Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Methods Mol Biol. 2024;2805:213-228. doi: 10.1007/978-1-0716-3854-5_15.
Imaging the spatiotemporal dynamics of host-microbiota interactions is of particular interest for augmenting our understanding of these complex systems. This is especially true of plant-microbe interactions happening around, on, and inside plant roots where relatively little is understood about the dynamics of these systems. Over the past decade, a number of microfluidic devices have been developed to grow plants hydroponically in gnotobiotic conditions and image morphogenesis of the root and/or dynamics with fluorescently labeled bacteria from the plant root microbiome. Here we describe the construction and use of our Arabidopsis Root Microbiome Microfluidic (ARMM) device for imaging fluorescent protein expressing bacteria and their colonization of Arabidopsis roots. In contrast to other plant root imaging devices, we designed this device to have a larger chamber for observing Arabidopsis root elongation and plant-microbe interactions with older seedlings (between 1.5 and 4 weeks after germination) and a 200 μm chamber depth to specifically maintain thin Arabidopsis roots within the focal distance of the confocal microscope. Our device incorporates a new approach to growing Arabidopsis seedlings in screw-top tube caps for simplified germination and transfer to the device. We present representative images from the ARMM device including high resolution cross section images of bacterial colonization at the root surface.
研究宿主-微生物相互作用的时空动态对于增强我们对这些复杂系统的理解特别有意义。在植物-微生物相互作用方面更是如此，这些相互作用发生在植物根部周围、表面和内部，而对于这些系统的动态，我们相对了解较少。在过去的十年中，已经开发出许多微流控设备，用于在无菌条件下水培种植植物，并对根的形态发生和/或用植物根微生物组中的荧光标记细菌进行成像。在这里，我们描述了 Arabidopsis Root Microbiome Microfluidic (ARMM) 设备的构建和使用，用于对表达荧光蛋白的细菌及其对拟南芥根的定殖进行成像。与其他植物根成像设备不同，我们设计该设备的目的是拥有更大的腔室来观察拟南芥根的伸长和植物-微生物相互作用，同时也可以容纳年龄较大的幼苗（萌发后 1.5 到 4 周之间），并具有 200μm 的腔室深度，以专门将薄的拟南芥根保持在共聚焦显微镜的焦平面内。我们的设备采用了一种新的方法，即在螺旋盖管帽中种植拟南芥幼苗，以简化萌发和转移到设备中的过程。我们展示了来自 ARMM 设备的代表性图像，包括根表面细菌定殖的高分辨率横截面图像。
