Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution

Biological systems are the sum of their dynamic three-dimensional(3D)parts.Therefore,it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions.Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution.However,many of these methods require specialized equipment and personnel to complete them.Here,we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution,focusing on Arabidopsis clathrin-coated vesicles(CCVs).While CCVs are essential trafficking organelles,their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments.First,we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography,providing sufficient resolution to define the clathrin coat arrangements.Critically,the samples are prepared directly on electron microscopy grids,removing the requirement to use extremely corrosive acids,thereby enabling the use of this method in any electron microscopy lab.Secondly,we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells.Finally,to facilitate the high-throughput and robust screening of metal replicated samples,we provide a deep learning analysis method to screen the“pseudo 3D”morphologies of CCVs imaged with 2D modalities.Collectively,our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.