A tilting device for three‐dimensional microscopy: Application to in situ imaging of interphase cell nuclei

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Abstract
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°, 90° and 180°; the volumes and positions of the labelled regions (spots) were calculated. The results also confirm that, in principle, any angle of a 2p geometry can be fixed for data acquisition with a high reproducibility. This indicates the feasibility of axiotomographical microscopy of cell nuclei.