Krebs Mark P, Xiao Mei, Sheppard Keith, Hicks Wanda, Nishina Patsy M
The Jackson Laboratory, 600 Main Street, Bar Harbor, ME, 04609, USA.
Methods Mol Biol. 2016;1438:395-415. doi: 10.1007/978-1-4939-3661-8_20.
Noninvasive live imaging has been used extensively for ocular phenotyping in mouse vision research. Bright-field imaging and optical coherence tomography (OCT) are two methods that are particularly useful for assessing the posterior mouse eye (fundus), including the retina, retinal pigment epithelium, and choroid, and are widely applied due to the commercial availability of sophisticated instruments and software. Here, we provide a guide to using these approaches with an emphasis on post-acquisition image processing using Fiji, a bundled version of the Java-based public domain software ImageJ. A bright-field fundus imaging protocol is described for acquisition of multi-frame videos, followed by image registration to reduce motion artifacts, averaging to reduce noise, shading correction to compensate for uneven illumination, filtering to improve image detail, and rotation to adjust orientation. An OCT imaging protocol is described for acquiring replicate volume scans, with subsequent registration and averaging to yield three-dimensional datasets that show reduced motion artifacts and enhanced detail. The Fiji algorithms used in these protocols are designed for batch processing and are freely available. The image acquisition and processing approaches described here may facilitate quantitative phenotyping of the mouse eye in drug discovery, mutagenesis screening, and the functional cataloging of mouse genes by individual laboratories and large-scale projects, such as the Knockout Mouse Phenotyping Project and International Mouse Phenotyping Consortium.
在小鼠视觉研究中，无创活体成像已被广泛用于眼部表型分析。明场成像和光学相干断层扫描（OCT）是两种特别有助于评估小鼠眼球后部（眼底）的方法，包括视网膜、视网膜色素上皮和脉络膜，由于先进仪器和软件的商业可用性，它们被广泛应用。在这里，我们提供了使用这些方法的指南，重点是使用Fiji进行采集后图像处理，Fiji是基于Java的公共领域软件ImageJ的捆绑版本。描述了一种明场眼底成像协议，用于采集多帧视频，随后进行图像配准以减少运动伪影、平均以降低噪声、阴影校正以补偿不均匀照明、滤波以改善图像细节以及旋转以调整方向。描述了一种OCT成像协议，用于获取重复的体积扫描，随后进行配准和平均以生成三维数据集，该数据集显示出减少的运动伪影和增强的细节。这些协议中使用的Fiji算法设计用于批量处理且可免费获得。本文所述的图像采集和处理方法可能有助于各个实验室和大型项目（如基因敲除小鼠表型分析项目和国际小鼠表型分析联盟）在药物发现、诱变筛选以及小鼠基因功能编目中对小鼠眼睛进行定量表型分析。
