An Integrated Method for Tracking and Monitoring Stomata Dynamics from Microscope Videos

Patchy stomata are a common and characteristic phenomenon in plants. Understanding and studying the regulation mechanism of patchy stomata are of great significance to further supplement and improve the stomatal theory. Currently, the common methods for stomatal behavior observation are based on static images, which makes it difficult to reflect dynamic changes of stomata. With the rapid development of portable microscopes and computer vision algorithms, it brings new chances for stomatal movement observation. In this study, a stomatal behavior observation system (SBOS) was proposed for real-time observation and automatic analysis of each single stoma in wheat leaf using object tracking and semantic segmentation methods. The SBOS includes two modules: the real-time observation module and the automatic analysis module. The real-time observation module can shoot videos of stomatal dynamic changes. In the automatic analysis module, object tracking locates every single stoma accurately to obtain stomatal pictures arranged in time-series; semantic segmentation can precisely quantify the stomatal opening area (SOA), with a mean pixel accuracy (MPA) of 0.8305 and a mean intersection over union (MIoU) of 0.5590 in the testing set. Moreover, we designed a graphical user interface (GUI) so that researchers could use this automatic analysis module smoothly. To verify the performance of the SBOS, the dynamic changes of stomata were observed and analyzed under chilling. Finally, we analyzed the correlation between gas exchange and SOA under drought stress, and the correlation coefficients between mean SOA and net photosynthetic rate (Pn), intercellular CO₂ concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr) are 0.93, 0.96, 0.96, and 0.97.