John F. Hardesty, MD Department of Ophthalmology and Visual Sciences, Washington University School of Medicine; Graduate Program in Neuroscience, Washington University School of Medicine.
John F. Hardesty, MD Department of Ophthalmology and Visual Sciences, Washington University School of Medicine; Department of Neuroscience, Washington University School of Medicine; Hope Center for Neurological Disorders, Washington University School of Medicine;
J Vis Exp. 2021 Feb 13(168). doi: 10.3791/61970.
The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer's disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access. This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.
视网膜将环境中的光信号转化为电信号，这些信号被传播到大脑。视网膜疾病很常见，会导致视力障碍和失明。了解这些疾病的进展对于制定新的治疗方法至关重要。在疾病动物模型中的活体显微镜检查是了解神经退行性变的有力工具，并为从阿尔茨海默病到中风等疾病的治疗带来了重要进展。鉴于视网膜是唯一可通过光学方法内在获得的中枢神经系统结构，它自然适合进行活体成像。然而，晶状体和角膜的固有光学特性给有效的成像访问带来了一些挑战。本方案概述了在细胞分辨率下对小鼠视网膜中细胞群体和结构进行体内双光子成像的方法，适用于急性和慢性成像实验。它展示了使用腺相关病毒 (AAV) 载体、转基因小鼠和无机染料等一系列标记技术对视网膜神经节细胞 (RGC)、无长突细胞、小胶质细胞和血管成像的示例。重要的是，这些技术适用于视网膜的所有细胞类型，并且描述了访问其他感兴趣的细胞群体的建议方法。还详细介绍了用于显示和量化的手动图像后处理的示例策略。这些技术可直接应用于健康和疾病状态下视网膜功能的研究。
