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基于双光子共聚焦成像技术的小梁网通道原位成像研究

张静  钱秀清  张海霞  时梅林  刘志成   

  1. 221004江苏,徐州医科大学医学影像学院(张静、时梅林);100069首都医科大学生物医学工程学院 临床生物力学应用基础研究北京市重点实验室(钱秀清、张海霞、刘志成)
  • 收稿日期:2019-04-10 出版日期:2019-05-25 发布日期:2019-06-06
  • 通讯作者: 刘志成,Email:zcliu@ccmu.edu.cn E-mail:zcliu@ccmu.edu.cn

In situ imaging of trabecular meshwork outflow pathways basing on two-photon confocal microscope

ZHANG Jing1, QIAN Xiu-qing2, ZHANG Hai-xia2, SHI Mei-lin1, LIU Zhi-cheng2   

  1. In situ imaging of trabecular meshwork outflow pathways basing on two-photon confocal microscope
  • Received:2019-04-10 Online:2019-05-25 Published:2019-06-06
  • Contact: LIU Zhi-cheng, Email: zcliu@ccmu.edu.cn E-mail:zcliu@ccmu.edu.cn

摘要:

目的 观察基于双光子共聚焦成像技术在不进行组织固定和染色的情况下获取大鼠小梁网外流通道微观结构图像的应用效果。设计 实验研究。研究对象 SD大鼠6只。方法 将6只大鼠随机分成两组(A组和B组),A组大鼠的完整眼球用于小梁网通道纵截面成像数据的获取;B组大鼠左眼球剖开用于小梁网通道横截面及眼球矢状面成像,右眼球为对照组。采用自行搭建的双光子共聚焦成像系统(激发波长950 nm、侧向分辨率0.3 μm、轴向分辨率1.5 μm),从前房角处进行成像,获取不同方位小梁网及其周围组织的微观结构信息。通过图像处理方法,定量分析Schlemm管截面直径随成像深度的变化情况。主要指标 不同方位小梁网通道成像深度、小梁网微观结构特点及Schlemm管平均截面直径。结果 基于双光子共聚焦成像技术分别获取到小梁网通道纵截面、横截面以及眼球矢状面的图像数据。不同深度的小梁网通道组织形态结构各异,深层小梁网纤维排列致密,形成的孔隙较小;浅层小梁网的纤维相互交错,形成的孔隙较大,利于房水流出。在角巩膜缘下方190~215 μm成像深度范围内,Schlemm管的平均截面直径在34~68 μm之间变化。结论 双光子共聚焦成像可观察小梁网通道的微观结构,为进一步小梁网通道房水外流动力学研究奠定了基础。

关键词: 小梁网, 双光子共聚焦显微镜, Schlemm管, 青光眼

Abstract:

Objective To observed the microstructures of the trabecular meshwork (TM) outflow pathways of rats using the two-photon confocal imaging technique without tissue fixation and staining. Design Experimental study. Participants The six SD rats. Methods Six rats were randomly divided into two groups (group A and group B). After being sacrificed, the intact eyeball of the rat (group A) was used to acquire the longitudinal section imaging data of the TM outflow pathways. The left eyes of the SD rats in group B were dissected for the cross section and sagittal plane of the TM outflow pathways imaging,and the other eyes was used as the control group. Through the anterior chamber of the eye, the self-built two-photon confocal imaging system (the excitation wavelength of 950 nm, lateral resolution of 0.3 μm and axial resolution of 1.5 μm) was used to image the microstructures of the TM and adjacent tissues from different directions. And the relation between the average cross-sectional diameter of Schlemm’s canal and the imaging depth based on image processing was quantitatively investigated. Main Outcome Measures The imaging depth of the TM outflow pathways from different directions, the structural features of the TM and the average cross-sectional diameter of Schlemm’s canal. Results The imagings of longitudinal section, cross section and sagittal structural features of the TM and the adjacent tissues were obtained based on two-photon confocal microscope. The fibers of TM seem to be heterogeneous with depth increases. The fibers of the deep TM were densely arranged, and the formed pores were small; the fibers of the shallow trabecular meshwork were interlaced, and the formed pores were large to facilitate the outflow of aqueous humor. The average cross-sectional diameter of the Schlemm’s canal was varied from 34 μm to 68 μm in the imaging depth range from 190 μm to 215 μm below the limbus. Conclusion Our research provides a trans-scleral imaging method to visualize the microstructure of the TM outflow pathways, which laid a methodological basis for further understanding of aqueous humor outflow dynamics in the TM outflow pathways.

Key words: trabecular meshwork, two-photon confocal microscope, Schlemm’s canal, glaucoma