A simple and accessible method for whole-leaf stomatal mapping using a flatbed scanner

Abstract Stomata regulate plant gas exchange, photosynthesis, and transpiration. Their spatial distribution is an important functional trait, but most studies are limited to microscopic fields of view that cover only a small part of the leaf surface. Here, we present a simple scanner-based approach for mapping stomata at the whole-leaf scale. Using a 4800-dpi (189 pixels mm-1) scan of a Sonneratia apetala leaf, we identified 48,772 stomata across 645 mm2 and generated a whole-leaf stomatal distribution map. We validated scanner-derived stomatal locations against light microscopy using 853 matched stomata. Pairwise distances derived from the two methods showed strong agreement, with an R2 = 0.983. This method provides a low-cost and accessible way to map stomatal abundance, density, and spatial distribution in leaves with suitable epidermal features. We also developed a preliminary semi-automated workflow for candidate stomatal detection and localization. We emphasize that this approach is not designed to measure stomatal area, guard-cell size, or pore aperture, and that it should be validated with microscopy before use in new species. Although the method is not suitable for all leaf types, this scanner-based framework can extend stomatal research beyond local microscopic observations and support future studies of leaf functional traits.