Mapping of Leaf Pigments in Lettuce via Hyperspectral Imaging and Machine Learning

The nutritional and commercial value of lettuce (Lactuca sativa L.) is determined by its foliar pigment and phenolic composition, which varies among cultivars. This study aimed to assess the capacity of hyperspectral and applied multispectral imaging, combined with machine learning algorithms, to predict and map key biochemical traits, such as chloroplastidic pigments (chlorophylls and carotenoids) and extrachloroplastidic pigments (anthocyanins, flavonoids, and phenolic compounds). Eleven cultivars exhibiting contrasting pigmentation profiles were grown under controlled greenhouse conditions, and their chlorophyll a and b, carotenoid, anthocyanin, flavonoid, and total phenolic contents were evaluated. Spectral reflectance data were acquired via a Headwall hyperspectral sensor and a MicaSense multispectral sensor, and the pigment contents were quantified via solvent extraction and a UV microplate reader. We developed predictive models via seven machine learning approaches, with partial least squares regression (PLSR) and random forest (RF) emerging as the most robust algorithms for pigment estimation. Chlorophyll a and b are highly and positively correlated (r > 0.9), which is consistent with their hyperspectral reflectance imaging results. The hyperspectral data consistently outperformed the multispectral data in terms of predictive accuracy (e.g., R2 = 0.91 and 0.76 for anthocyanins and flavonoids via RF) and phenolic compounds with R2 = 0.79, capturing subtle spectral features linked to biochemical variation. Spatial maps revealed strong genotype-dependent heterogeneity in pigment and phenolic distributions, supporting the potential of this approach for cultivar discrimination and pigment phenotyping. These findings demonstrate that hyperspectral imaging integrated with data-driven modelling offers a powerful, nondestructive framework for the biochemical monitoring of leafy vegetables, supporting breeding, precision agriculture, and food quality assessment.