Tissue-resolved photosynthetic responses in living leaves revealed by microscopic imaging-pulse-amplitude-modulation

Leaves of terrestrial plants possess heterogeneous anatomical structures composed of multiple cell layers. Although biochemical differences among leaf tissues have been inferred from protein analyses and anatomical studies, direct comparisons of dynamic photochemical responses among tissues while preserving their spatial context remain challenging. Leaves experience intrinsically heterogeneous environments because incident light enters primarily from above and propagates through complex internal leaf structures. Therefore, analyzing photosynthetic activity at the tissue and cellular levels is essential for understanding how photosynthesis operates within structurally heterogeneous leaves. Here, we combined live leaf-section imaging with microscopic Imaging-PAM chlorophyll fluorescence measurements to analyze photochemical responses at the tissue level in living leaf sections. In dorsiventral dicot leaves, palisade tissues exhibited a higher effective PSII quantum yield [Y(II)] and more rapid induction of regulated energy dissipation [Y(NPQ)] than spongy tissues, indicating higher photosynthetic capacity and photoprotective activity. In contrast, rice leaves, which lack palisade-spongy differentiation, showed uniform photochemical responses along the adaxial-abaxial axis. In the C4 plant finger millet, mesophyll and bundle sheath cells displayed distinct photochemical responses consistent with their functional differentiation in C4 photosynthesis. These results demonstrate that photosynthetic responses are spatially organized according to leaf anatomical structure. Although section-based measurements do not reproduce the native optical and gas environments of intact leaves, they enable the comparison of intrinsic tissue-specific photochemical properties under approximately equivalent illumination. The present approach enables tissue-resolved chlorophyll fluorescence analysis in living leaves, providing a new framework for investigating how leaf architecture shapes the spatial organization of photosynthetic activity.