2025/02/18 by Matthias Pöge, Marcel Dickmanns, Peng Xu +16 · 1 voice · 6 citations
Biochemistry, Genetics and Molecular Biology · #Computer science #Cryo-electron tomography #Electron #Electron tomography #Magnetic and Electromagnetic Effects #Materials science #Nanotechnology #Nuclear physics #Optics #Physics #Tomography
paper · pdf · doi:10.1101/2025.02.14.638237
published in bioRxiv (Cold Spring Harbor Laboratory) (Cold Spring Harbor Laboratory)
openalex publication_date 2025/02/18 · openalex created_date 2025/02/19 · openalex updated_date 2026/07/14
Abstract Cryo-Electron Tomography (cryo-ET) allows to visualize the molecular architecture of pristinely preserved cells and tissues. The workflow of sample preparation for cryo-ET is rather complex; it involves vitrification by rapid freezing followed by cryo-Focused Ion Beam (FIB) milling rendering the volumes of interest thin enough for cryo-ET data acquisition. The established protocols for single cells grown on or deposited on EM-grids are not suitable for multicellular plant tissues. Plunge-freezing does not yield vitrified samples in most cases and must be replaced by high-pressure freezing. This, in turn, necessitates extensive modifications of the subsequent FIB milling procedures. In this communication we describe procedures for sample screening, targeted FIB milling guided by cryo-fluorescence microscopy and a novel lamella trimming step that allows to obtain homogenously thin lamellae suitable for cryo-ET. We have tested all the steps along the workflow with a variety of plant tissues including the moss Physcomitrium patens and tissues of Arabidopsis thaliana and Limonium bicolor . We could demonstrate that the workflow optimized for plant tissues allows to attain subnanometer resolution in cases where subtomogram averaging is applicable.