Arsenic (As) is a widely spread soil contaminant which can cause toxicity in plants. Although, many studies have investigated the spectral characteristics of metal affected plants, the extent to which different toxicities may result in correspondingly different spectral signatures has received little attention. Quantitative analyses of structural changes in the arrangement of mesophyll and their relationship with spectral and physiological changes may help to identify spectral signatures related to arsenic stress. The objectives of this study are to identify spectral and physiological changes caused by arsenic toxicity and to relate these changes to leaf internal structure. Hydroponically-grown spinach (Spinacia oleracea L. cv. Babyleaf) was treated with 0, 5, 10 and 15 μmol L−1 As (0, 0.37, 0.72 and 1.12 mg As L−1) for 4 weeks in a growth chamber. Spectral properties of leaves were obtained for visible and infrared frequencies. Physiological properties such as net photosynthetic rate and stomatal conductance were measured. 3D confocal stacks were acquired for 6 samples. Using special software, different virtual test probes with an arbitrary predefined (e.g. random) position and orientation were generated within the stack of sections and measured mean mesophyll cell volume and internal surface area using the fakir method. Significant differences between treatments were found in at least some of the measured parameters with arsenic accumulation.