Cryo-electron tomography of plant chloroplasts: preserving native thylakoid architecture for 3D imaging

Abstract

Cryo-focused ion beam scanning electron microscopy (cryo-FIB-SEM) combined with cryo-electron tomography (cryo-ET) enables visualization of chloroplast architecture close to its native state, but vitrifying isolated organelles remains challenging. We outline practical considerations for applying cryo-FIB-SEM and cryo-ET to plant chloroplasts and compare osmolyte conditions that support vitrification without distorting membranes. Using isolated chloroplasts from an AtVIPP1-GFP transplastomic Nicotiana tabacum line to aid fluorescence-guided targeting, we found that trehalose (0.33 M) and betaine (0.66 M) maintained lamella quality and preserved grana organization, whereas mannitol and sucrose frequently produced ice artifacts. Tomograms from trehalose-treated samples showed well-resolved grana stacks and intact thylakoid networks consistent with native architecture. These observations offer practical guidance for laboratories adopting chloroplast cryo-ET and highlight sample preparation choices that improve reproducible 3D imaging at nanometer resolution.