ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.
ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.
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高级成像 内吞和细胞骨架动力学的扩展分辨率结构照明成像
DOI：
10.1126/science.aab3500
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发表时间：
2015-08-28
期刊：
影响因子：
--
通讯作者：
Betzig E
中科院分区：
文献类型：
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作者：
Li D;Shao L;Chen BC;Zhang X;Zhang M;Moses B;Milkie DE;Beach JR;Hammer JA 3rd;Pasham M;Kirchhausen T;Baird MA;Davidson MW;Xu P;Betzig E
超分辨率荧光显微镜在纳米成像工具中的独特之处在于它能够成像活细胞中的蛋白质动力学。在这方面，结构照明显微镜(SIM)因其高速和低照度而脱颖而出，但通常只提供两倍的分辨率增益。我们通过两种方法扩展了活细胞SIM的分辨率：84纳米横向分辨率的超高数值孔径SIM用于100多个多色帧，以及图案化激活的45到62纳米分辨率的非线性SIM大约20到40帧。我们将这些方法应用到细胞膜附近的图像动力学中，这些细胞膜是由网状蛋白和小窝蛋白、早期内体中的Rab5a和a-肌动蛋白组成的空间分辨组件，通常与皮质肌动蛋白有关。此外，我们还研究了线粒体、肌动蛋白和高尔基体的三维动力学。
Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and a-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.