Abstract:

Objective The investigation of plant photosynthetic phenotypes is essential for unlocking insights into plant physiological characteristics and dissecting morphological traits. However, traditional two-dimensional chlorophyll fluorescence imaging methods struggle to capture the complex three-dimensional spatial variations inherent in plant photosynthetic processes. To boost the efficacy of plant phenotyping and meet the increasingly demand for high-throughput analysis of photosynthetic phenotypes, the development and validation of a novel plant photosynthetic phenotype imaging system was explored, which uniquely combines three-dimensional structured light techniques with chlorophyll fluorescence technology. Methods The plant photosynthetic phenotype imaging system was composed of three primary parts: A tailored light source and projector, a camera, and a motorized filter wheel fitted with filters of various bandwidths, in addition to a terminal unit equipped with a development board and a touchscreen interface. The system was based on the principles and unique characteristics of chlorophyll fluorescence and structured light phase-shifted streak 3D reconstruction techniques. It utilized the custom-designed light source and projector, together with the camera's capability to choose specific wavelength bands, to its full potential. The system employed low-intensity white light within the 400–700 nm spectrum to elicit stable fluorescence, with blue light in the 440–450 nm range optimally triggering the fluorescence response. A projector was used to project dual-frequency, twelve-step phase-shifted stripes onto the plant, enabling the capture of both planar and stripe images, which were essential for the reconstruction of the plant's three-dimensional structure. An motorized filter wheel containing filters for red, green, blue, and near-infrared light, augmented by a filter less wheel for camera collaboration, facilitated the collection of images of plants at different wavelengths under varying lighting conditions. When illuminated with white light, filters corresponding to the red, green, and blue bands were applied to capture multiband images, resulting in color photographs that provides a comprehensive documentation of the plant's visual features. Upon exposure to blue light, the near-infrared filter was employed to capture near-infrared images, yielding data on chlorophyll fluorescence intensity. During the structured light streak projection, no filter was applied to obtain both planar and streak images of the plant, which were then employed in the 3D morphological reconstruction of the plant. The terminal, incorporating a development board and a touch screen, served as the control hub for the data acquisition and subsequent image processing within the plant photosynthetic phenotypic imaging system. It enabled the switching of light sources and the selection of camera bands through a combination of command and serial port control circuits. Following image acquisition, the data were transmitted back to the development board for analysis, processing, storage, and presentation. To validate the accuracy of 3D reconstruction and the reliability of photosynthetic efficiency assessments by the system, a prototype of the plant photosynthetic phenotypic imaging system was developed using 3D structured light and chlorophyll fluorescence technology, in accordance with the aforementioned methods, serving as an experimental validation platform. The accuracy of 3D reconstruction and the effectiveness of photosynthetic analysis capabilities of this imaging system were further confirmed through the analysis and processing of the experimental results, with comparative evaluations conducted against conventional 3D reconstruction methods and traditional chlorophyll fluorescence-based photosynthetic efficiency analyses. Results and Discussions The imaging system utilized for plant photosynthetic phenotypes incorporates a dual-frequency phase-shift algorithm to facilitate the reconstruction of three-dimensional (3D) plant phenotypes. Simultaneously, plant chlorophyll fluorescence images were employed to evaluate the plant's photosynthetic efficiency. This method enabled the analysis of the distribution of photosynthetic efficiency within a 3D space, offering a significant advancement over traditional plant photosynthetic imaging techniques. The 3D phenotype reconstructed using this method exhibits high precision, with an overall reconstruction accuracy of 96.69%. The total error was merely 3.31%, and the time required for 3D reconstruction was only 1.11 s. A comprehensive comparison of the 3D reconstruction approach presented with conventional methods had validated the accuracy of this technique, laying a robust foundation for the precise estimation of a plant's 3D photosynthetic efficiency. In the realm of photosynthetic efficiency analysis, the correlation coefficient between the photosynthetic efficiency values inferred from the chlorophyll fluorescence image analysis and those determined by conventional analysis exceeded 0.9. The experimental findings suggest a significant correlation between the photosynthetic efficiency values obtained using the proposed method and those from traditional methods, which could be characterized by a linear relationship, thereby providing a basis for more precise predictions of plant photosynthetic efficiency. Conclusions The method melds the 3D phenotype of plants with an analysis of photosynthetic efficiency, allowing for a more holistic assessment of the spatial heterogeneity in photosynthetic efficiency among plants by examining the pseudo-color images of chlorophyll fluorescence's spatial distribution. This approach elucidates the discrepancies in photosynthetic efficiency across various regions. The plant photosynthetic phenotype imaging system affords an intuitive and comprehensive view of the photosynthetic efficiency in plants under diverse stress conditions. Additionally, It provides technical support for the analysis of the spatial heterogeneity of high-throughput photosynthetic efficiency in plants.

Key words: structured light streak projection, chlorophyll fluorescence, plant phenotype, three-dimensional reconstruction, photosynthetic efficiency, heterogeneity analysis