TY - JOUR
AU - Pennington, Jannice, G
AB - 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. 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) 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). Fig. 1. View largeDownload slide (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. Fig. 1. View largeDownload slide (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. 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, which can introduce artifacts and unwanted changes to the cellular structure. Three-dimensional reconstructions based on EM imaging can be obtained using different approaches, such as serial section TEM (ssTEM), in which serial sections made along a biological sample are imaged sequentially or serial surface imaging in which the surface of a block containing a biological sample is imaged by scanning EM (SEM) as thin layers are removed from the surface either by ultramicrotomy (serial blockface SEM or SBF-SEM) or 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 real-space 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 cryo-imaging 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 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]. Tomographic reconstructions can be viewed from any angle and sliced at intervals 50–100 times thinner than the thickness of original section. Organelles and structures of interest can be manually or automatically traced in tomographic (segmentation) to render 3D tomographic models that can then be used for qualitative and quantitative analyses [18]. In cell biology, ET is applied to two main type of samples, plastic-embedded and cryofixed samples (cryo-ET). ET of plastic-embedded samples In this case, sections of 150–300 nm in thickness are made from fixed and resin-embedded biological materials. This approach is commonly used for cell biological studies of tissues, organs and even whole multicellular organisms. Typically, imaging is done in 200–300 kV TEM at room temperature. For most applications in cell biology, imaging of semi-thick plastic sections over 300 nm results in poor resolution. For larger samples, it is possible to combine serial tomograms from serial sections to obtain tomographic reconstructions of larger cellular volumes without compromising resolution. However, this approach is very laborious, as it requires the careful collection of serial sections, ideally on the same grid. In addition, it leads to a 15–25-nm gap of missing material between the serial sections, difficulting the alignment of serial tomograms and segmentation of complex cellular structures [19]. An alternative approach is scanning TEM (STEM)-ET. STEM-ET can render 3D reconstructions of thick (0.5–1.5 μm) sections at a resolution comparable to that of conventional ET of ~300-nm thick sections [20–22]. The quality of any tomographic reconstruction depends on the preservation of the specimen. Cryofixation, either at atmospheric or under high pressure, followed by cryo-substitution achieves much better preservation of the cellular structure than chemical fixation. In practice, freezing with no detectable ice crystal damage at atmospheric pressure can only be achieved in samples up to 10-μm thick. For larger specimens up to 200-μm thick, such as dissected root tips, leaf tissues, developing seeds or even plant cell cultures, high-pressure freezing should be used [23,24]. High-pressure frozen samples can then be dehydrated and fixed through the process of freeze-substitution, in which the frozen samples are placed in cryovials containing solvents with fixatives at −80°C for either 2–3 days or for a few hours under agitation [25]. After the frozen water is completely substituted with solvents, the samples are embedded in resin and sectioned in an ultramicrotome. Contrast enhancement can be achieved by staining plastic sections with heavy metal-containing reagents, such as lead citrate and uranyl acetate. After section staining, 10- or 15-nm colloidal gold particles are applied to each side of the sections to be used as fiducials that facilitate the fine alignment of the tilt images during the tomographic reconstruction [26]. This ET approach provides very valuable 3D information of cellular structures but faces some problems, such as the need for samples to be fixed, dehydrated and plastic-embedded, which can introduce changes to the cellular structure. In addition, most of the information contained in the images derives from the electron-dense stains used to enhance contrast (e.g. osmium, lead) and not from the cellular components themselves. Cryo-ET Cryo-EM refers to the visualization of frozen-hydrated molecules, organelles or cells. Samples are in vitreous ice (i.e. frozen without ice crystals) and in native state (neither chemically fixed nor stained with heavy metals), providing the most faithful structural preservation possible [27–30]. There are two main modalities in cryo-EM, single-particle analysis (SPA) and cryo-ET. For SPA, a thin layer of a solution containing isolated particles (e.g. proteins, molecular complexes, viruses) is cryo-plunged into liquid ethane or liquid propane. These particles now embedded in a thin layer of vitreous ice, ideally in random orientations, are imaged in a cryo-EM. Thousands of particles in the collected images are then sorted according to their orientation and averaged into classes to calculate 3D reconstructions that can reach atomic or near-atomic resolution [27,31]. Different from SPA, cryo-ET does not depend on the imaging of thousands of repetitive structures but it can generate 3D reconstructions of variable objects such as organelles, although at lower resolution than those achieved by SPA. In addition, it is possible to apply cryo-ET to whole cells or cell slices, allowing for the analysis of molecular assemblies and membranes in their cellular context [27]. The development of new and improved devices to cryofix and process samples for cryo-ET together with a new generation of direct electron detectors and algorithms for image analysis have enabled a true revolution in the cell biology field, broadening our understanding on the architecture and dynamic of molecular assemblies in their native cellular context [27,30,32]. Just as described for ET at room temperature, sample thickness is a limiting factor for cryo-ET as vitreous sections thicker than 300–500 nm often result in poor resolution tomographic reconstructions [33]. This means that the analysis of most eukaryotic cells and tissues requires difficult procedures to produce thinner frozen slices or lamellas. The sensitivity of ice-embedded material used for cryo-ET generates important challenges related to (1) sample preparation/handling and (2) imaging under an electron beam. Sample preparation for cryo-ET Whereas direct cryo-ET imaging of bacterial [34,35] and archeal [36] cells, isolated eukaryotic organelles [37], cell edges and organelles protruding from the main cell body [38] is possible, similar studies from most eukaryotic cells, including plant cells, require sectioning or thinning of the sample prior to imaging. Producing thin specimens (ideally 300-nm thick or thinner) of frozen eukaryotic cells is a technical challenge. Traditionally, this has been achieved by cryo-ultramicrotomy or vitrified sectioning [39,40]. Here, samples can be vitrified in a high-pressure freezer in specialized dome-shaped carriers or copper tubes that can then be inserted into a cryo-ultramicrotome for sectioning under cryogenic temperatures. Although a number of accessories to facilitate sample manipulation for vitrified sectioning have become available in recent years [39,41–43] (Fig. 2a–d), this is still a very demanding technique. In addition, vitrified sectioning often causes cutting artifacts such as knife marks, deformation by compression and crevasses [39,44,45]. Fig. 2. View largeDownload slide Vitreous sectioning of yeast cells. (a) High-pressure frozen yeast cells are sectioned in a Leica UC7 cryo-ultramicrotome with a Diatome double micromanipulator. 1, UC7 cryo-chamber; 2, Electrostatic discharge device; 3, Right micromanipulator with grid holder; 4, Left micromanipulator with gold-coated hair; 5, Grid; 6, Sample; 7, cryo-knife. (b) Ribbon of 50-nm vitreous cryo-sections of high-pressure frozen yeast cells. The ribbon was attached to the grid using a discharge device. (c) Cryo-sections of yeast cells imaged at −178˚C using a side-entry 626 Gatan Cryo-holder on a Tecnai F30 cryo-electron microscope. M, mitochondria; N, nucleus; V, vacuole. (d) Details of a mitochondrion from panel (c). Scale bars: 500 nm. Fig. 2. View largeDownload slide Vitreous sectioning of yeast cells. (a) High-pressure frozen yeast cells are sectioned in a Leica UC7 cryo-ultramicrotome with a Diatome double micromanipulator. 