Precision thin sectioning of silica phytoliths by Focused Ion Beam (FIB-SEM) v1

Phytoliths are microbodies of biogenic silica made by many plant species in all ecosystems over the globe. They serve critical functions in alleviating plant stresses and influencing the carbon and silicon biogeochemical cycles. The investigation of carbon occlusion within phytolith, as a potential source of long-term soil carbon storage, has long been hampered by a lack of direct experimental evidence. In this protocol, we employed a Focused Ion Beam coupled to a Scanning Electron Microscopy (FIB-SEM) to produce thin lamellas (approximately 15 × 10 µm2 size, with thickness below 200 nm) enabling synchrotron scanning transmission X-ray microspectroscopy (STXM) analysis with ≈100–200 nm pixel size resolution at energies near the silicon and carbon K-absorption edges. Our results revealed the spatial distributions of carbon within phytoliths, highlighting its presence at lamella borders, within islands, and dispersed in extended regions. This protocol of phytolith slicing into thin lamella provides unprecedented insights into the spatial and chemical characteristics of carbon within phytoliths, offering a low-invasive alternative to wet-chemical digestion methods.