Thermal lensing effects in two-photon light-sheet microscopy.

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.