Overcoming thermal constraints in two-photon imaging using thermoelectric cooling

Two-photon laser scanning microscopy is widely used for deep-tissue imaging but is increasingly constrained by bulk tissue heating arising from linear absorption of excitation light. This volumetric heating accumulates with imaging depth, imposing conservative safe power limits that restrict achievable signal-to-noise ratio and imaging performance. While passive mitigation strategies such as objective are commonly employed, their effectiveness in suppressing bulk heating is limited. Here, we present a coupled numerical and experimental framework to quantify laser-induced bulk tissue heating during two-photon imaging and to evaluate active thermoelectric cooling strategies under realistic imaging conditions. Optical energy deposition is modeled using Monte Carlo simulations of photon transport and coupled to transient bioheat transfer calculations solved using an implicit finite difference scheme. The model is formulated in a two-dimensional domain and incorporates generalized thermal boundary conditions to represent localized thermoelectric cooling. The framework is validated against contact thermometry measurements performed in tissue-mimicking phantoms during 1035 nm point-scanning excitation at imaging depths of 300 μm and 1000 μm. Lateral temperature profiling demonstrates strong agreement between simulated and measured temperature rise, with an average error below 8%. Using a peak temperature rise criterion of 2°C, depth-dependent safe average power thresholds are quantified in the absence of active cooling. Applying thermoelectric cooling under otherwise identical imaging conditions substantially relaxes these thermal constraints, enabling up to a 4× increase in deliverable average power at shallow imaging depths and a 3.67× increase at deeper imaging depths. These results demonstrate the potential of active thermoelectric cooling to mitigate bulk tissue heating and extend the operational envelope of high-power two-photon microscopy.