Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.
Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.
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DOI：
10.1091/mbc.e14-08-1287
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发表时间：
2015-05-01
影响因子：
3.3
通讯作者：
Okada Y
中科院分区：
文献类型：
--
作者：
Hayashi S;Okada Y
介绍了一种以自旋盘共聚焦显微镜为基础的新型超分辨荧光显微镜。一种特殊设计的磁盘模式将分辨率提高了一倍，达到120纳米，时间分辨率为100赫兹，这对于实时成像来说足够快。目前大多数超分辨率（SR）显微镜技术以牺牲时间分辨率为代价超过了衍射极限，从而影响了它们在活细胞成像中的应用。本文介绍了一种基于共聚焦显微镜光学原理的新型SR荧光显微镜，我们将其命名为自旋盘超分辨显微镜（SDSRM）。理论上，SDSRM相当于一个结构照明显微镜（SIM），达到120 nm的空间分辨率，是宽视场荧光显微镜衍射极限的两倍。然而，SDSRM比传统SIM快10倍，因为SR信号是通过磁盘的条纹模式通过光学解调恢复的。因此，单个SR图像只需要通过旋转磁盘的单个平均图像。在此理论的基础上，我们改进了商用旋转盘共聚焦显微镜。生物样品证实了120 nm左右分辨率的提高。观察微管、线粒体、溶酶体和核内体的快速动态，时间分辨率为30-100帧/秒。因为我们的方法只需要很小的光学修改，它可以很容易地从现有的旋转盘共聚焦显微镜升级到用于活细胞成像的SR显微镜。
A new superresolution fluorescence microscope is described that is based on the spinning disk confocal microscope. A specially designed pattern of disk doubles the resolving power to 120 nm with 100-Hz temporal resolution, which is fast enough for live imaging. Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of microtubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30–100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging.