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、非生物胁迫或有益或有害微生物的存在都可以触发改变基因表达、触发生化反应和改变生长模式的反应。这些反应涉及整合信号的受体、驱动特定基因活性的激素、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。在真实的时间内记录这些反应对于理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，这可能发生在几分钟到几小时到几天，是由“之前和之后”的图像记录。通常采用此选项是因为重复对样本进行成像，特别是当需要将样本从受控环境中取出或记录在触发触摸或重力反应的位置时，可能会产生严重的副作用，使准确测量生理反应变得具有挑战性。** 该提案概述了一个新的研究平台，该平台优化了立体荧光显微镜，用于在受控环境和未扰动位置下对植物生长反应进行长期成像。该系统的关键创新是一个垂直阶段。尽管立体显微镜的设计是为了用放置在水平台上的样品上方的透镜观察样品，但植物器官--特别是根和茎--的反应是分别向下或向上弯曲，甚至不可能进行短期成像。为了克服这个问题，我们的解决方案是将显微镜翻转，使载物台垂直放置。植物将以自然方向生长，以便在它们发生时和长时间内捕获它们的反应。电动载物台将用于随时间调整器官生长，并最大限度地提高多个标本的高通量采集。显微镜的设计优化了荧光检测，使基因表达，激素传感器和蛋白质分布模式可以在空间和时间上记录的生长参数。为了使植物在长时间的培育期内保持健康状态，显微镜和标本将被安置在一个环境室中，该环境室将允许调节气体、温度和湿度。通过这些创新，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.**