Kirchhelle Charlotte, Moore Ian
Department of Plant Sciences, University of Oxford.
Department of Plant Sciences, University of Oxford;
J Vis Exp. 2017 May 17(123):55331. doi: 10.3791/55331.
Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.
植物发育的几个方面，如侧根形态发生，会在几天的时间跨度内发生。为了研究潜在的细胞和亚细胞过程，需要能够保持生理条件的高分辨率延时显微镜技术。植物组织必须有充足的养分和水分供应，并持续进行气体交换，但是当它们被淹没并固定在盖玻片下时，特别容易缺氧。一种已成功采用的策略是使用灌注系统来维持氧气和养分的持续供应。然而，这种装置可能很复杂、麻烦，并且需要专门的设备。这里介绍一种使用全氟萘烷作为浸没介质的简单成像系统的替代策略。该系统易于设置，所需设备最少，并且易于安装在显微镜载物台上，允许多个成像室并行设置和成像。在这个系统中，前两天侧根的生长速度与在琼脂平板上的标准条件下的生长速度没有区别，并且侧根生长至少还会以降低的速度持续一天。植物组织通过琼脂块获得养分，琼脂块也可用于施用一系列药理化合物。该系统是为监测侧根发育而建立的，但很容易适用于对其他植物器官如胚轴和主根进行成像。
