A vertical stage stereofluorescence microscope for documenting plant responses to environmental signals

对于植物来说，表现和生存取决于它们精致的发育可塑性。不同的环境条件、非生物压力或有益或有害微生物的存在都可以触发反应，改变基因表达，触发生化反应，并改变生长模式。这些反应涉及整合信号的受体、驱动特定基因活动的激素、产生化学防御的代谢途径，以及协调生长方向和形态发生变化的细胞骨架系统。实时记录这些反应对于了解植物优化其发育的机制至关重要。然而，通常情况下，这些反应可能会在几分钟到几小时到几天的时间内发生，并通过“之前和之后”的图像进行记录。这一选择通常被采用，因为对标本的重复成像，特别是当它们需要从受控环境中移走或记录在触发触摸或重力反应的位置时，可能会产生深远的副作用，使准确测量生理反应具有挑战性(如果不是不可能的话)。*这项建议概述了一个新的研究平台，它优化了立体荧光显微镜，用于在受控环境和不受干扰的位置下对植物生长反应进行长期成像。这一制度的关键创新是垂直舞台。立体显微镜的设计是为了让人们用放在水平舞台上的标本上方的透镜来观察标本，而植物器官--尤其是根和茎--分别通过向下或向上弯曲来做出反应，使得即使是短期成像也不可能。为了克服这一点，我们的解决方案是将显微镜倒置，使舞台垂直放置。植物将以它们的自然方位生长，这样它们的反应就可以在发生时被捕捉到，并在很长一段时间内被捕捉到。机动化阶段将用于随着时间的推移调整器官生长，并最大限度地从多个样本中获得高通量数据。显微镜设计优化了荧光检测，以便可以在空间和时间上记录与生长参数相关的基因表达、激素传感器和蛋白质分布模式。为了长期保持植物的健康状态，显微镜和标本将被安置在一个环境室内，允许调节气体、温度和湿度。有了这些创新，研究人员将能够记录和量化植物在接近模拟其正常生长条件的条件下如何对不同的环境信号做出反应。

For plants, performance and survival depends on their exquisite developmental plasticity. Varied environmental conditions, abiotic stress, or the presence of beneficial or harmful microbes can all trigger responses that alter gene expression, trigger biochemical reactions, and modify growth patterns. These responses involve receptors that integrate signals, hormones that drive specific gene activity, metabolic pathways that produce chemical defenses, and cytoskeletal systems that coordinate changes in growth directions and morphogenesis. Documenting these responses in real time is critical to understanding the mechanisms by which plants optimize their development. Typically, however, these responses, which can take place over minutes to hours to days, are documented by "before and after" images. This option is usually adopted because repeated imaging of specimens, especially when they need to be removed from a controlled environment or documented in positions that trigger touch or gravity responses, can have profound side effects, making it challenging if not impossible to measure physiological responses accurately. ******This proposal outlines a new research platform that optimizes stereo-fluorescence microscopy for long-term imaging of plant growth responses under controlled environments and unperturbed positions. The key innovation to this system is a vertical stage. Whereas stereo microscopes are designed so that specimens are viewed with the lens above the samples, which are placed on a horizontal stage, plant organ - especially roots and stems - respond by bending downward or upward respectively, rendering impossible even short-term imaging. To overcome this, our solution is to turn the microscope on its back so that the stage is positioned vertically. Plants will be grown in their natural orientation so that their responses can be captured as they occur and over long periods. The motorized stage will be used to adjust for organ growth over time and maximize high throughput acquisition from multiple specimens. The microscope design optimizes fluorescence detection so that gene expression, hormone sensors, and protein distribution patterns can be spatially and temporally documented in relation to the growth parameters. To keep the plants in a healthy state for long incubation periods, the microscope and specimens will be housed in an environmental chamber that will allow for gas, temperature and humidity to be regulated. With these innovations, researchers will be able to document and quantify how plants respond to different environmental signals under conditions that closely simulate their normal growing conditions.**