1, UC7 cryo-chamber; 2, Electrostatic discharge device; 3, Right micromanipulator with grid holder; 4, Left micromanipulator with gold-coated hair; 5, Grid; 6, Sample; 7, cryo-knife. (b) Ribbon of 50-nm vitreous cryo-sections of high-pressure frozen yeast cells. The ribbon was attached to the grid using a discharge device. (c) Cryo-sections of yeast cells imaged at −178˚C using a side-entry 626 Gatan Cryo-holder on a Tecnai F30 cryo-electron microscope. M, mitochondria; N, nucleus; V, vacuole. (d) Details of a mitochondrion from panel (c). Scale bars: 500 nm. To eliminate compression artifacts and minimize sample handling, specimen can be thinned by cryo-focused ion beam SEM (cryo-FIB SEM) milling [46]. Here, a beam of Gallium ions removes material from a vitrified specimen (e.g. a Chlamydomonas cell) and leaves behind a thin cryo-lamella of the cell. The milling process is monitored by SEM imaging under vacuum. Samples remain vitreously frozen during FIB milling [47]. In a typical cryo-FIB-SEM milling procedure, cells are plunge-frozen on EM grids and placed on a specialized cryo-holder within the cryo-FIB SEM. For in situ cryo-lamella milling (or ‘on-grid thinning’), the resulting lamella remains attached to the EM grid and can be directly imaged for cryo-ET without further handling [46]. However, to achieve proper vitrification by plunge freezing and to avoid damage caused by long milling times, cells processed by this approach have to be thinner than 10 μm, [33]. This works well for small cells (bacteria and small eukaryotic cells) but it is not ideal for larger cells, tissues and whole organisms. For larger specimens that require high-pressure freezing, other cryo-FIB strategies are available. For example, it is possible to combine trimming of high-pressure frozen samples by cryo-ultramicrotomy, followed by cryo-FIB milling to obtain up to 300-nm thick lamellas that can be used for cryo-ET [47,48]. Another approach uses a lamella lift-out strategy to collect potentially multiple lamellas from one organism or tissue. In this case, with the help of a micromanipulator within the SEM, the vitreous lamellas are transferred to a grid for cryo-ET imaging [33]. This technique is often assisted by the visualization of areas of interest by cryo-fluorescence light microscopy [49] prior to cryo-FIB milling and has been successfully applied to Caenorhabditis elegans embryos [33]. There are only a few studies in plants and algae using cryo-ET approaches. Cryo-FIB SEM milling has been successfully applied to Chlamydomonas reinhardtii for cryo-ET to visualize chloroplast architecture [50], nuclear pore organization [51], COPI coats on Golgi-derived vesicles [52], protein arrays within Golgi cisternae [53] and tethering of 26S proteasome complexes to nuclear pores [54]. However, most plant cells and tissues are not amenable to in situ cryo-lamella milling (just as a reference, an Arabidopsis root is 100-μm thick). For cryo-ET of cryo-FIB milled lamellas, high-pressure frozen plant samples could be processed by two approaches, either initial trimming by cryo-ultramicrotomy followed by in situ cryo-lamella milling or a lamella lift-out procedure and transfer of the resulting lamellas to a grid for imaging. Data collection and image analysis in cryo-ET Radiation damage, low signal-to-noise ratio and beam-induced sample movement are major factors limiting resolution in cryo-EM. Single tilt series are collected using low electron dose mode focusing and tracking a region adjacent to the target area to reduce irradiation damage to the area of interest. The total electron dose of a ±60° tilt series must be calculated considering that each image should derived from only 1–2 electrons/Å2, which often results in poor signal-to-noise ratio. Loss in resolution due to radiaton damage can be further reduced by using a dose-symmetric tilt scheme [55]. Here, low-tilt images are collected early in the series before radiation damage accumulates and maximizing high-resolution information whereas high-tilt images are collected later in the series. The inherent poor signal-to-noise ratio resulting from low electron dose imaging in cryo-ET has been partially overcome by (1) the development of more sensitive detectors, (2) contrast enhancement methods and (3) the application of averaging-based noise reduction algorithms (e.g. sub-tomogram averaging in cryo-ET). Digital imaging using charged-coupled device (CCD) cameras in TEM offers limited resolution as CCD cameras do not collect the information carried by the electrons directly. A phosphorescent scintillation screen converts the electron image to photons that are subsequently captured by the CCD camera. This electron-to-photon conversion step poses a limitation to the resolution of images captured by the CCD system. In recent years, the development of more sensitive, direct electron detection devices based on complementary metal–oxide–semiconductor (CMOS) detectors have revolutionized the field of cryo-EM [56]. Direct electron detectors/cameras can also improve resolution by minimizing beam-induced drift during imaging. Each tilt may be recorded in movie mode and the individual move slices can be aligned to remove sample drift. To collect high-resolution images, the sample must be 300-nm thick or thinner to insure that enough elastically-scattered electrons that interact with the sample reach the detector. Elastically-scattered electrons interact