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

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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.**