Whole-animal functional and developmental imaging with isotropic spatial resolution
Whole-animal functional and developmental imaging with isotropic spatial resolution
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DOI：
10.1038/nmeth.3632
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发表时间：
2015-12-01
期刊：
影响因子：
48
通讯作者：
Kellerbeams, Philipp J.
中科院分区：
文献类型：
--
作者：
Chhetri, Raghav K.;Amat, Fernando;Kellerbeams, Philipp J.
在大型标本上成像快速细胞动力学需要在所有维度上的高分辨率、高成像速度、良好的物理覆盖和低光损伤。为了满足这些要求，我们开发了各向同性多视图（IsoView）光片显微镜，通过同时光片照明和荧光检测沿着四个正交方向快速图像大标本。通过高通量多视图反卷积将这四个视图组合在一起，产生在所有三个维度上具有高分辨率的图像。我们展示了果蝇幼虫在1.1-2.5的空间分辨率下的全动物功能成像。m和2 Hz的时间分辨率几个小时。我们还提出了空间各向同性的全脑功能成像在斑马鱼幼虫和空间各向同性的快速细胞动力学跨原肠胚果蝇胚胎成像。与传统的光片显微镜相比，IsoView显微镜提高了空间分辨率至少七倍，降低了分辨率各向异性至少三倍。与现有的高分辨率光片技术相比，IsoView显微镜有效地增加了一倍的穿透深度，并提供亚秒级的时间分辨率为标本400倍以上，以前可以成像。
Imaging fast cellular dynamics across large specimens requires high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To meet these requirements, we developed isotropic multiview (IsoView) light-sheet microscopy, which rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. Combining these four views by means of high-throughput multiview deconvolution yields images with high resolution in all three dimensions. We demonstrate whole-animal functional imaging of Drosophila larvae at a spatial resolution of 1.1-2.5. m and temporal resolution of 2 Hz for several hours. We also present spatially isotropic whole-brain functional imaging in Danio rerio larvae and spatially isotropic multicolor imaging of fast cellular dynamics across gastrulating Drosophila embryos. Compared with conventional light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.