Whole-brain block-face serial microscopy tomography at subcellular resolution using FAST
Whole-brain block-face serial microscopy tomography at subcellular resolution using FAST
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DOI：
10.1038/s41596-019-0148-4
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发表时间：
2019-05-01
期刊：
影响因子：
14.8
通讯作者：
Hashimoto, Hitoshi
中科院分区：
文献类型：
--
作者：
Seiriki, Kaoru;Kasai, Atsushi;Hashimoto, Hitoshi
在这里，我们描述了一个优化的和详细的协议块面连续显微镜断层扫描（FAST）。FAST能够以轴突空间分辨率对固定大脑进行高速连续切片荧光成像，并进行后续图像数据处理。它提供全脑解剖和功能分析，包括单细胞水平的核染色脑的结构分析，报告动物脑的细胞类型特异性映射和顺行/逆行标记的神经元追踪。光片荧光显微镜的清除大脑是有利的成像速度方面，但其空间分辨率一般是有限的，而相反的是真实的传统的共聚焦显微镜。FAST提供了一种解决方案来克服这些技术限制。本方案描述了组装FAST硬件、样品制备、成像和图像处理的详细程序。一个单一的成像会话需要少至2.4小时，每个小鼠大脑，样品制备需要1至几天，这取决于预处理;然而，多个样品可以同时制备。我们预计，FAST将有助于更好地了解大脑系统的公正和无假设的方法。
Here, we describe an optimized and detailed protocol for block-face serial microscopy tomography (FAST). FAST enables high-speed serial section fluorescence imaging of fixed brains at an axonal spatial resolution and subsequent image data processing. It renders brain-wide anatomical and functional analyses, including structural profiling of nuclear-stained brain at the single-cell level, cell-type-specific mapping with reporter animal brains and neuronal tracing with anterograde/retrograde labeling. Light-sheet fluorescence microscopy of cleared brains is advantageous in regard to imaging speed, but its spatial resolution is generally limited, whereas the opposite is true for conventional confocal microscopy. FAST offers a solution to overcome these technical limitations. This protocol describes detailed procedures for assembling the FAST hardware, sample preparation, imaging and image processing. A single imaging session takes as little as 2.4 h per mouse brain, and sample preparation requires 1 to several days, depending on pretreatments; however, multiple samples can be prepared simultaneously. We anticipate that FAST will contribute to unbiased and hypothesis-free approaches for a better understanding of brain systems.