Yufeng Gao1,2,†, Xianyuan Xia1,2,†, Lina Liu1,2, Ting Wu1,2..., Tingai Chen1,2, Jia Yu1,2, Zhili Xu3, Liang Wang1,2, Fei Yan3, Zhuo Du4,5, Jun Chu1,2, Yang Zhan6, Bo Peng7, Hui Li1,2,8,* and Wei Zheng1,2,9,*|Show fewer author(s)
Author Affiliations
1Research Center for Biomedical Optics and Molecular Imaging, Shenzhen Key Laboratory for Molecular Imaging, Guangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China2CAS Key Laboratory of Health Informatics, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China3Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China4State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China5Key Laboratory of Genetic Network Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China6Brain Cognition and Brain Disease Institute, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China7Centre for Micro Nano Systems and Bionic Medicine, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China8e-mail: hui.li@siat.ac.cn9e-mail: zhengwei@siat.ac.cnshow less
DOI: 10.1364/PRJ.441778
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Yufeng Gao, Xianyuan Xia, Lina Liu, Ting Wu, Tingai Chen, Jia Yu, Zhili Xu, Liang Wang, Fei Yan, Zhuo Du, Jun Chu, Yang Zhan, Bo Peng, Hui Li, Wei Zheng, "Axial gradient excitation accelerates volumetric imaging of two-photon microscopy," Photonics Res. 10, 687 (2022)
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Abstract
Two-photon excitation fluorescence microscopy (TPM), owing to its capacity for subcellular resolution, intrinsic optical sectioning, and superior penetration depth in turbid samples, has revolutionized biomedical research. However, its layer-by-layer scanning to form a three-dimensional image inherently limits the volumetric imaging speed and increases phototoxicity significantly. In this study, we develop a gradient excitation technique to accelerate TPM volumetric imaging. The axial positions of the fluorophores can be decoded from the intensity ratio of the paired images obtained by sequentially exciting the specimen with two axially elongated two-photon beams of complementary gradient intensities. We achieved a 0.63 μm axial localization precision and demonstrate the flexibility of the gradient TPM on various sparsely labeled samples, including bead phantoms, mouse brain tissues, and live macrophages. Compared with traditional TPM, our technique improves volumetric imaging speed (by at least sixfold), decreases photobleaching (i.e., less than in 25 min), and minimizes photodamages.