PDF(Pubmed)
Abstract：
Since its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in deeper layers (30 - 50 µm) of biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the image acquisition settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. By measuring the full width at half maximum of the thinnest filamentous structures in our preparation, we achieved a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed visualisation of putative GABAergic horizontal cell synapses in the outer retina. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina at depths between 30 and 50 µm, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.
摘要：
自从它的发明,超分辨率显微镜已成为生物结构高级成像的流行工具，允许在低于衍射极限的空间尺度下可视化亚细胞结构。因此，最近，这并不奇怪，不同的超分辨率技术正在应用于神经科学，例如，解决神经递质受体和蛋白质复合物组成在突触前终末的聚集。尽管如此，这些实验绝大多数都是在细胞培养或非常薄的组织切片中进行的，而在生物样品的较深层（30-50μm）中只有少数超分辨率成像的例子。在这种情况下，哺乳动物的全视网膜已很少被研究与超分辨率显微镜。这里,我们的目标是建立一个受激发射损耗(STED)显微镜对整个视网膜成像方案.为此,我们开发了样品制备，包括视网膜组织的水平切片，与STED兼容的荧光团的免疫标记方案，并优化了图像采集设置。我们标记了躯体中的亚细胞结构，树突,和小鼠内部视网膜中的视网膜神经节细胞轴突。通过测量我们制备中最薄的丝状结构的半峰全宽，与传统的共焦图像相比，我们实现了两个或更高的分辨率增强。当与视网膜的水平切片相结合时，这些设置允许可视化外视网膜中推定的GABA能水平细胞突触。一起来看,我们成功地建立了一个STED协议，用于在30到50µm深度的全装鼠标视网膜中进行可靠的超分辨率成像，这使得调查，例如,健康和疾病中视网膜突触的蛋白质复合物组成和细胞骨架超微结构。
