• Photonics Research
  • Vol. 10, Issue 3, 687 (2022)
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、*
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, China
  • 2CAS Key Laboratory of Health Informatics, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
  • 3Paul C. Lauterbur Research Center for Biomedical Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
  • 4State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
  • 5Key Laboratory of Genetic Network Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
  • 6Brain Cognition and Brain Disease Institute, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
  • 7Centre for Micro Nano Systems and Bionic Medicine, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
  • 8e-mail: hui.li@siat.ac.cn
  • 9e-mail: zhengwei@siat.ac.cn
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    DOI: 10.1364/PRJ.441778 Cite this Article
    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[J]. Photonics Research, 2022, 10(3): 687 Copy Citation Text show less

    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 2.07±2.89% in 25 min), and minimizes photodamages.

    1. INTRODUCTION

    Two-photon excitation fluorescence microscopy (TPM) is a powerful tool for the in vivo three-dimensional (3D) imaging of cellular and subcellular structures and functions deep in turbid tissues [1]. Owing to its nonlinear excitation property, TPM provides compelling performance of near-diffraction-limited spatial resolution in relatively thick samples. However, the conventional TPM, i.e., Gaussian-focus TPM (Gauss-TPM), captures volumetric images by serially scanning a 3D space with a Gaussian focus, which significantly limits the imaging speed. Owing to its importance in capturing rapid biological events such as calcium transients in neurons, considerable effort has been devoted to improving the focus scanning rate [2,3], such as by applying resonant scanning [4], acoustic scanning [57], ultrasound lenses [8,9], electrotunable lenses [10,11], remote focusing [12,13], multifocal excitation [1417], and multi-angle line scanning [18]. Other strategies, including temporal focusing [19,20] and multiplane imaging [21], have been developed to increase the volumetric imaging speed by capturing a two-dimensional (2D) image within a single exposure. Although these methods can partially alleviate the problem by improving the scanning speed, the implementation of layer-by-layer scanning to visualize 3D structures inherently limits the volumetric imaging speed. Moreover, the layer-by-layer scanning strategy repeatedly exposes the tissues above or beneath the focal plane to the excitation light, thereby aggravating photodamage and phototoxicity.

    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[J]. Photonics Research, 2022, 10(3): 687
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