In-situ Observation of Fast Chloroplast Dynamics in Intact Leaves

Abstract

Chloroplast relocation is a hallmark of plant photoprotection, yet has long been regarded as a slow, minute-scale process. Here we overturn this view by directly visualizing chloroplast motility in intact leaves with the second near-infrared (NIR-II, 900-1880 nm) fluorescence confocal microscopy, an optical strategy uniquely suited for deep, in situ imaging. By harnessing the intrinsic long-wavelength fluorescence tail of chloroplasts, this method minimizes scattering, extends imaging depth, and enables simultaneous stimulation-imaging with subcellular resolution--capabilities not attainable with conventional visible fluorescence or multiphoton fluorescence microscopy. Using this approach, we discover that submerged leaves of amphibious plants exhibit remarkably rapid avoidance responses to red light, whereas aerial leaves show negligible relocation. Control experiments exclude influence of the physical structure of leaves, confirming that these contrasting responses reflect distinct physiological adaptations to environmental light regimes. Beyond revealing unexpected speed and flexibility in chloroplast dynamics, our findings establish the second near-infrared fluorescence confocal microscopy as an important tool for direct observation of fast subcellular processes in deep photosynthetic tissues and advancing plant photobiology.