Shedding new light on the hidden complexity of seeds: chemically selective imaging of seed coats with stimulated Raman scattering microscopy

The seed coat plays a pivotal role in seed development and germination, acting as a protective barrier and mediating interac-tions with the external environment. Traditional histochemical techniques and analytical methods have provided valuable insights into seed coat composition and function. However, these methods often suffer from limitations such as indirect chemical signatures and lack of spatial resolution. Here, we introduce stimulated Raman scattering (SRS) microscopy as a novel analytical tool for non-destructive, label-free, high-resolution mapping of biopolymers, water and applied active ingre-dients (AIs) in intact seed coats. We demonstrate the capability of SRS microscopy to perform depth-resolved, chemically selective imaging of major seed coat biopolymers (pectin, tannin, and suberin). By comparing wild type arabidopsis thali-ana seeds with genetically modified mutants deficient in suberin and tannin, we illustrate the potential for semi-quantitative analysis of biopolymer content. Furthermore, we show that SRS microscopy can track the permeability of seed coats to wa-ter using deuterated water (D2O) uptake studies. Real-time imaging reveals differences in water permeation between wild type and suberin deficient seeds, highlighting the importance of seed coat composition in regulating water uptake during germina-tion. Additionally, we extend the application of SRS microscopy to large seeds, such as brassica oleracea, utilizing epi-detected imaging for surface studies. Finally, using a deuterated insecticide (clothianidin-d3), we demonstrate the capability of SRS microscopy to visualize the incorporation of AIs into seed coats. Our study presents SRS microscopy as a powerful tool for characterizing seed coat composition and understanding the diffusion of low molecular weight compounds into seeds. This technique offers new opportunities for designing seeds with tailored properties for improved germination and resilience to environmental stressors.