贝塞尔双光子光片显微镜在活体生物成像中的应用  

In vivo biological imaging applications of Bessel two-photon light-sheet microscopy

作　　者：卜庆盼 罗芳琳 张杏云[2] BU Qingpan;LUO Fanglin;ZHANG Xingyun(School of Life Sciences,Changchun Normal University,Changchun 130032,China;Key Laboratory of Optical System Advanced Manufacturing Technology,Changchun Institute of Optics,Fine Mechanics and Physics,Chinese Academy of Sciences,Changchun 130033,China)

机构地区：[1]长春师范大学生命科学学院,吉林长春130032 [2]中国科学院长春光学精密机械与物理研究所光学系统先进制造重点实验室,吉林长春130033

出　　处：《液晶与显示》2024年第11期1477-1482,共6页Chinese Journal of Liquid Crystals and Displays

基　　金：吉林省科技厅发展计划(No.YDZJ202301ZYTS348);吉林省教育厅科学技术研究规划(No.JJKH20220833KJ);长春师范大学自然科学项目(No.CSJJ2022002ZK)。

摘　　要：双光子光片显微镜具有分辨率高、光漂白低等优势,在生物学领域有着广泛的应用。但由于照明光片厚度与长度之间的取舍矛盾问题,导致轴向分辨率和成像视场无法兼顾,因此在应用中受到限制。为了解决这一问题,本文采用液晶空间光调制器将普通高斯照明光束调制为贝塞尔光束,利用贝塞尔光束的“无衍射”特性实现了大且薄的光片照明,光片长度与厚度之比达到了163。在此基础上,设计并搭建了一套贝塞尔双光子光片显微镜,在310μm的视场范围内实现了横向440 nm、轴向1.9μm的三维高分辨率成像。本文成功实现了斑马鱼胚胎的活体成像,可以做到血液细胞流速的测量和脊柱骨细胞的分割计数,验证了贝塞尔双光子光片显微镜在活体生物成像中的应用潜力。Two-photon light-sheet microscopy offers high resolution and low photobleaching,making it widely used in biological research.However,a trade-off between light sheet thickness and length limits its axial resolution and field of view,restricting its applications.To address this issue,this paper employs a liquid crystal spatial light modulator(SLM)to modulate conventional Gaussian illumination beam into Bessel beam.The“non-diffracting”property of the Bessel beam enables the generation of a large and thin light sheet,achieving a length-to-thickness ratio of 163.Based on this,a Bessel two-photon light-sheet microscope is constructed,achieving three-dimensional high-resolution imaging with a lateral resolution of 440 nm and an axial resolution of 1.9μm within a 310μm field of view.Successful in vivo imaging of zebrafish embryos is demonstrated,including the measurement of blood cell flow velocity and the segmentation and counting of spinal cord cells,validating the application potential of the Bessel two-photon light-sheet microscope in live biological imaging.

关 键 词：液晶空间光调制器 双光子光片显微镜 贝塞尔光束 活体成像 

分 类 号：Q334[生物学—遗传学]