Large-FOV, dual-region, two-photon fluorescence lifetime imaging microscopy (LD-2P-FLIM) for multiparameter and quantitative brain function imaging

• 成果类型: Article
• 署名作者: Ye, Shiwei; Gao, Yufeng; Deng, Mengying; Sheng, Yaozhang; Xu, Xingyun; Cao, Xueming; Liu, Lianjian; Xiao, Na; Zou, Junjie; Xie, Minghan; Zeng, Long; Xiang, Huachuang; Yu, Jia; Wu, Ting; He, Yuezhi; Yao, Jing; Li, Hui; Guo, Yanwu; Chu, Jun; Zheng, Hairong; Liu, Chengbo; Zheng, Wei
• 署名单位: Chinese Academy of Sciences; Shenzhen Institute of Advanced Technology, CAS; Chinese Academy of Sciences; Shenzhen Institute of Advanced Technology, CAS; Southern Medical University - China; Chinese Academy of Sciences; Shenzhen Institute of Advanced Technology, CAS
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2536213123
• 发表日期: 2026-08-11
• 页码: e2536213123
• 关键词: two-photon microscope; fluorescence lifetime imaging; large imaging throughput; multiparameter brain function imaging; quantitative calcium imaging; FIELD-OF-VIEW; MULTI-PLANE

摘要

Large-scale imaging of multiple dynamic behaviors and quantitative neurochemical concentrations with high spatiotemporal resolution is essential for understanding complex brain functions. Two-photon microscopy (TPM) is ideally suited for in vivo brain function imaging because of its high resolution and deep tissue penetration. However, conventional TPM is limited by a restricted field-of-view (FOV), an inherent trade-off between the imaging area and temporal resolution, and an insufficient amount of information obtained using only intensity recording. Here, we propose large-FOV, dual-region, two-photon fluorescence lifetime imaging microscopy (LD-2P-FLIM) for multiparameter and quantitative brain function imaging, with a 3 & times; 3 mm2 FOV, a uniform lateral resolution of 0.7 mu m, and a FLIM throughput of up to 15.73 megapixels/s (512 & times; 512 pixels, 30 Hz, two regions). We extend the FOV by breaking the limit of commercial objectives with an effective adaptive optics strategy. To alleviate the trade-off between the imaging area and temporal resolution, we use a temporal multiplexing system that enables simultaneous and flexible two-region imaging across the large FOV. Furthermore, we develop a field programmable gate array module to demultiplex fluorescence signals from different regions and perform high-throughput, two-region FLIM. We demonstrate the superior performance of LD-2P-FLIM by simultaneous monitoring of neural activities across multiple cortical areas, synchronous recording of neurovascular coupling under both physiological and pathological conditions, long-term observation of the microglial response to local neuron injury, and quantitative imaging of calcium concentrations across a large neuronal population in vivo.