Abstract
Two-photon excitation fluorescence microscopy has become an effective tool for tracking neural activity in the brain at high resolutions thanks to its intrinsic optical sectioning and deep penetration capabilities. However, advanced two-photon microscopy modalities enabling high-speed and/or deep-tissue imaging necessitate high average laser powers, thus increasing the susceptibility of tissue heating due to out-of-focus absorption. Despite cooling the cranial window by maintaining the objective at a fixed temperature, average laser powers exceeding 100-200 mW have been shown to exhibit the potential for altering physiological responses of the brain. This paper proposes an enhanced cooling technique for inducing a laminar flow to the objective immersion layer while implementing duty cycles. Through a numerical study, we analyze the efficacy of heat dissipation of the proposed method and compare it with that of the conventional, fixed-temperature objective cooling technique. The results show that improved cooling could be achieved by choosing appropriate flow rates and physiologically relevant immersion cooling temperatures, potentially increasing safe laser power levels by up to three times (3×). The proposed active cooling method can provide an opportunity for faster scan speeds and enhanced signals in nonlinear deep brain imaging.