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The two-photon all-optical physiology system has attracted great interest in deciphering neuronal circuits in vivo , benefiting from its advantages in recording and modulating neuronal activities at single neuron resolutions. However, the interference, or crosstalk, between the imaging and photostimulation beams introduces a significant challenge and may impede the future application of voltage indicators in two-photon all-optical physiology system. Here, we propose the time multiplexed excitation method to distinguish signals from neuronal activities and crosstalks from photostimulation. In our system, the laser pulses of the imaging beam and photostimulation beam are synchronized, and a time delay is introduced into these pulses to separate the fluorescence signal generated by these two beams. We demonstrate the efficacy of our system in eliminating crosstalk signals from photostimulation and evaluate its influence on both genetically encoded calcium indicators (GECIs) and genetically encoded voltage indicators (GEVIs) through in vivo experiments., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2024 Optica Publishing Group.)