Precise tracking of nanoparticles in plant roots

One of the foremost challenges in nanobiotechnology is obtaining direct evidence of nanoparticles' absorption and internalization in plants. Although confocal laser scanning microscopy (CLSM) or transmission electron microscopy (TEM) are currently the most commonly used tools to characterize nanoparticles in plants, subjectivity of researchers, incorrect sample handling, inevitable fluorescence leakage and limitations of imaging instruments lead to false positives and non-reproducibility of experimental results. This protocol provides an easy-to-operate dual-step method, combining CLSM for macroscopic tissue examination and TEM for cellular-level analysis, to effectively trace single particles in plant roots with accuracy and precision. In addition, we also provide detailed methods for processing plant materials before imaging, including cleaning, and staining, to maximize the accuracy and reliability of imaging. This protocol involves currently commonly used nanomaterial types, such as metal-based and doped carbon-based materials, and enables accurate localization of nanoparticles with different sizes at the cell level in Arabidopsis thaliana root samples either through contrast or element mapping analysis. It serves as a valuable reference and benchmark for scholars in plant science, chemistry and environmental studies to understand the interaction between plant roots and nanomaterials and to detect the distribution of nanomaterials in plants. Excluding plant culture time, the protocol can be completed in 4-5 d.