Simultaneous thermal and large field-of-view two-photon imaging enables optimization of signal-to-noise.

Significance: Although large field-of-view two-photon microscopy (LF-TPM) is a powerful neuroimaging tool, low signal-to-noise (SNR) poses challenges for high-speed imaging. Increasing the average laser power improves the SNR, but the thermal effect of high laser power on the cortex is not well studied. Further, capturing the curvature of the cortex requires creating an image stack, which also reduces the temporal resolution. Solving these problems would enable mesoscopic mapping with LF-TPM. Aim: We aim to study the temperature dynamic of the mouse cortex as a function of the field of view and the average laser power and demonstrate the feasibility of relatively high illumination power. Combining the higher illumination intensity and the curved scanning, we aim to showcase the capability of the LF-TPM system in both stimulated and spontaneous mesoscopic mapping. Approach: We developed a combined thermal imaging and LF-TPM system to measure the spatial-temporal dynamics of heat during TPM imaging. We used an electrically tunable lens to vary the focusing depth and track the cortical curvature as a function of the medial distance. We then used the optimized system to image functional activations and resting-state functional connectivity patterns across both hemispheres. Results: The steady-state maximum cortical temperature declines as the FOV increases. For a Conclusions: Concurrent thermal and LF-TPM imaging enables a quantitative optimization of laser power and SNR in LF-TPM systems. We demonstrated the feasibility of bilateral brain mapping using LF-TPM. These findings will help expand the utility of LF-TPMs for mesoscopic brain mapping applications.