3D Clearing and Molecular Labeling in Plant Tissues.

Plant histology and imaging traditionally involve the transformation of tissues into thin sections to minimize light scatter in opaque material, allowing optical clarity and high-resolution microscopy. Recently, new techniques in 3D tissue clearing, including PEA-CLARITY, have been developed to minimize light scatter within intact, whole samples. These techniques can achieve equivalent microscopic resolution to that of thin section imaging with the added benefit of maintaining the original 3D structure and position of biomolecules of interest. Furthermore, PEA-CLARITY is compatible with standard stains and immunohistochemistry, allowing molecular interrogation of intact, 3D tissues. This chapter outlines the current methods available for 3D histology in plants and details the materials, equipment, reagents, and procedure for the PEA-CLARITY technique.

PEA-CLARITY: 3D molecular imaging of whole plant organs.

Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.