Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain
Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain
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DOI：
10.1364/oe.20.020582
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发表时间：
2012-08-27
期刊：
影响因子：
3.8
通讯作者：
Pavone, F. S.
中科院分区：
文献类型：
--
作者：
Silvestri, L.;Bria, A.;Pavone, F. S.
阐明大脑功能的神经通路是神经科学中最大的挑战之一。基于光片的显微镜是一种通过对透明小鼠大脑进行光学切片来绘制脑回路的尖端方法。然而，该方法提供的图像对比度不足以解析和重建整个神经元网络。在这里，我们结合了光片照明和共焦狭缝检测的优点，以提高图像的对比度在真实的时间，帧速率为10 Hz。事实上，在共焦光片显微镜（CLSM）中，离焦和散射光在检测之前被过滤掉，而无需多次采集或对采集的数据进行任何后处理。通过与结构化照明方法进行比较，在透明小鼠脑中验证了CLSM的背景抑制能力。我们表明，CLSM允许重建宏观脑体积与亚细胞分辨率。我们获得了L7-GFP转基因小鼠小脑中浦肯野细胞的全面图谱。此外，我们能够追踪thy 1-GFP-M转基因小鼠大脑中的神经元投射。CLSM保证的全脑高分辨率荧光成像可能是通过神经元通路导航大脑的强大工具。虽然这项工作的重点是大脑成像，但CLSM负担得起的宏观尺度高分辨率断层扫描非常适合以微米级分辨率探索不同标本的解剖结构，如小鼠器官，胚胎或苍蝇。(C)2012美国光学学会
Elucidating the neural pathways that underlie brain function is one of the greatest challenges in neuroscience. Light sheet based microscopy is a cutting edge method to map cerebral circuitry through optical sectioning of cleared mouse brains. However, the image contrast provided by this method is not sufficient to resolve and reconstruct the entire neuronal network. Here we combined the advantages of light sheet illumination and confocal slit detection to increase the image contrast in real time, with a frame rate of 10 Hz. In fact, in confocal light sheet microscopy (CLSM), the out-of-focus and scattered light is filtered out before detection, without multiple acquisitions or any post-processing of the acquired data. The background rejection capabilities of CLSM were validated in cleared mouse brains by comparison with a structured illumination approach. We show that CLSM allows reconstructing macroscopic brain volumes with sub-cellular resolution. We obtained a comprehensive map of Purkinje cells in the cerebellum of L7-GFP transgenic mice. Further, we were able to trace neuronal projections across brain of thy1-GFP-M transgenic mice. The whole-brain high-resolution fluorescence imaging assured by CLSM may represent a powerful tool to navigate the brain through neuronal pathways. Although this work is focused on brain imaging, the macro-scale high-resolution tomographies affordable with CLSM are ideally suited to explore, at micron-scale resolution, the anatomy of different specimens like murine organs, embryos or flies. (C) 2012 Optical Society of America