3D Reconstruction of Mature Arabidopsis Ovules Using FIB-SEM to Study Filiform Apparatus Morphology

Volume electron microscopy based on serial sectioning allows for three-dimensional (3D) visualization and analysis of the internal structures of tissues, cells, and organelles. One such technique, focused ion beam (FIB) scanning electron microscopy (SEM), has the advantages of nanoscale sectioning and high z-resolution, but the disadvantage of limited volume processing. Because of this limitation, targeting localized objects by FIB-SEM is difficult. Here, we developed a FIB-SEM observation workflow that enables the analysis of the filiform apparatus of synergid cells enclosed in the Arabidopsis ovule. In this protocol, plant samples are stained, embedded, trimmed, and carbon-coated while maintaining their orientation within the tissue. Then, sequential observations are performed using Cut & See function of FIB-SEM, followed by image processing for 3D reconstruction. Utilization of multi-scanning and image cropping from high-resolution data helps to identify localized targets within plant tissue. The filiform apparatus, which is an invaginated cell wall structure of the synergid cells, shows distinct contrast in each image, allowing for segmentation using brightness-based binarization. Such segmentation avoids the need to manually trace complex structures and facilitates 3D reconstruction by volume electron microscopy. Key features • Sampling and trimming of the resin block enable directionally loading in FIB-SEM. • Multi-scanning by FIB-SEM and target extraction by image processing software enable 3D reconstruction of local areas within the sample block. • Binarization using distinctive brightness of cellular structures enables segmentation without manual tracing of complex structures such as the filiform apparatus cell wall.