolution is governed by the illumination beam profile, motivating the development of advanced beam-shaping techniques to enhance imaging performance. Two-photon LSFM (2P-LSFM), in particular, improves the signal-to-background ratio by reducing laser scattering and distortion in biological specimens. However, we report a potentially detrimental thermal effect in 2P-LSFM: the high laser powers required for two-photon excitation induce localized heating, which alters the refractive index of the medium and effectively forms a divergent thermal lens in water. At 500 mW the light-sheet waist broadens by 25\% and shifts by 300 μm before stabilizing several seconds after the laser shutter is opened. Both experiments and simulations reveal that this thermal lensing effect scales with laser power and the path length the beam travels through water. The resulting degradation in resolution and signal-to-noise ratio may compromise imaging applications that require high laser powers for rapid volumetric imaging of large specimens or functional brain imaging. This limitation is particularly critical in dynamic sample environments, such as during stepwise repositioning or flow-based delivery of chemical or hydrodynamic sensory stimuli, where changes occur on timescales comparable to the thermal settling time.},
url = {https://doi.org/10.1364/BOE.564339},
doi = {https://doi.org/10.1364/BOE.564339},
}TY - JOUR
AU - Hubert, Antoine
AU - Trentesaux, Hugo
AU - Pujol, Thomas
AU - Debrégeas, Georges
AU - Bormuth, Volker
PY - 2026
DA - 2026/01/29
TI - Thermal Lensing Effects in Two-Photon Light-Sheet Microscopy
JO - Biomedical Optics express
VL - 17
IS - 2
AB - In light-sheet fluorescence microscopy (LSFM), the axial resolution is governed by the illumination beam profile, motivating the development of advanced beam-shaping techniques to enhance imaging performance. Two-photon LSFM (2P-LSFM), in particular, improves the signal-to-background ratio by reducing laser scattering and distortion in biological specimens. However, we report a potentially detrimental thermal effect in 2P-LSFM: the high laser powers required for two-photon excitation induce localized heating, which alters the refractive index of the medium and effectively forms a divergent thermal lens in water. At 500 mW the light-sheet waist broadens by 25% and shifts by 300 μm before stabilizing several seconds after the laser shutter is opened. Both experiments and simulations reveal that this thermal lensing effect scales with laser power and the path length the beam travels through water. The resulting degradation in resolution and signal-to-noise ratio may compromise imaging applications that require high laser powers for rapid volumetric imaging of large specimens or functional brain imaging. This limitation is particularly critical in dynamic sample environments, such as during stepwise repositioning or flow-based delivery of chemical or hydrodynamic sensory stimuli, where changes occur on timescales comparable to the thermal settling time.
SP - 1064
EP - 1073
DO - https://doi.org/10.1364/BOE.564339
UR - https://doi.org/10.1364/BOE.564339
ER -