视网膜色素上皮细胞高分辨率稳定动态成像技术

It is reported that congenital retinal diseases are associated with abnormal function of retinal pigment epithelial cells (RPE cells), and RPE cells are affected in retinal diseases including a variety of macular diseases leading to irreversible blindness. One of the difficulties in imaging RPE cells in vivo at high resolution is that autofluorescence images are blurred by retinal motions during the long integration times required given the weak autofluorescence signal. Currently combined with adaptive optics confocal scanning laser ophthalmoscope (AOSLO), single RPE cells had been resolved in vivo allowing the integration of very weak signals across many frames, but were unable to achieve the dynamic and microscopic observation of RPE cells characteristics. Therefore, this project uses a line scanning confocal technology to extract retinal motions, and feedback controls a tilt mirror to execute beam tracking in real-time as compensation for image blurs caused by retinal motions. In case of no loss of energy utilization, the aim of this project is to develop a high-precision retinal motions correcting device, and this device can be directly applied to AOSLO system, achieving high resolution, stability dynamic imaging of RPE cells using autofluorescence. Based on these researches, the instrument to be constructed should be seen as an stability dynamic imaging platform with a cellular level resolution for the study of microscopic structures and associations with visual functions of RPE cells under physiological conditions, as well as for the study of the pathogenesis, disease progression, efficacy surveillance and intervention associated with RPE cells under pathological conditions.

据报道先天性视网膜疾病都与视网膜色素上皮细胞（RPE细胞）功能异常相关, RPE细胞功能异常也将导致多种不可逆致盲性黄斑疾病。由于RPE细胞自发荧光信号微弱，长曝光时间内视网膜抖动引起的图像模糊影响在体高分辨率成像。目前结合自适应光学共焦扫描技术，通过多帧图像平均消除视网膜抖动后能够分辨出单个RPE细胞，但是无法实现RPE细胞的动态性微观特征观察。有鉴于此，本项目利用线扫描共焦技术提取视网膜抖动反馈控制倾斜反射镜进行实时的光束跟踪补偿视网膜抖动造成的图像模糊。在不损失光能利用率的情况下，研究开发一套高精度视网膜抖动校正装置，可以直接应用于自适应光学共焦扫描成像技术，实现RPE细胞自发荧光的高分辨率稳定动态成像。从而为研究RPE细胞在生理状态下的微观结构与视觉功能关联，以及病理状态下的发病机理、病程进展以及疗效监视与干预提供一种细胞分辨率水平的、稳定动态成像新工具。

为了克服视网膜抖动造成的高分辨率成像细胞图像模糊，以实现活体视网膜RPE细胞高分辨率、稳定动态成像的目标。项目成功将线光束共焦扫描技术与自适应光学共焦扫描技术集成出一套视网膜综合成像实验系统，利用线光束共焦扫描200Hz高速成像功能测量视网膜抖动，并反馈控制倾斜镜实现自适应光学共焦扫描成像中的实时光束跟踪校正视网膜抖动，实现了活体视网膜包括RPE细胞、视细胞在内的2μm级高分辨率、30Hz稳定动态成像。该系统成像功能丰富，可以同时实现三种功能：（1）采集视网膜30°大视场、200Hz高速成像图像，（2）采集视网膜细胞2°视场、2μm高分辨率成像图像，（3）完成视网膜2°视场的视网膜抖动校正。项目研制的成像系统和方法已经申报发明专利，其中共光束扫描成像、波前测量、大视场高分辨率成像等核心技术已经申报PCT国际发明。基于此成像系统，团队开展了多种视网膜高分辨率成像实验研究，相关成果发表于光学、生物光学、眼科医学等领域的重要期刊上，并与国内知名的临床眼科研究团队建立了紧密合作关系。项目取得的研究成果为将来新型、高速、宽视场、高分辨率视网膜成像系统研发奠定了基础，将有效促进自适应光学高分辨率成像技术在视网膜相关病理研究领域的深入应用。