Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.
Nat Methods. 2015 Dec;12(12):1171-8. doi: 10.1038/nmeth.3632. Epub 2015 Oct 26.
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 μm 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.
要在大样本中实现快速细胞动力学的成像，需要在所有维度上具有高分辨率、高成像速度、良好的物理覆盖范围和低光损伤。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，它通过同时沿四个正交方向进行光片照明和荧光检测，快速对大样本进行成像。通过高通量多视图反卷积对这四个视图进行组合，可在所有三个维度上获得高分辨率的图像。我们在果蝇幼虫中以 1.1-2.5μm 的空间分辨率和数小时的 2Hz 时间分辨率进行了整个动物的功能成像。我们还在斑马鱼幼虫中进行了空间各向同性的全脑功能成像，并在正在原肠胚形成的果蝇胚胎中进行了快速细胞动力学的空间各向同性多色成像。与传统的光片显微镜相比，IsoView 显微镜将空间分辨率提高了至少 7 倍，将分辨率各向异性降低了至少 3 倍。与现有的高分辨率光片技术相比，IsoView 显微镜将有效增加穿透深度，并为之前无法成像的 400 倍大的样本提供亚秒级的时间分辨率。
