High-Resolution Field Emission Scanning Electron Microscopy (FESEM) Imaging of Cellulose Microfibril Organization in Plant Primary Cell Walls

Abstract

Abstract We have used field emission scanning electron microscopy (FESEM) to study the high-resolution organization of cellulose microfibrils in onion epidermal cell walls. We frequently found that conventional “rule of thumb” conditions for imaging of biological samples did not yield high-resolution images of cellulose organization and often resulted in artifacts or distortions of cell wall structure. Here we detail our method of one-step fixation and dehydration with 100% ethanol, followed by critical point drying, ultrathin iridium (Ir) sputter coating (3 s), and FESEM imaging at a moderate accelerating voltage (10 kV) with an In-lens detector. We compare results obtained with our improved protocol with images obtained with samples processed by conventional aldehyde fixation, graded dehydration, sputter coating with Au, Au/Pd, or carbon, and low-voltage FESEM imaging. The results demonstrated that our protocol is simpler, causes little artifact, and is more suitable for high-resolution imaging of cell wall cellulose microfibrils whereas such imaging is very challenging by conventional methods.

Plant phenotyping relevance

植物細胞壁のセルロース微繊維構造を高解像度で取得するFESEM試料調製・撮像法を開発し、従来法と比較検証しており、植物形態・構造の表現型取得が中心である。

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