De Mey J R, Kessler P, Dompierre J, Cordelières F P, Dieterlen A, Vonesch J-L, Sibarita J-B
Ecole Supérieure de Biotechnologie de Strasbourg, UMR-7175 CNRS/Université Louis Pasteur (Strasbourg I), BP10413, 67412 IllKIRCH Cedex, France.
Methods Cell Biol. 2008;85:83-112. doi: 10.1016/S0091-679X(08)85005-4.
Many cellular processes involve fast movements of weakly labeled cellular structures in all directions, which should be recorded in 3D time-lapse microscopy (4D microscopy). This chapter introduces fast 4D imaging, which is used for sampling the cell's volume by collecting focal planes in time-lapse mode as rapidly as possible, without perturbing the sample by strong illumination. The final images should contain sufficient contrast allowing for the isolation of structures of interest by segmentation and the analysis of their intracellular movements by tracking. Because they are the most sensitive, systems using wide-field microscopy and deconvolution techniques are discussed in greater depth. We discuss important points to consider, including system components and multifunctionality, spatial resolution and sampling conditions, and mechanical and optical stability and how to test for it. We consider image formation using high numerical aperture optics and discuss the influence of optical blur and noise on image formation of living cells. Spherical aberrations, their consequences for axial image quality, and their impact on the success of deconvolution of low intensity image stacks are explained in detail. Simple protocols for acquiring and treating point spread functions (PSFs) and live cells are provided. A compromise for counteracting spherical aberration involving the use of a kit of immersion oils for PSF and cell acquisition is illustrated. Recommendations for evaluating acquisition conditions and deconvolution parameters are given. Finally, we discuss future developments based on the use of adaptive optics which will push back many of today's limits.
许多细胞过程涉及弱标记细胞结构在各个方向上的快速移动，这些过程需要在三维延时显微镜（四维显微镜）中进行记录。本章介绍快速四维成像，它通过在延时模式下尽可能快速地采集焦平面来对细胞体积进行采样，同时不会因强光照射而干扰样本。最终图像应具有足够的对比度，以便通过分割分离出感兴趣的结构，并通过跟踪分析其细胞内运动。由于使用宽场显微镜和去卷积技术的系统最为灵敏，因此将对其进行更深入的讨论。我们将讨论需要考虑的要点，包括系统组件和多功能性、空间分辨率和采样条件，以及机械和光学稳定性及其测试方法。我们将考虑使用高数值孔径光学器件的图像形成，并讨论光学模糊和噪声对活细胞图像形成的影响。将详细解释球差、其对轴向图像质量的影响以及对低强度图像堆栈去卷积成功的影响。还将提供获取和处理点扩散函数（PSF）及活细胞的简单方案。展示了一种通过使用一套用于PSF和细胞采集的浸油来抵消球差的折衷方法。给出了评估采集条件和去卷积参数的建议。最后，我们将讨论基于自适应光学的未来发展，这将突破当今的许多限制。
