Stomatalia: a deep learning-based platform for quantitative stomata and pavement cell analysis

Stomata and pavement cells are fundamental components of the leaf epidermis, jointly regulating gas exchange, water loss, and leaf surface expansion. Stomata size, aperture, density, and pavement cell morphology are critical parameters for assessing plant transpiration efficiency, epidermal growth dynamics, and adaptive responses to environmental constraints. Despite their biological importance, quantifying stomatal and pavement-cell traits remains seldom not generalized, simple, and fast enough . Manual or semi-automated approaches limit large-scale phenotyping and restrict the integration of epidermal morphology into crop-improvement pipelines aimed at developing climate-resilient varieties with optimised stomatal patterning. To address such limitations, we developed Stomatalia, a deep learning-based platform designed to automate and standardise the quantification of stomatal and pavement cell traits. The algorithm was trained on epidermal images of cultivated and wild potato and tomato genotypes grown under optimal and abiotic-stress conditions. Stomatalia automatically detects stomata and pavement cells and extracts a broad range of morphological and integrative epidermal parameters, enabling high-throughput phenotyping within a unified workflow.

Prior to platform development, we optimised a rapid, minimally-destructive leaf-printing protocol that generates negative impressions of the leaf surface within 40-100 s. Transparent positive prints were subsequently produced and imaged under a light microscope at scale settings ranging from 20 to 200 μm. The resulting images are analysed using Stomatalia's user-friendly web-based interface, which runs an instance-segmentation deep learning algorithm to detect, count, and calculate stomatal and pavement cell parameters. The platform outputs structured files containing raw measurements, derived integrative traits, and associated metadata, facilitating downstream statistical and physiological analyses. Algorithm evaluation on independent datasets demonstrated high performance within the validated dicot imaging domain, with F₁-scores ranging from 0.86 to 0.94 depending on image scale, species, and resolution, and high segmentation overlap for both stomata and pavement cells. The generality of stomatal detection was also tested on spring onion, chickpea, balsam poplar, and wheat in cross-species feasibility tests, although performance was more variable in monocots, and pavement-cell segmentation remained species- and architecture-dependent. Benchmarking against another publicly available app further showed that, under the tested web interface settings and image types, Stomatalia exhibited closer agreement with manual counts and substantially faster processing times. The practical performance of Stomatalia was further tested in a proof-of-concept trial with potato plants subjected to optimal irrigation and a long, gradual drought. The platform reliably quantified epidermal traits despite variations in leaf morphology and image quality, supporting the integrated interpretation of stomatal and pavement-cell responses under stress.

We developed Stomatalia as a robust, user-friendly deep learning platform for automated, high-throughput analysis of bright-field leaf epidermal images across varying magnifications and resolutions. Stomatalia facilitates rapid, reproducible, and coordinated phenotyping of stomatal and pavement cells by integrating methodological standardisation, computational automation, and multi-trait extraction in a single analytical workflow. Its strongest current application is the analysis of high-quality dicot leaf-print images, particularly in species and imaging conditions similar to those used for model training and validation. Cross-species and benchmark analyses further define its current scope: stomatal detection can be transferred to some additional epidermal architectures, whereas robust pavement-cell segmentation in monocots or highly divergent species will require further annotation and model retraining. Within these defined boundaries, Stomatalia provides a flexible and extensible framework for studying stomatal and pavement cell morphology and environmental plasticity, while also supporting broader efforts to dissect and optimise plant responses to abiotic stress.