Leaf Area Estimation by Photographing Leaves Sandwiched between Transparent Clear File Folder Sheets

Image analysis is a promising method for in situ leaf area measurement. However, as leaves are three-dimensional, the use of two-dimensional images captured using a digital camera can result in underestimation. To overcome this problem, we tested a clear folder method. Before photographing leaves with a digital camera, we flattened the leaves by sandwiching them between a pair of transparent plastic clear file folder sheets, which are stationery implements for carrying documents. Although similar methods have been previously proposed, their applicability to species with different leaf shapes has never been investigated. We tested the efficacy of this method using 12 species from various taxa (monocots, magnoliids, and basal and core eudicots) and leaf morphology (entire vs. lobed, simple vs. compound leaves, small and large leaves). Individual leaf areas and the Montgomery parameters obtained using this method were then compared with those obtained using the standard method, which employs a flatbed digital scanner. We observed strong correlations (R2 > 0.98) between the camera and scanner data. The regression slopes were close to unity (0.96–1.01) and the intercepts were close to zero. These findings suggest that the clear folder method can be used as an inexpensive alternative method to estimate the area of leaves in situ with acceptable accuracy. An introductory manual for readers unfamiliar with image analysis using ImageJ is presented in the end of the paper.