3D Plant cell architecture of Arabidopsis thaliana (Brassicaceae) using focused ion beam–scanning electron microscopy

Abstract

• Premise of the study: Focused ion beam–scanning electron microscopy (FIB-SEM) combines the ability to sequentially mill the sample surface and obtain SEM images that can be used to create 3D renderings with micron-level resolution. We have applied FIB-SEM to study Arabidopsis cell architecture. The goal was to determine the efficacy of this technique in plant tissue and cellular studies and to demonstrate its usefulness in studying cell and organelle architecture and distribution.• Methods: Seed aleurone, leaf mesophyll, stem cortex, root cortex, and petal lamina from Arabidopsis were fixed and embedded for electron microscopy using protocols developed for animal tissues and modified for use with plant cells. Each sample was sectioned using the FIB and imaged with SEM. These serial images were assembled to produce 3D renderings of each cell type.• Results: Organelles such as nuclei and chloroplasts were easily identifiable, and other structures such as endoplasmic reticula, lipid bodies, and starch grains were distinguishable in each tissue.• Discussion: The application of FIB-SEM produced 3D renderings of five plant cell types and offered unique views of their shapes and internal content. These results demonstrate the usefulness of FIB-SEM for organelle distribution and cell architecture studies.

Correlations between lignin content and structural robustness in plants revealed by X-ray ptychography

“…Several imaging techniques have provided three-dimensional information on plant cell anatomy. Focused ion beam-scanning electron microscopy (FIB-SEM) allowed us to study the architecture of 20 µm 3 fragments of Arabidopsis, creating three-dimensional renderings of organelle identification 22 . Confocal microscopy 23 , X-ray computed tomography 24 and transmission electron microscopy (TEM) 25 opened the path to obtain high resolution images of biological systems.…”

A Protocol for Endophyte-­Free Callus Tissue of the Grape <i>Vitis aestivalis</i> “Norton/Cynthiana” (Vitaceae)

“…SEM fixation protocol-Tissues were fixed following protocols developed from Bhwana et al [23] and Anderson and Miller [24]. Modifications to the protocols were as follows: tissues selected from the fungal incubation plates were fixed with 2.5% glutaraldehyde and 2.0% osmium.…”

“…SEM imaging protocol-Fixed tissues were imaged for visual analysis with a Hitachi 3400N scanning electron microscope, using imaging protocols modified from Bhawana et al [23] and Anderson and Miller [24]. Accelerating voltages of 8, 10, 12 and 15 kV were used in the imaging procedure.…”

Exploring the interior of cuticles and compressions of fossil plants by FIB‐SEM milling and image microscopy

“…FIB-SEM instruments were initially developed for the study of in situ nanostructures in industrial materials (Orloff et al, 2003). They eventually found applications in a great variety of research areas, such as lithography and nanodevice developments (Fujii & Kaito, 2005;Moberly et al, 2007;Volkert & Minor, 2007;Santschi et al, 2009;Javed et al, 2014), petrology and mineralogy (Heaney et al, 2001;Lee et al, 2003;Wirth, 2004;de Winter et al, 2009;Stokes & Hayles, 2009), biological materials (Drobne et al, 2005;Nalla et al, 2005;Orso, 2005;Milani et al, 2007;Moberly et al, 2007;de Winter et al, 2009;Marko, 2010;Grandfield & Engqvist, 2012;Rigort et al, 2012;Hoffmann et al, 2015;Wagenknecht et al, 2015), extant pollen grains and plant tissues (House & Balkwill, 2013;Bhawana et al, 2014), and the analysis of forensic data (Milani et al, 2012).…”