BACKGROUND Ex vivo, whole-mount explant culture of the rodent retina has proved to be a valuable approach for studying retinal development. In a limited number of recent studies, this method has been coupled to live fluorescent microscopy with the goal of directly observing dynamic cellular events. However, retinal tissue thickness imposes significant technical limitations. To obtain 3-dimensional images with high quality axial resolution, investigators are restricted to specific areas of the retina and require microscopes, such as 2-photon, with a higher level of depth penetrance. Here, we report a retinal live imaging method that is more amenable to a wider array of imaging systems and does not compromise resolution of retinal cross-sectional area. RESULTS Mouse retinal slice cultures were prepared and standard, inverted confocal microscopy was used to generate movies with high quality resolution of retinal cross-sections. To illustrate the ability of this method to capture discrete, physiologically relevant events during retinal development, we imaged the dynamics of the Fucci cell cycle reporter in both wild type and Cyclin D1 mutant retinal progenitor cells (RPCs) undergoing interkinetic nuclear migration (INM). Like previously reported for the zebrafish, mouse RPCs in G1 phase migrated stochastically and exhibited overall basal drift during development. In contrast, mouse RPCs in G2 phase displayed directed, apical migration toward the ventricular zone prior to mitosis. We also determined that Cyclin D1 knockout RPCs in G2 exhibited a slower apical velocity as compared to wild type. These data are consistent with previous IdU/BrdU window labeling experiments on Cyclin D1 knockout RPCs indicating an elongated cell cycle. Finally, to illustrate the ability to monitor retinal neuron differentiation, we imaged early postnatal horizontal cells (HCs). Time lapse movies uncovered specific HC neurite dynamics consistent with previously published data showing an instructive role for transient vertical neurites in HC mosaic formation. CONCLUSIONS We have detailed a straightforward method to image mouse retinal slice culture preparations that, due to its relative ease, extends live retinal imaging capabilities to a more diverse group of scientists. We have also shown that, by using a slice technique, we can achieve excellent lateral resolution, which is advantageous for capturing intracellular dynamics and overall cell movements during retinal development and differentiation.

中文翻译：

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发育中的小鼠视网膜切片的实时成像。

背景技术离体的啮齿类动物视网膜的全量外植体培养已被证明是研究视网膜发育的有价值的方法。在有限的最新研究中，此方法已与实时荧光显微镜结合，目的是直接观察动态细胞事件。然而，视网膜组织厚度强加了明显的技术限制。为了获得具有高质量轴向分辨率的3维图像，研究人员仅限于视网膜的特定区域，并且需要显微镜（例如2光子）进行更高深度的渗透。在这里，我们报告了一种视网膜实时成像方法，该方法更适合更广泛的成像系统，并且不影响视网膜横截面积的分辨率。结果制备了小鼠视网膜切片培养物并进行了标准处理，倒置共聚焦显微镜用于产生具有高质量的视网膜横截面分辨率的电影。为了说明这种方法在视网膜发育过程中捕获离散的，生理学相关事件的能力，我们在野生型和Cyclin D1突变型视网膜祖细胞（RPCs）经历动力学间核迁移（INM）的过程中，对Fucci细胞周期报告基因的动力学进行了成像。像以前报道的斑马鱼一样，G1期的小鼠RPC随机迁移并在发育过程中表现出整体基底漂移。相反，在有丝分裂之前，处于G2期的小鼠RPC表现出定向的，朝心室的顶端迁移。我们还确定，与野生型相比，G2中的Cyclin D1基因敲除RPC的根尖速度较慢。这些数据与先前对Cyclin D1敲除RPC的IdU / BrdU窗口标记实验一致，表明细胞周期延长。最后，为了说明监测视网膜神经元分化的能力，我们对早期产后水平细胞（HCs）进行了成像。延时电影揭示了特定的HC神经突动力学，与先前发表的数据一致，该数据显示了在HC镶嵌形成过程中瞬态垂直神经突具有指导作用。结论我们已经详细描述了一种简单的方法来对小鼠视网膜切片培养物进行成像，由于其相对容易，将活体视网膜成像功能扩展到了更多的科学家群体。我们还表明，通过使用切片技术，我们可以获得出色的横向分辨率，