with the sample and alter the phase of the electron wave function without energy exchange; these electrons can be used for high-resolution imaging. Energy filters are often employed so that only zero-loss electrons contribute to the image. Cryo-samples thicker that 300 nm can produce inelastically scattered electron events that reduce resolution [33]. Since in-focus images have low contrast [57], frozen unstained biological samples are imaged off-focus using defocus phase contrast. This results in a phase contrast transfer function that provides selective contrast enhancement only in certain frequency ranges within the specimen [58]. In addition, defocus phase contrast entails a compromise between image contrast and resolution as the increased contrast results in lower resolution. To further improve contrast in cryo-EM, it is also possible to use phase plates that introduce a phase shift between the scattered and unscattered waves without the need of defocusing and over a wide range of frequencies [59,60]. Averaging-based algorithms applied to cryo-ET data can improve signal-to-noise ratio and therefore, resolution. If cryo-electron tomograms contain repetitive structural features, they can be extracted, averaged and classified using algorithms similar to those used in SPA. This approach is called sub-tomogram averaging [11,12]. However, different from SPA methods, sub-tomogram averaging needs to consider the anisotropic resolution inherent to ET [11]. Visualizing plant cells by electron tomography In combination with other imaging and molecular techniques, the ability to resolve the 3D architecture of cellular components at the nanoscale resolution by ET and cryo-ET provides powerful insights into fundamental plant cellular processes. ET was first applied in the plant cell biology field in 2001 by the Laboratory of Andrew Staehelin at the University of Colorado in Boulder for the study of plant cytokinesis [61]. Since then, ET of plastic sections and a few cryo-ET studies of plant cells and algae have been published reporting on a variety of cellular structures and processes, such as preprophase band assembly [62–64], phragmoplast organization [65] and cell plate maturation [66,67]; organelle dynamics during the cell cycle [68]; Golgi and trans Golgi Network (TGN) structure and dynamics [53,69–75] and COPI coat structure on Golgi-derived vesicles [52]; organization of the endoplasmic reticulum [76], its association with lipid droplets in leaves [77], and its contact sites with the plasma membrane [78] and other organelles [79]; formation of vacuoles and endosomes/prevacuolar compartments [69]; ultrastructural changes of plasmodesmata during development [80]; nuclear pore structure [51] and associations between 26S proteasome particles and nuclear pores [54]; the vesiculation process in multivesicular endosomes [81]; membrane dynamics in autophagy [82,83] (Fig. 3); membrane remodeling during viral infections [84–86]; formation and organization of thylakoid membranes [50,87–90]; mitochondrial structure under iron deficiency [86,91]; cell wall architecture [92]; flagellar architecture and function in Chlamydomonas [93,94]. Fig. 3. View largeDownload slide Electron tomographic reconstruction of vacuoles and prevacuolar compartment in maize endosperm cells in maize at 14 (a, a′) and 22 (b, b′) days after pollination. (a) and (a′) Tomographic slices and corresponding tomographic models of aleurone cells showing the distribution of storage protein-rich inclusions (asterisks), and intravacuolar autophagic membranes (depicted in green). LB, lipid body; M, mitochondrion; N, nucleus; P, plastid; PSV, protein storage vacuole. (b) and (b′) Tomographic reconstruction and tomographic model of an endosperm vacuole. Note the presence of a large electron-dense inclusion (asterisk), intravacuolar membranes (IM) and a globoid (GL). Bars = 500 nm in (a, a′), 100 nm (b, b′). Reprinted from Reyes et al. (2011) Plant Cell 23:769-84 (Copyright 2011 American Society of Plant Biologists). Fig. 3. View largeDownload slide Electron tomographic reconstruction of vacuoles and prevacuolar compartment in maize endosperm cells in maize at 14 (a, a′) and 22 (b, b′) days after pollination. (a) and (a′) Tomographic slices and corresponding tomographic models of aleurone cells showing the distribution of storage protein-rich inclusions (asterisks), and intravacuolar autophagic membranes (depicted in green). LB, lipid body; M, mitochondrion; N, nucleus; P, plastid; PSV, protein storage vacuole. (b) and (b′) Tomographic reconstruction and tomographic model of an endosperm vacuole. Note the presence of a large electron-dense inclusion (asterisk), intravacuolar membranes (IM) and a globoid (GL). Bars = 500 nm in (a, a′), 100 nm (b, b′). Reprinted from Reyes et al. (2011) Plant Cell 23:769-84 (Copyright 2011 American Society of Plant Biologists). ET has contributed critical insights on the mechanisms underlying dynamic processes in plant and algal cells. Moreover, ET has provided evidence of novel structures whose molecular identities are not entirely known. For example, ET studies of plant cells undergoing cytokinesis revealed the occurrence of a ribosome-depleted zone around by the forming cell plate and phragmoplast mid-zone. This zone also called cell plate assembly matrix is predicted to contain factors that promote membrane fusion and stabilization of microtubule plus ends during cytokinesis [66]. Another example is the recent visualization of intracisternal contents in the Chamydomonas Golgi apparatus, including an array of protein linkers that maintain the narrow luminal spacing of the trans cisternae. The molecular identity of these linkers is currently unknown but it has been speculated that they promote cargo exit from the Golgi by pushing cargo to the cisterna periphery [53]. Another fascinating example of how a detailed analysis of the 3D architecture of an organelle is critical to understand its basic functions comes from chloroplasts and their thylakoid membranes. A model of thylakoid organization developed in the 60’s and 7’0s postulated that each grana associates with multiple, right-handed helically-arranged stroma thylakoids, with both stacked and unstacked thylakoid membranes. In the context of this model, the fact that thylakoids are a continuous membranous and lumenal system has important implications for our understanding on the light-dependent photosynthetic reactions [95,96]. However, due to the complexity of the thylakoids arrays, it was challenging to fully appreciate their 3D organization. Only recently, ET studies on both plastic-embedded sections and cryofixed plant and Chlamydomonas samples were able to provide 3D structural evidence supporting the helical arrangement of thylakoid membranes [50,87,90,95]. Because segmented tomographic models contain quantitative spatial information, it is possible to use them to calculate distances between cellular structures, changes in membrane surface area and volume of complex organelles during maturation or other cellular events captured in electron tomograms. Thus, for example, it has been estimated that in the cellularizing Arabidopsis endosperm, ∼500,000 vesicles fuse together during the assembly of an individual cell plate but 75% of the total membrane is removed from cell plates during maturation [61], providing a notion of the magnitude of membrane trafficking fluxes during plant cytokinesis. Tomographic models can also be used as platforms for mathematical simulations. For example, geometries derived from multivesicular endosomes undergoing vesiculation have been used for calculating diffusion of membrane proteins on endosomes [81]. Other tomography imaging approaches As noted, an important constrains in ET is sample thickness, 300 nm for ET and 1.5 μm for STEM-ET. As a consequence, the analysis of a whole eukaryotic cells (5–100-μm thick) and tissues by ET requires laborious and time-consuming serial sectioning and acquisition of multiple tilt-series. An alternative tomographic approach suitable for whole cell imaging is cryo-soft X-ray tomography (cryo-SXT). Here, whole cells with a thickness of up to 15 μm can be imaged in single tomograms using soft X-ray radiation produced by a synchrotron. There is tradeoff in resolution, however, as cryo-SXT is restricted to a resolution of 25 nm. Image contrast is achieved by using the differential absorption of soft X-ray of biological molecules at the ‘water window’ (the spectral region defined by the X-ray absorption of carbon and oxygen) [97]. At this energy range, X-rays within this region are absorbed an order of magnitude more strongly by biological materials than by water; this difference can be translated into image contrast [97]. Soft X-ray tomography offers some unique advantages: the samples are in a frozen-hydrated state so there is no need for fixation and embedding, and it can be imaged directly without staining with heavy metals. In addition, as samples can be freely rotated under the soft X-ray beam, the derived tomographic reconstructions do not suffer from the missing wedge of information as seen in ET. X-ray tomography has been applied to the analysis of intact Chlamydomonas cells [98]. Future perspectives The field of ET has undergone amazing changes in the last decade, with new approaches for direct visualization of frozen-hydrated biological samples, the development of direct electron detectors, and methods for fully automated collection of tilt