Most leaf development studies at the cell and organ levels have been limited to the leaf surface, with data referring to the leaf surface area and to the number and surface area of epidermal cells. However, leaf sub-epidermal tissues, the palisade and spongy mesophyll, contain the main actors in photosynthesis. The number and thickness of palisade cell layers and the volume occupied by spongy mesophyll (cells and intercellular spaces) affect the accumulation of photosynthates and, as such, whole plant growth. Studies into the leaf phenotype of growth-affected Arabidopsis thaliana mutants have revealed a higher variability in leaf thickness than in leaf surface area. In general, there is no correlation between these two variables, which means that to describe a leaf phenotype, leaf volume has to be taken into account. A method has been developed for high-resolution imaging of leaves in three dimensions usingmultiphoton laser scanning microscopy, and for the analysis of images, providing data on volumes and volumetric proportions of cells and tissues and cell density. The method has been used in the study of A. thaliana leaf expansion from emergence to the onset of senescence for leaves located at different nodal positions in the rosette, completing our knowledge of individual leaf development processes with their volumetric component. The method will further be applied in the study of leaf plasticity in response to the environment for both A. thaliana and apple tree, a model and an agronomic species, respectively. (Texte intégral)