Different Imaging Techniques for the 2 and 3D Characterization of Plant Cell Ultrastructure in the SEM and TEM

Abstract

Two and three-dimensional (2D and 3D) imaging of plant samples with the scanning and transmission electron microscope (SEM, TEM) can reveal important information regarding physiological and anatomical adaptations of plants to environmental (stress) situations [1-3]. This study provides an overview of SEM and TEM techniques for the rapid evaluation of 2D ultrastructural changes in plants. Additionally, methods for the 3D reconstruction and volume extraction of plant cells based on serial section TEM (ssTEM), focused ion beam SEM (FIB-SEM) are demonstrated. Leaves of Nicotiana tabacum and Cucurbita pepo were prepared conventionally and with the help of microwave irradiation [1]. Additionally, for SEM investigations leaf replicas were made by applying dental putty [2]. SEM investigations were performed with a Versa 3D SEM (FEI, Hillsboro, OR, USA). For TEM investigations ultrathin sections (80 nm) were imaged with a JEOL 1010 TEM (JEOL, Akishima, Japan). For ssTEM, 71 sections of tobacco cells were imaged with a Zeiss EM 902 TEM (Zeiss, Oberkochen, Germany) while FIB-SEM was used to image 126 slices of pumpkin cells. Track EM (Image J) was used for 3D reconstructions and volume extractions. The use of microwave irradiation strongly reduced sample preparation time from 6 to 2h for SEM and from 3d to 5h for TEM investigations. SEM revealed that the surface of samples prepared with microwave irradiation was well preserved and comparable to those prepared conventionally (Figure 1 a & b). Stomatal and epidermal cells could be clearly distinguished (Figure 1 a & b). Samples showed signs of shrinkage (Figure 1 b) which was not observed on leaf replicas which showed a smooth surface (Figure 1 c). TEM revealed that the ultrastructure of samples prepared with microwave irradiation was well preserved and similar to those prepared conventionally (Figure 2 a & b). The cytoplasm contained chloroplasts with thylakoids and starch grains, nuclei with eu- and hetero-chromatin, mitochondria, peroxisomes, vacuoles and cell walls (Figure 2a & b). 3D reconstruction by FIB-SEM was faster and less sophisticated than 3D reconstruction by ssTEM [3]. Nevertheless, both methods delivered adequate results (Figure 2 c & d). Volume extraction revealed that tobacco cells were larger (31410 μm3) than pumpkin cells (20697 μm3) and contained more chloroplasts (175 vs. 124), mitochondria (1317 vs. 291) and peroxisomes (745 vs. 79). While individual chloroplasts, mitochondria, peroxisomes were larger in pumpkin plants (25, 53, and 50%) they covered more total volume in tobacco plants (5390, 395, 374 μm3) when compared to pumpkin plants (4762, 134, 59 μm3). Summing up, microwave-assisted sample preparation and the production of leaf replicas enabled the rapid evaluation of 2D ultrastructure of plant cells for TEM and SEM investigations. 3D reconstructions based on FIB-SEM and TEM were well suited to extract volume data of whole plant cells. These techniques are well suited to study the effects of environmental stress situations on plant ultrastructure. SEM micrographs of the surface of tobacco leaves showing stomatal (arrows) and epidermal cells. While samples prepared with the help of microwave irradiation (a) and conventionally (b) showed signs of shrinkage (arrowheads in b), leaf surface replicas with dental putty (c) showed a smooth surface. Bars = 50 μm. TEM micrographs of the 2D ultrastructure of pumpkin (a) and tobacco (b) plant leaf cells with chloroplasts (C), mitochondria (M), nuclei (N), and vacuoles (V). 3D reconstructions [modified according to 4] of pumpkin (c) and tobacco (d) plant cells using FIB-SEM (c) and ssTEM (d). Cell wall (gray), chloroplasts (green), mitochondria (red), nucleus (brown), peroxisomes (purple), and vacuole (blue). Bars=1 μm. Cubes = 3 and 4 μm3.

Plant phenotyping relevance

植物細胞の2D/3D画像取得、再構成、体積抽出を中心に、SEM/TEMおよびFIB-SEM手法と試料調製法を比較・評価しているため、植物フェノタイピング手法として適格。

Code and data availability

This is a two-page meeting abstract describing SEM/TEM imaging and 3D reconstructions of tobacco and pumpkin cells. No public phenotype/trait datasets, image deposits, author analysis code, or trained models are mentioned; the only tool referenced (Track EM, an ImageJ plugin) is a generic third-party library, and no de

No evidence-backed public reproduction asset is currently recorded.