Entomological Research •• (2016) •• –••
RES EAR CH P APE R
Techniques and applications of three-dimensional electron microscopy in entomological research Min Kyo JUNG1,2*, Min Soo KANG3* and Ji Young MUN4,5 1
School of Life Sciences and Biotechnology, Korea University, Korea Department of Convergence Medicine, University of Ulsan College of Medicine and Asan Institute for Life Sciences, Asan Medical Center, Seoul, South Korea 3 Department of Medical IT Marketing, College of Health Industry, Eulji University, Korea 4 Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Korea 5 BK21 Plus Program, Department of Senior Healthcare, Graduate School, Eulji University, Korea 2
Correspondence Ji Young Mun, Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam-Si, Gyeonggi-Do 13135, Republic of Korea. Email:
[email protected] Received 10 January 2016; accepted 17 May 2016. *Equal first author contributions. doi: 10.1111/1748-5967.12180
Abstract Structural studies using two-dimensional (2D) images show limitations in understanding the structure and functions of cellular organelle and protein. To overcome the difficulty, over the last few years 3D reconstruction techniques using electron microscopy have been developed at extremely high speed. In this paper, currently available 3D reconstruction techniques of electron microscopy (such as electron tomography, serial section analysis and single particle analysis) are introduced using our data as examples of the application. The 3D structure of mitochondria with the defect of mitochondrial protein in round worm, Caenorhabditis elegans, through electron tomography, the cell–cell interaction in lamina of Drosophila melanogaster by serial-section using ultramicrotome and high-voltage electron microscopy and a thin filament related to muscle contraction in Drosophila melanogaster were used for examples of the application. These results through 3D reconstruction reveal the structural changes in a cellular organelle and protein that had not been shown by 2D structure. Key words: 3D structure, cellular structure, electron microscopy, protein.
Introduction A general method for observation of cellular ultrastructure using transmission electron microscopy (TEM) includes embedding a sample in plastic resin, ultra-thin sectioning (60–70 nm) and double staining with uranyl acetate and lead citrate (Cheville & Stasko 2014). However, these thin sections only allow observation of a limited area, and the original structure can not be seen (Hall 1995). Even though the limitation of the sectioning process can be avoided in the study of macromolecules, it is still possible to misunderstand their structure because of the direction of loading on grid (Sorzano et al. 2004). Three-dimensional (3D) reconstruction through electron microscopy (3DEM) techniques have been developed to overcome this issue. These techniques can be divided into three main types:
© 2016 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
(i) electron tomography; (ii) the serial-section method; and (iii) the single-particle method (Fig. 1). Specially, tomography and the single-particle method is not only used to reconstruct the 3D structure but also to greatly improve the resolution of image. Each method will be explained with our data in this paper.
Materials and methods Sample preparation of animal tissues Adult worms and flies were selected under a stereoscope and transferred to sample carriers. To prevent cryo-damage, we added one drop of 1-hexadecene to each sample to fill any
M. K. Jung et al.
Figure 1 3D reconstruction method for electron microscopy. Tomography for cellular organelle, serial-section technique using transmission and scanning electron microscopy for tissue and cell structure, and Single-particle analysis for protein monomer and complex.
air cavities, and then loaded the samples carefully into specimen carriers. After loading, the samples were cryoimmobilized in a jet freezer (JF 8000, RMC; Boekeler, Tucson, AZ, USA) and the frozen samples were transferred to a homemade freeze-substitution unit. Freeze-substitution was proceeded in dry acetone containing 2% osmium tetroxide and 0.2% uranyl acetate through serial incubations of 72 h at –80 °C, 3 h at –20 °C and 3 h at 4 °C. The samples were then transferred to room temperature and embedded using a Spurr kit (EMS, Hatfield, PA, USA) (Mun et al. 2010). Electron tomography based 3D reconstruction For electron tomography, samples were sectioned (60 nm) with an ultra-microtome (RMC MTXL) and double-stained with uranyl acetate and lead citrate. The sections were then viewed under a Tecnai 12 electron microscope (FEI, Eindhoven, Netherlands) at 120 kV. Colloidal gold particles were applied to both sides of sections as fiducial markers. A tilt series of 61 images was recorded around one tilt axis over an angular range of 120° with 2° tilt intervals (Mun et al. 2010). Images were aligned by ETOMO software. Surfacerendered models were produced using the IMOD program (Coquelle et al. 2011).
formvar carbon, and double-stained with 3% uranyl acetate and lead citrate. The serial sections were then imaged under a JEM-ARM1300S high-voltage transmission electron microscope (JEOL, Tokyo, Japan, installed at Korea Basic Science Institute, Daejeon, Korea) at 1,250 kV (Lee et al. 2005). Aligned serial images were rendered by IMOD.
