Root phenotype analysis is a challenging task that requires monitoring the growth of roots in darkness to simulate natural conditions while allowing the above-ground parts to grow under light. Most existing methods involve exposing the roots to light, which significantly alters their growth and function. In this paper, we propose an improved imaging system that can overcome this limitation present in laboratory settings. The Dynamic Dark Root Chamber (DDrC) is capable of continuously monitoring and capturing images to track the dynamic development of root structures under controlled growth conditions. Our imaging system is based on a Raspberry Pi camera module and infrared LEDs, which do not induce any stress responses in the roots. The DDrC is easy to set up, reasonably priced, and suitable for dynamic phenotyping experiments. We provide a detailed tutorial on assembling and adjusting the imaging chamber. The results indicate that our system is a valuable tool for studying the genetic and environmental factors affecting root structure and development, and for identifying root traits associated with plant adaptability and performance.

Figure 1 An imaging chamber powered by Raspberry Pi and equipped with infrared LEDs for precise monitoring and analysis of dynamic plant growth and root development. A) The image outlines the basic components inside the imaging chamber. The structural framework serves as the main support for the entire device and can be individually adjusted based on materials and dimensions. The infrared LEDs emit light at 880nm, allowing for the acquisition of root images obscured by white light without affecting their physiology. The integrated camera is positioned within the body relative to the sample. The Raspberry Pi is placed outside the imaging chamber, serving as the computational center for data processing and control. B) A detailed component diagram shows each component and how they are interconnected. It illustrates the electrical connections of the camera, infrared LEDs, and Raspberry Pi, indicating their physical arrangement within the chamber. C) In this view, the imaging chamber is closed, enclosing a petri dish filled with soil, serving as a small root box. The root box is positioned so that seedlings can grow out of the imaging chamber, allowing for normal lighting in any growth chamber. The display screen of the Raspberry Pi is visible at the front, showing images captured from the roots. D) The image displays the internal components of the Raspberry Pi computer module. E) The image zooms in on the camera module inserted into the body, further showcasing the cables required to connect the camera module and infrared LEDs to the Raspberry Pi, including the detailed wiring needed for seamless operation.

Figure 2 A) shows selected images of barley seedlings taken from a time series (captured at 10-minute intervals, representing a 48-hour growth cycle). The dynamic changes in root angle, number of seminal roots, and root growth rate can be evaluated simultaneously. Further features that are easily extracted include the growth rate of the coleoptile and adaptability to different soil textures and compositions. B) The images obtained from the imaging chamber are suitable for batch segmentation using RhizoVision Explorer. The segmentation process is more effective when the soil is darker and has lower humidity, resulting in a high contrast level between the barley roots and the background. C) The images capture the emergence of barley cotyledons. The imaging chamber allows for the above-ground parts to be exposed to light for long-term experiments. D) The image shows the roots growing after 5 days of germination in the imaging chamber. The images are captured under a few seconds of infrared light (880nm). E) The imaging setup allows for continuous assessment of the growth status of germinating seedlings. The touchscreen can select captured images, providing qualitative control over the experimental setup during experiments. Depending on the selected size of the imaging chamber, growth dynamics can be observed continuously over several days to weeks without interfering with seedling growth.

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