Staier Florian, Eipel Heinz, Matula Petr, Evsikov Alexei V, Kozubek Michal, Cremer Christoph, Hausmann Michael
Kirchhoff Institute for Physics, University of Heidelberg, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany.
Rev Sci Instrum. 2011 Sep;82(9):093701. doi: 10.1063/1.3632115.
With the development of novel fluorescence techniques, high resolution light microscopy has become a challenging technique for investigations of the three-dimensional (3D) micro-cosmos in cells and sub-cellular components. So far, all fluorescence microscopes applied for 3D imaging in biosciences show a spatially anisotropic point spread function resulting in an anisotropic optical resolution or point localization precision. To overcome this shortcoming, micro axial tomography was suggested which allows object tilting on the microscopic stage and leads to an improvement in localization precision and spatial resolution. Here, we present a miniaturized device which can be implemented in a motor driven microscope stage. The footprint of this device corresponds to a standard microscope slide. A special glass fiber can manually be adjusted in the object space of the microscope lens. A stepwise fiber rotation can be controlled by a miniaturized stepping motor incorporated into the device. By means of a special mounting device, test particles were fixed onto glass fibers, optically localized with high precision, and automatically rotated to obtain views from different perspective angles under which distances of corresponding pairs of objects were determined. From these angle dependent distance values, the real 3D distance was calculated with a precision in the ten nanometer range (corresponding here to an optical resolution of 10-30 nm) using standard microscopic equipment. As a proof of concept, the spindle apparatus of a mature mouse oocyte was imaged during metaphase II meiotic arrest under different perspectives. Only very few images registered under different rotation angles are sufficient for full 3D reconstruction. The results indicate the principal advantage of the micro axial tomography approach for many microscopic setups therein and also those of improved resolutions as obtained by high precision localization determination.
随着新型荧光技术的发展，高分辨率光学显微镜已成为研究细胞和亚细胞成分中三维（3D）微观世界的一项具有挑战性的技术。到目前为止，所有应用于生物科学3D成像的荧光显微镜都显示出空间各向异性的点扩散函数，导致光学分辨率或点定位精度各向异性。为克服这一缺点，有人提出了微轴断层扫描技术，该技术允许在显微镜载物台上倾斜样品，从而提高定位精度和空间分辨率。在此，我们展示了一种可安装在电动显微镜载物台上的小型化设备。该设备的占地面积相当于一张标准显微镜载玻片。一根特殊的玻璃纤维可在显微镜物镜的物空间中手动调节。通过内置在设备中的小型步进电机可控制玻璃纤维的逐步旋转。借助特殊的安装装置，将测试颗粒固定在玻璃纤维上，进行高精度光学定位，并自动旋转以从不同视角获取图像，在此视角下确定相应物体对之间的距离。利用标准显微镜设备，根据这些与角度相关的距离值，计算出实际的3D距离，精度在十纳米范围内（此处对应10 - 30 nm的光学分辨率）。作为概念验证，在不同视角下对处于减数分裂中期II停滞期的成熟小鼠卵母细胞的纺锤体装置进行了成像。仅需在不同旋转角度下记录的极少数图像就足以进行完整的3D重建。结果表明了微轴断层扫描方法在许多显微镜设置中的主要优势，以及通过高精度定位测定所获得的分辨率提升的优势。
