Shaw P J
Department of Cell Biology, John Innes Institute, Norwich, U.K.
J Microsc. 1990 May;158(Pt 2):165-72. doi: 10.1111/j.1365-2818.1990.tb02989.x.
The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the x-y plane. This is true of conventional or confocal microscopes. To alleviate this problem we used multiple tilted views to supply the 'missing data' and thus increase the resolution in z. A special tilting stage was constructed which allowed specimens to be rotated through large angles. The relative, translation, rotation and z-spacing between data sets were determined by a novel Wiener/phase cross-correlation function. Once brought to a common coordinate system the data sets can be combined by Fourier space techniques similar to those used in X-ray crystallography. We applied this technique to metaphase chromosomes from intact embryos of Drosophila melanogaster. As determined from significant intensity in the Fourier transform, the resolution of the final reconstruction was about 0.25 micron in x and y, and 0.4 micron in z.
光学显微镜在光轴（z）方向上的分辨率比在x - y平面上要低得多。传统显微镜或共聚焦显微镜都是如此。为了缓解这个问题，我们使用多个倾斜视角来提供“缺失数据”，从而提高z方向上的分辨率。我们构建了一个特殊的倾斜载物台，它能使标本进行大幅度旋转。数据集之间的相对平移、旋转和z轴间距通过一种新颖的维纳/相位互相关函数来确定。一旦将数据集置于一个公共坐标系中，就可以通过类似于X射线晶体学中使用的傅里叶空间技术将它们合并起来。我们将这项技术应用于黑腹果蝇完整胚胎的中期染色体。根据傅里叶变换中的显著强度确定，最终重建的分辨率在x和y方向约为0.25微米，在z方向为0.4微米。
