Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination
Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination
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DOI：
10.1529/biophysj.107.120345
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发表时间：
2008-06-15
影响因子：
3.4
通讯作者：
Sedat, John W.
中科院分区：
文献类型：
--
作者：
Gustafsson, Mats G. L.;Shao, Lin;Sedat, John W.
结构照明显微术是一种利用空间结构照明光提高宽视场荧光显微术空间分辨率的方法。在这里，我们描述了这种方法如何可以应用于三维轴向以及横向分辨率的两倍，真正的光学切片。光栅用于产生三个相互相干的光束，其在样品中干涉以形成横向和轴向变化的照明图案。空间结构化的激发强度导致通常不可达的高分辨率信息通过空间频率混合被编码到观察到的图像中。这种新的信息是计算提取和用于生成一个三维重建与两倍的高分辨率，在所有三个维度，是可能的，在传统的宽视场显微镜。该方法已被证明在测试对象和生物标本，并产生了第一个光学显微镜图像的联会复合体中的横向元素被清楚地解决。
Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.