Exploring two-photon optogenetics beyond 1100 nm for specific and effective all-optical physiology

Two-photon (2-P) all-optical approaches combine in vivo 2-P calcium imaging and 2-P optogenetic modulations. Here, firstly, we combined in vivo juxtacellular recordings and GCaMP6f-based 2-P calcium imaging in mouse visual cortex to tune our detection algorithm towards a 100% specific identification of action potential-related calcium transients. Secondly, we minimized photostimulation artifacts by using extended-wavelength-spectrum laser sources for optogenetic stimulation. We achieved artifact-free all-optical experiments performing optogenetic stimulation from 1100 nm to 1300 nm. Thirdly, we determined the spectral range for maximizing efficacy until 1300 nm. The rate of evoked transients in GCaMP6f/C1V1-co-expressing cortical neurons peaked already at 1100 nm. By refining spike detection and defining 1100 nm as the optimal wavelength for artifact-free and effective GCaMP6f/C1V1-based all-optical physiology, we increased the translational value of these approaches, e.g., for the development of network-based therapies.双光子（Two-photon，2-P）全光学方法将在体双光子钙成像与双光子光遗传调控相结合。本研究首先在小鼠视觉皮层中整合了在体胞旁记录与基于GCaMP6f的双光子钙成像，以优化检测算法，实现对动作电位相关钙瞬变的100%特异性识别。其次，本研究采用宽波长光谱激光光源开展光遗传刺激，从而最大限度降低了光刺激伪迹。最终在1100 nm至1300 nm的光遗传刺激范围内，成功实现了无伪迹的全光学实验。第三，本研究明确了可实现最优调控效能的光谱范围，直至1300 nm。在共表达GCaMP6f与C1V1的皮层神经元中，诱发钙瞬变的发生率在1100 nm时即已达到峰值。通过优化动作电位检测流程，并将1100 nm确定为实现无伪迹且高效的基于GCaMP6f/C1V1的全光学生理学研究的最优波长，本研究提升了该类技术的转化应用价值，例如在基于神经网络的治疗手段开发领域。