Long-working-distance high-collection-efficiency three-photon microscopy for in vivo long-term imaging of zebrafish and organoids
Peng Deng,
Shoupei Liu,
Yaoguang Zhao,
Xinxin Zhang,
Yufei Kong,
Linlin Liu,
Yujie Xiao,
Shasha Yang,
Jiahao Hu,
Jixiong Su,
Ang Xuan,
Jinhong Xu,
Huijuan Li,
Xiaoman Su,
Jingchuan Wu,
Yuli Jiang,
Yu Mu,
Zhicheng Shao,
Cihang Kong,
Bo Li
Affiliations
Peng Deng
Department of Neurosurgery, Huashan Hospital, MOE Frontiers Center for Brain Science, State Key Laboratory of Medical Neurobiology, Institutes for Translational Brain Research, Fudan University, Shanghai 200032, China
Shoupei Liu
Department of Neurosurgery, Huashan Hospital, MOE Frontiers Center for Brain Science, State Key Laboratory of Medical Neurobiology, Institutes for Translational Brain Research, Fudan University, Shanghai 200032, China
Yaoguang Zhao
Department of Neurosurgery, Huashan Hospital, MOE Frontiers Center for Brain Science, State Key Laboratory of Medical Neurobiology, Institutes for Translational Brain Research, Fudan University, Shanghai 200032, China
Xinxin Zhang
Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China
Yufei Kong
Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Institute of Pediatrics, National Children’s Medical Center, Children’s Hospital, Fudan University, Shanghai 200032, China
Linlin Liu
Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Institute of Pediatrics, National Children’s Medical Center, Children’s Hospital, Fudan University, Shanghai 200032, China
Yujie Xiao
Department of Neurosurgery, Huashan Hospital, MOE Frontiers Center for Brain Science, State Key Laboratory of Medical Neurobiology, Institutes for Translational Brain Research, Fudan University, Shanghai 200032, China
Shasha Yang
Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai 200032, China
Jiahao Hu
Department of Neurosurgery, Huashan Hospital, MOE Frontiers Center for Brain Science, State Key Laboratory of Medical Neurobiology, Institutes for Translational Brain Research, Fudan University, Shanghai 200032, China
Jixiong Su
Department of Neurosurgery, Huashan Hospital, MOE Frontiers Center for Brain Science, State Key Laboratory of Medical Neurobiology, Institutes for Translational Brain Research, Fudan University, Shanghai 200032, China
Ang Xuan
Department of Neurosurgery, Huashan Hospital, MOE Frontiers Center for Brain Science, State Key Laboratory of Medical Neurobiology, Institutes for Translational Brain Research, Fudan University, Shanghai 200032, China; Academy for Engineering and Technology, Fudan University, Shanghai 200433, China
Jinhong Xu
Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Institute of Pediatrics, National Children’s Medical Center, Children’s Hospital, Fudan University, Shanghai 200032, China
Huijuan Li
Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Institute of Pediatrics, National Children’s Medical Center, Children’s Hospital, Fudan University, Shanghai 200032, China
Xiaoman Su
State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, MOE Frontiers Center for Brain Science, Department of Neurology of Zhongshan Hospital, Fudan University, Shanghai 200032, China
Jingchuan Wu
Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai 200032, China
Yuli Jiang
Department of Neurosurgery, Huashan Hospital, MOE Frontiers Center for Brain Science, State Key Laboratory of Medical Neurobiology, Institutes for Translational Brain Research, Fudan University, Shanghai 200032, China
Yu Mu
Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China
Zhicheng Shao
Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Institute of Pediatrics, National Children’s Medical Center, Children’s Hospital, Fudan University, Shanghai 200032, China; Corresponding author
Cihang Kong
Department of Neurosurgery, Huashan Hospital, MOE Frontiers Center for Brain Science, State Key Laboratory of Medical Neurobiology, Institutes for Translational Brain Research, Fudan University, Shanghai 200032, China; Corresponding author
Bo Li
Department of Neurosurgery, Huashan Hospital, MOE Frontiers Center for Brain Science, State Key Laboratory of Medical Neurobiology, Institutes for Translational Brain Research, Fudan University, Shanghai 200032, China; Corresponding author
Summary: Zebrafish and organoids, crucial for complex biological studies, necessitate an imaging system with deep tissue penetration, sample protection from environmental interference, and ample operational space. Traditional three-photon microscopy is constrained by short-working-distance objectives and falls short. Our long-working-distance high-collection-efficiency three-photon microscopy (LH-3PM) addresses these challenges, achieving a 58% fluorescence collection efficiency at a 20 mm working distance. LH-3PM significantly outperforms existing three-photon systems equipped with the same long working distance objective, enhancing fluorescence collection and dramatically reducing phototoxicity and photobleaching. These improvements facilitate accurate capture of neuronal activity and an enhanced detection of activity spikes, which are vital for comprehensive, long-term imaging. LH-3PM’s imaging of epileptic zebrafish not only showed sustained neuron activity over an hour but also highlighted increased neural synchronization and spike numbers, marking a notable shift in neural coding mechanisms. This breakthrough paves the way for new explorations of biological phenomena in small model organisms.
