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Two-photon Ca 2+ imaging in vivo using a cranial window and specific neuronal labeling enables real-time, in situ, and long-term imaging of the living brain. neuronal activity in vivo is critical to understanding the physiological or pathological functions of the brain. 9 Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China. 8 Biomedical Center, Huazhong Agricultural University, Wuhan, 430070, China. 7 College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China. 6 State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, China.5 Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.4 Biomedical Center, Huazhong Agricultural University, Wuhan, 430070, China.3 College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China.2 State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, China.1 Academician Workstation of Animal Disease Control and Nutrition Immunity in Henan Province, Henan Joint International Research Laboratory of Veterinary Biologics Research and Application, Anyang Institute of Technology, Anyang, 455000, China.