Bradl J, Hausmann M, Ehemann V, Komitowski D, Cremer C
Institute of Applied Physics, University of Heidelberg, Germany.
J Microsc. 1992 Oct;168(Pt 1):47-57. doi: 10.1111/j.1365-2818.1992.tb03249.x.
The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the focal plane (x-y plane). This is true of conventional as well as confocal microscopes. For quantitative microscopy, for instance studies of the three-dimensional (3-D) organization of chromosomes in human interphase cell nuclei, the 3-D image must be reconstructed by a point spread function or an optical transfer function with careful consideration of the properties of the imaging system. To alleviate the reconstruction problem, a tilting device was developed so that several data sets of the same cell nucleus under different views could be registered. The 3-D information was obtained from a series of optical sections with a Zeiss transmission light microscope Axiomat using a stage with a computer-controlled stepping motor for movement in the z-axis. The tilting device on the Axiomat stage could turn a cell nucleus through any desired angle and also provide movement in the x-y direction. The technique was applied to 3-D imaging of human lymphocyte cell nuclei, which were labelled by in situ hybridization with the DNA probe pUC 1.77 (mainly specific for chromosome 1). For each nucleus, 3-D data sets were registered at viewing angles of 0 degrees, 90 degrees and 180 degrees; the volumes and positions of the labelled regions (spots) were calculated. The results also confirm that, in principle, any angle of a 2 pi geometry can be fixed for data acquisition with a high reproducibility. This indicates the feasibility of axiotomographical microscopy of cell nuclei.
光学显微镜在光轴方向（z轴）上的分辨率远低于焦平面（x-y平面）。传统显微镜和共聚焦显微镜都是如此。对于定量显微镜检查，例如对人类间期细胞核中染色体三维（3-D）组织的研究，必须通过点扩散函数或光学传递函数来重建3-D图像，并仔细考虑成像系统的特性。为了缓解重建问题，开发了一种倾斜装置，以便可以记录同一细胞核在不同视角下的多个数据集。使用带有计算机控制步进电机的载物台在z轴上移动的蔡司透射光显微镜Axiomat，从一系列光学切片中获取3-D信息。Axiomat载物台上的倾斜装置可以将细胞核旋转到任何所需角度，还能在x-y方向上移动。该技术应用于人类淋巴细胞细胞核的3-D成像，这些细胞核通过与DNA探针pUC 1.77（主要对1号染色体具有特异性）的原位杂交进行标记。对于每个细胞核，在0度、90度和180度的视角下记录3-D数据集；计算标记区域（斑点）的体积和位置。结果还证实，原则上可以固定2π几何形状的任何角度进行数据采集，且具有很高的重现性。这表明细胞核轴断层显微镜检查的可行性。
