BACKGROUND Duckweed has been considered as an alternative future food material as a result of its high nutritional values, although it also has a high moisture content resulting in a short shelf life. Moisture sorption isotherms are used to design dehydration and storage conditions to prolong the shelf life food products. To date, information regarding the sorption isotherm of duckweed has not been reported. Scanning electron microscopy (SEM) is frequently used to study food microstructure. However, this technique has to be performed under high-vacuum conditions and takes a long time. In the present study, two-photon imaging microscopy was selected to investigate the microstructure of dried duckweed instead of SEM. RESULTS Among five sorption isotherm models, the Peleg model gave the highest goodness of fit. The monolayer moisture content (M-0) of duckweed was in the range 7.43-7.92% dry basis (d.b.) and 8.87-8.86% d.b. for the GAB and BET multilayer kinetic models, respectively. The moisture changing behavior at each relative humidity step could be described by two exponential and reaction order kinetics. A clear cell structure (hexagonal shape) and stomata, as well as structural images (both 2D and 3D), were obtained using the two-photon microscopy technique. CONCLUSION The Peleg model best described the moisture sorption behaviors of dried duckweed and the shape of sorption isotherms were classified as type III isotherm. The M-0 of dried duckweed ranged from 7.43 to 8.86% d.b. Two-photon microscopy was a potent tool for investigating the microstructure and composition of dried duckweed. (c) 2021 Society of Chemical Industry.