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

用于记录植物对环境信号的反应的立式立体荧光显微镜

• 批准号: RTI-2020-00469
• 负责人: Wasteneys, Geoffrey
• 金额: $ 10.52万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=693584
• 关键词: vertical; stage; stereofluorescence; microscope; documenting

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.**

对于植物来说，性能和生存取决于其精致的发育可塑性。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引发改变基因表达、引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。实时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，这些反应可能会发生几分钟、几小时甚至几天，并通过“之前和之后”图像记录。通常采用此选项是因为对样本进行重复成像，特别是当它们需要从受控环境中移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，这使得准确测量生理反应即使不是不可能也具有挑战性。 ******该提案概述了一个新的研究平台，该平台优化立体荧光显微镜，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。该系统的关键创新是垂直舞台。立体显微镜的设计是通过放置在水平平台上的样品上方的镜头来观察标本，而植物器官（尤其是根和茎）会分别向下或向上弯曲，从而导致甚至无法进行短期成像。为了克服这个问题，我们的解决方案是将显微镜翻转过来，使载物台垂直放置。植物将按照其自然方向生长，以便在其发生时和长时间内捕捉它们的反应。电动载物台将用于随着时间的推移调整器官生长，并最大限度地提高多个样本的高通量采集。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。为了使植物在较长的培养期内保持健康状态，显微镜和标本将被安置在环境室中，以调节气体、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非常模拟其正常生长条件的条件下如何响应不同的环境信号。**