series. On the sample preparation side, there are three areas that offer new exciting possibilities for the plant cell biology field: (a) new and improved approaches to generate thin lamellas from high-pressure frozen tissues; (b) correlative light and cryo-electron microscopy methods to map the location of fluorescent signals into cryo-EM images and (c) the development of genetically-encoded EM-tags. As mentioned above, the application of cryo-ET to plant cells and tissues will require either cryo-ultramicrotomy followed by in situ cryo-lamella or cryo-lamella lift-out approaches. Although these two approaches remain technically challenging at present and restricted to a few laboratories with the required expertise, the development of new accessories and micromanipulators are making these methods more broadly accessible. The imaging of vitreous cryo-lamellas [33] and cryo-sections [99] by cryo-fluorescent microscopy makes possible the integration of light and electron microscopy data. This approach called correlative light and cryo-electron microscopy (cryo-CLEM) entails the visualization of fluorescent signals (e.g. fluorescently-tagged proteins) in vitrified cells using a light microscope. The vitrified specimen is kept in a specialized cryo-stage at cryogenic temperatures during fluorescence imaging. The location of the fluorescence signal is registered to identify regions of interest that can be later mapped in cryo-sections or cryo-lamellas for cryo-ET imaging [100,101]. One of the most challenging tasks in ET is the identification of molecules and protein complexes within the tomographic reconstructions. One approach for protein identification is the development of genetically-encoded, electron-dense protein tags that can be visualized by TEM. Although a robust and general tag for EM detection has not been developed yet, some peptides based on metallothionein, a small protein of ~60 amino acids that can bind ~20 gold atoms, seem to hold some promise [102,103]. In addition, other tags that can be visualized by both light and electron microscopy (CLEM tags) are constantly being developed. Many of them use diaminobenzidine (DAB) to induce electron-dense precipitates through polymerization either using peroxidase activity [104–106] or photo-oxidation [107,108]. However, DAB needs to be introduced into the biological samples in the presence of detergents and often results into a diffuse precipitates. A newly developed CLEM tag called FerriTag [109] is based on the ability of ferritin particles to sequester iron atoms and can be induced to oligomerize with the protein of interest using the FK506-binding protein (FKBP)–rapamycin–FRB (FKBP-rapamycin binding) heterodimerization system [110]. FerriTag consists of an untagged ferritin light chain and FRB–mCherry–ferritin heavy chain and needs to be co-expressed with the protein of interest fused to FKBP-GFP. In the presence of rapamycin, the FKBP and FRB domains heterodimerize and the target protein becomes FerriTagged. So far, this tag has only been tested in mammalian cells resulting in tightly defined electron densities ideal for nanoscale mapping of protein distribution. However, although FerriTag offers some advantages compared with DAB-based CLEM tags, it is also limited in its application since it may not be compatible with iron-sensitive systems and it is only effective when the FKBP and the FRP domains are in the same subcellular compartment. Funding The research conducted in the Otegui Laboratory on plant cell imaging is supported by the National Science Foundation grants MCB1614965, IOS1457123, IOS 1329956 and the National Institute of Food and Agriculture, United States Department of Agriculture, Hatch Act Formula Fund WIS01791 to M.S.O. References 1 Jin D , Xi P , Wang B , Zhang L , Enderlein J , and van Oijen A M ( 2018 ) Nanoparticles for super-resolution microscopy and single-molecule tracking . Nat. Methods 15 : 415 – 423 . Google Scholar Crossref Search ADS PubMed 2 Huang B , Babcock H , and Zhuang X ( 2010 ) Breaking the diffraction barrier: super-resolution imaging of cells . Cell 143 : 1047 – 1058 . Google Scholar Crossref Search ADS PubMed 3 Sahl S J , Hell S W , and Jakobs S ( 2017 ) Fluorescence nanoscopy in cell biology . Nat. Rev. Mol. Cell Biol. 18 : 685 . Google Scholar Crossref Search ADS PubMed 4 Komis G , Samajova O , Ovecka M , and Samaj J ( 2015 ) Super-resolution microscopy in plant cell imaging . 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TI - Electron tomography in plant cell biology
JF - Microscopy
DO - 10.1093/jmicro/dfy133
DA - 2019-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/electron-tomography-in-plant-cell-biology-JniBKwZ3Jv
SP - 69
VL - 68
IS - 1
DP - DeepDyve
ER -