A rapid and effective optical-clearing technique for deep tissue fluorescence imaging in trees

Key message This non-invasive clearing technique allows for deep clear imaging of plant tissue and 3D structural analysis of leaf cells. Optical-clearing technology allows the reduction of light scattering and absorption in internal biological tissues. This technology allows microscopy techniques, such as confocal laser scanning microscopy, to obtain structural information deep within the internal tissue and also allows for three-dimensional (3D) tissue analysis. However, the transparent method in plants remains to be further elucidated. In this study, we first report on a non-invasive, rapid and effective clearing technique that increases the transmission of light through plants. Importantly, we found that the leaf terminal veins of four species had different characteristics based on fluorescence imaging observations. Moreover, through the combination of transparent technology and microscopic observation, we clearly observed the three-dimensional structure of plant leaf cells. Our simple and rapid clearing method is useful for fluorescence imaging the intact internal structure. Taken together, the results of this study will provide valuable information for further microscopic experimental investigations in trees.