Background Understanding the environmental impacts on root growth and root health is essential for effective agricultural and environmental management. Hyperspectral imaging (HSI) technology provides a non-destructive method for detailed analysis and monitoring of plant tissues and organ development, but unfortunately examples for its application to root systems and the root-soil interface are very scarce. There is also a notable lack of standardized guidelines for image acquisition and data analysis pipelines.

Methods This study investigated HSI techniques for analyzing rhizobox-grown root systems across various imaging configurations, from the macro- to micro-scale, using the imec VNIR SNAPSCAN camera. Focusing on three graminoid species with different root architectures allowed us to evaluate the influence of key image acquisition parameters and data processing techniques on the differentiation of root, soil, and root-soil interface/rhizosheath spectral signatures. We compared two image classification methods, Spectral Angle Mapper (SAM) and K-Means clustering, and two machine learning approaches, Random Forest (RF) and Support Vector Machine (SVM), to assess their efficiency in automating root system image classification.

Results Our study demonstrated that training a RF model using SAM classifications, coupled with wavelength reduction using the second derivative spectra with Savitzky-Golay (SG) smoothing, provided reliable classification between root, soil, and the root-soil interface, achieving 88-91% accuracy across all configurations and scales. Although the root-soil interface was not clearly resolved, it helped to improve the distinction between root and soil classes. This approach effectively highlighted spectral differences resulting from the different configurations, image acquisition settings, and among the three species. Utilizing this classification method can facilitate the monitoring of root biomass and future work investigating root adaptations to harsh environmental conditions.

Conclusions Our study addressed the key challenges in HSI acquisition and data processing for root system analysis and lays the groundwork for further exploration of VNIR HSI application across various scales of root system studies. This work provides a full data analysis pipeline that can be utilized as an online Python-based tool for the semi-automated analysis of root-soil HSI data.