Electron tomography in plant cell biology

Microscopy, 2019, 69–79 doi: 10.1093/jmicro/dfy133 Advance Access Publication Date: 17 November 2018 Review Electron tomography in plant cell biology Marisa S. Otegui1,2,3,* and Jannice G. Pennington4,5 Department of Botany, University of Wisconsin-Madison, 430 Lincoln Drive, Madison WI 53706, USA, Laboratory of Molecular and Cellular Biology, University of Wisconsin-Madison, 1525 Linden Drive, Madison WI 53706, USA, 3Department of Genetics, University of Wisconsin-Madison, 425 Henry Mall, Madison WI 53706, USA, 4Institute for Molecular Virology, University of Wisconsin-Madison, 1525 Linden Drive, Madison WI 53706, USA, and 5Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, USA 2 * To whom correspondence should be addressed. E-mail: Received 30 August 2018; Editorial Decision 23 October 2018; Accepted 31 October 2018 Abstract Electron tomography (ET) approaches are based on the imaging of a biological specimen at different tilt angles by transmission electron microscopy (TEM). ET can be applied to both plastic-embedded and frozen samples. Technological advancements in TEM, direct electron detection, automated image collection, and imaging processing algorithms allow for 2–7-nm scale axial resolution in tomographic reconstructions of cells and organelles. In this review, we discussed the application of ET in plant cell biology and new opportunities for imaging plant cells by cryo-ET and other 3D electron microscopy approaches. Key words: electron tomography, plant biology, cryo-electron microscopy Introduction Understanding the spatial and temporal distribution of macromolecules and organelles is an essential aspect of cell biology. Microscopy-based imaging approaches allow researchers to analyze the dynamic localization of cellular components, membrane remodeling events, the morphology and function of organelles, the structural features of proteins and molecular complexes and their interaction networks. The most commonly used microscopy imaging techniques employ either photons (light) or electrons to collect information on the various ways these forms of radiation interact with biological samples. Light microscopy allows for live imaging and offers unique opportunities to understand cellular dynamics in living systems during development, physiological responses, cell cycle, etc. The development of fluorescent probes (chemical and nanoparticle-based probes, genetically-encoded tags and combinations of both) [1] in the context of light microscopy has revolutionized the field of cell biology. The fluorescent probe toolkit for imaging selected molecules, membranes or organelles is continuously expanding and improving. However, fluorescence microscopy like other modalities of conventional light microscopy is limited by its resolution (≥200 nm in x–y and; ≥500 in z), imposed by the diffraction of light. Super-resolution microscopy imaging techniques (also called nanoscopy) such as stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM), photoactivated localization microscopy (PALM) and fluorescence photoactivation localization microscopy (FPALM) © The Author(s) 2018. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: 69 1 Microscopy, 2019, Vol. 68, No. 1 70 by milling with a focused-ion beam (FIB SEM) [7]. The resolution of these approaches based on serial imaging are limited to twice the section thickness [8] and therefore, commonly restricted to ~20–120 nm. Electron tomography (ET) approaches are instead based on collecting images of an individual sample at different angles and combining the individual 2D projections into a 3D reconstruction or tomogram via either Fourier or realspace methods [9,10]. ET of plastic-embedded samples and cryo-ET of vitrified material have allowed cell biologists to image macromolecular complexes and organelles in their native, 3D cellular context with an axial resolution of 2–7 nm, or even higher when combined with image analysis approaches, such as sub-tomogram averaging [11–13]. In this review, we discuss insights gained from ET of plastic-embedded samples as well as emerging techniques in cryo-ET. We also discuss other methodologies that use cryoimaging for mesoscale imaging of cells in their native state as well as current limitations and opportunities to expand and combine imaging modalities in plant cell biology. Electron tomography Fig. 1. (a) Comparison of axial resolutions achieved by different 3D EM approaches. ET, electron tomography; FIB SEM, focused-ion beam scanning electron microscopy; SPA, single-particle analysis; SBF-SEM, serial blockface scanning electron microscopy; SXT, soft X-rays tomography. (b) General principle of electron tomography. ET is based on similar principles as various tomographic techniques used in medical imaging such as computerized axial tomography (CAT-scan imaging). In a CAT-scan, the X-ray projections of the patient are collected over 180° or a full 360° rotation. For ET, the samples is placed into a holder that can be tilted under the electron beam and images are collected at angular intervals of 1–3°, generating a stack of 2D projections of a selected specimen area [14,15] (Fig. 1b). However, due to the thickness of the section (at 60° tilt, the path length of the electrons through the specimen is twice the specimen thickness whereas at 70°, it is three times the specimen thickness) [8] and the fact that the sample holder at high-tilt angles gets in the beam path, the angular range in ET is generally restricted to 60° or 70°, resulting in a wedge of missing information between the maximal tilt angle collected and 90°. This results in tomograms with anisotropic resolution. To improve the resolution isotropy, it is possible to collect tilt series along two orthogonal axes to generate dual-axis tomograms [16]. Typically, intermediate voltage (200–300 kV) TEM are used for data collection since lower voltages (e.g. 100 kV) do not provide good images of semi-thick sections at high-tilt angles. Once a stack of 2D projections is collected, the aligned images are used to calculate an electron tomogram using weighted back-projection algorithms [17]. The resolution and quality of the resulting tomogram depend on many factors, including sample preservation, magnification and quality of the detector, the angular interval, the angular range and section thickness [8]. have challenged the light diffraction limit by reaching practical lateral resolution in the 20–100-nm range [2–6]. However, when higher spatial resolution is needed, electron microscopy (EM) is the preferred imaging option (Fig. 1a). Conventional transmission EM (TEM) can resolve cellular macromolecules in their cellular context at a resolution of ~1–2 nm; however, it is often limited by the fact that it only generates 2D projections and it requires specimen fixation and processing, (...truncated)