Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.
Department of Genetics and Ophthalmology, Institute of Molecular and Clinical Ophthalmology Basel, Basel, Switzerland.
Adv Exp Med Biol. 2023;1415:395-402. doi: 10.1007/978-3-031-27681-1_58.
The small size of ciliary structures that underlies photoreceptor function and inherited ciliopathies requires imaging techniques adapted to visualizing them at the highest possible resolution. In addition to powerful super-resolution imaging modalities, emerging approaches to sample preparation, including expansion microscopy (ExM), can provide a robust route to imaging specific molecules at the nanoscale level in the retina. We describe a protocol for applying ExM to whole retinas in order to achieve nanoscale fluorescence imaging of ciliary markers, including tubulin, CEP290, centrin, and CEP164. The results are consistent with those from other super-resolution fluorescence techniques and reveal new insights into their arrangements with respect to the subcompartments of photoreceptor cilia. This technique is complimentary to other imaging modalities used in retinal imaging, and can be carried out in virtually any laboratory, without the need for expensive specialized equipment.
为了实现对纤毛标记物（包括微管蛋白、CEP290、中心体蛋白和 CEP164）的纳米级荧光成像，我们描述了一种将扩展显微镜（ExM）应用于整个视网膜的方案。该方案的结果与其他超分辨率荧光技术的结果一致，并揭示了它们在与光感受器纤毛的亚区室相对应的排列方面的新见解。该技术与视网膜成像中使用的其他成像模式互补，几乎可以在任何实验室中进行，而无需昂贵的专用设备。
