Kozubek M, Skalníková M, Matula Pe, Bártová E, Rauch J, Neuhaus F, Eipel H, Hausmann M
Laboratory of Optical Microscopy, Faculty of Informatics, Masaryk University, Botanická 68a, CZ-60200 Brno, Czech Republic.
Micron. 2002;33(7-8):655-65. doi: 10.1016/s0968-4328(02)00023-9.
Microaxial tomography provides a good means for microscopic image acquisition of cells or sub-cellular components like cell nuclei with an improved resolution, because shortcomings of spatial resolution anisotropy in optical microscopy can be overcome. Thus, spatial information of the object can be obtained without the necessity of confocal imaging. Since the very early developments of microaxial tomography, a considerable drawback of this method was a complicated image acquisition and processing procedure that requires much operator time. In order to solve this problem the Heidelberg 2pi-tilting device has been mounted on the Brno high-resolution cytometer as an attempt to bring together advanced microscopy and fast automated computer image acquisition and analysis. A special software module that drives all hardware components required for automated microaxial tomography and performs image acquisition and processing has been developed. First, a general image acquisition strategy is presented. Then the procedure for automation of axial tomography and the developed software module are described. The rotation precision has been experimentally proved followed by experiments with a specific biological example. For this application, also a method for the preparation of cell nuclei attached to glass fibres has been developed that allows for the first time imaging of three-dimensionally conserved, fluorescence in situ hybridisation-stained cell nuclei fixed to a glass fibre.
微轴断层扫描为细胞或细胞核等亚细胞成分的微观图像采集提供了一种良好手段，其分辨率有所提高，因为光学显微镜中空间分辨率各向异性的缺点可以被克服。因此，无需共聚焦成像就能获得物体的空间信息。自微轴断层扫描早期发展以来，该方法的一个相当大的缺点是图像采集和处理过程复杂，需要操作人员花费大量时间。为了解决这个问题，已将海德堡2π倾斜装置安装在布尔诺高分辨率细胞仪上，试图将先进的显微镜技术与快速自动计算机图像采集和分析结合起来。已开发出一个特殊的软件模块，该模块驱动自动微轴断层扫描所需的所有硬件组件，并执行图像采集和处理。首先，介绍一种通用的图像采集策略。然后描述轴向断层扫描自动化的过程以及所开发的软件模块。通过实验证明了旋转精度，随后以一个具体的生物学实例进行了实验。对于该应用，还开发了一种制备附着在玻璃纤维上的细胞核的方法，首次实现了对固定在玻璃纤维上的三维保存的荧光原位杂交染色细胞核的成像。
