The bias of a two-dimensional view: comparing two-dimensional and three-dimensional mesophyll surface area estimates...

The mesophyll surface area exposed to intercellular air space per leaf area (S_m ) is closely associated with CO₂ diffusion and photosynthetic rates. S_m is typically estimated from two-dimensional (2D) leaf sections and corrected for the three-dimensional (3D) geometry of mesophyll cells, leading to potential differences between the estimated and actual cell surface area. Here, we examined how 2D methods used for estimating S_m compare with 3D values obtained from high-resolution X-ray microcomputed tomography (microCT) for 23 plant species, with broad phylogenetic and anatomical coverage. Relative to 3D, uncorrected 2D S_m estimates were, on average, 15-30% lower. Two of the four 2D S_m methods typically fell within 10% of 3D values. For most species, only a few 2D slices were needed to accurately estimate S_m within 10% of the whole leaf sample median. However, leaves with reticulate vein networks required more sections because of a more heterogeneous vein coverage across slices. These results provide the first comparison of the accuracy of 2D methods in estimating the complex 3D geometry of internal leaf surfaces. Because microCT is not readily available, we provide guidance for using standard light microscopy techniques, as well as recommending standardization of reporting S_m values.