Negatively stained 3D electron microscopy for proteins Purified thin filaments in EGTA (7 μL) were applied to carbon-coated grids, negatively stained with 1% uranyl acetate and dried (Cammarato et al. 2004). Electron microscope images were recorded at 80 kV on a Tecnai 12 electron microscope using low-dose conditions. The images of filaments were selected and straightened using ImageJ (NIH, MD, USA), and converted to SPIDER format and cut into segments in SPIDER (Wadsworth Center, NY, USA). Iterative helical real-space reconstruction was carried out using SPIDER with F-actin as an initial reference model (Mun et al. 2014). UCSF Chimera (CA, USA) was used for visualization, analysis and atomic fitting of 3D volumes.
Results and discussion
Serial section based 3D reconstruction
Electron tomography
The sections with 250 nm thickness using an ultramicrotome (RMC MTXL) were collected on copper slot grids coated with
Electron tomography (ET) has been applied to 3D reconstruction of cells, cell organelles, and proteins (Frey et al.
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Entomological Research •• (2016) •• –•• © 2016 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
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2006; Heymann et al. 2013). The tilt-series of images can be computationally reconstructed into a 3D structure obtained from back projection. The sample thickness that the electron beam needs to pass through is increased when the tilt angle is large, and the range of tilt angles is generally known as ±60°. In order to minimize data loss, the tilt range can be expanded up to ±90°, and the double tilt method has been applied. Higher resolution can be obtained by subtomogram averaging of the multiple tomograms (Voortman et al. 2014). Similar to single-particle analysis, the subtomogram averages multiple copies of identical objects to increase the signal-tonoise ratio. In entomology, ET has been used for detailed structural change at high resolution. Eltsov et al. (2015) reported cytoskeletal reorganization during epithelial tissue sealing by large-volume ET in a Drosophila embryo, and Perkins et al. (2012) showed a honeycomb-like structure in the mitochondria of hypoxia-adapted Drosophila melanogaster through ET. Life cycles of viruses transmitted by insect cells (Miyazaki et al. 2013) and novel actin–myosin interactions in contracting insect flight muscle (Wu et al. 2010) were visualized by electron tomography. Our ET data also showed specific mitochondrial proteins take part in functions of mitochondria controlling cristae structure in worms (Han et al. 2006; Lee et al. 2009). Besides 2D images of the mitochondria with small and numerous vesicles in contrast to the tubular cristae of wild-type worms, 3D structure using electron tomography with high resolution revealed the factor controlling the structure of cristae and number of cristae junctions. In addition, 3D structures showed reduction of contact sites between the outer and inner membranes (Fig. 2). The contact sites, called cristae junctions, have been reported as factors controlling mitochondrial functions such as the channeling of metabolites, coordinated fusion and fission of
mitochondria, and protein transport (Reichert & Neupert 2002). These results suggest structural changes directly relate to function of cell organelles, and they emphasize the necessity of high-resolution imaging and electron tomography. Serial section method The equipment for serial imaging for cells and tissue can be divided into transmission electron microscopy (TEM) (Mishchenko 2009) and scanning electron microscopy (SEM) (Briggman & Denk 2006). Serial sections manually acquired by ultramicrotome were imaged by conventional TEM or high-voltage transmission electron microscopy (HVEM). Manual serial sectioning requires greater effort and time, but it has the big advantage of storing each section on grids for double staining or immune gold labeling. HVEM can help to decrease the effort and time for serial sectioning (Mun et al. 2009). HVEM can obtain images from 10 times thicker sections because it has 10 times stronger penetration than conventional TEM. For automatic serial sections, surface block-face SEM (SBF-SEM) comprising ultramicrotome (3VIEW, commercialized by Gatan; and Teneo VS, commercialized by FEI Co.) or focused ion beam (FIB), which is mounted inside the SEM vacuum chamber, and automatic tape-collecting ultra-microtome (ATUM) have been developed. Because the resin-embedded sample is directly imaged by detection of back-scattered electrons, serial sections are easy to align for 3D reconstruction without loss. In the ATUM technique, serial sections made by ultramicrotome can be collected automatically on the specific tape and can be stored on silicon wafers. The serial sections obtained through ATUM can be observed using SEM, but specific software package for mapping and imaging of select
Figure 2 Application of electron tomography. The structural changes in mitochondria due to defects of mitochondrial protein were investigated by electron tomography and 3D reconstruction. For tilt series, mitochondria in C. elegans were fixed by rapid freezing and freeze substitution for better preservation. The tilt images of mitochondria (±60°) were used for electron tomography and 3D reconstruction. The 3D structure shows the changes of cristae and cristae junction in mitochondria with deficiency of protein.
Entomological Research •• (2016) •• –•• © 2016 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
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regions within a library of ultrathin sections should be applied (Hayworth et al. 2014). SBF-SEM and ATUM are becoming prominent for collecting 3D data of large samples in relatively short times; this functionality is especially appropriate for the field of connectomics, which attempts to comprehensively understand the network of neurons in the human brain (Kasthuri et al. 2015; Wanner et al. 2015). The serial section based 3D structure of mitochondria and microtubules in D. melanogaster ommatidia showed that mitochondria are mainly located in the distal region near lens, and microtubules are mainly located in the distal and basal regions. The 3D reconstruction of these organelles can be used for critical evaluation of the dynamic change of cellular organelles caused by functional abnormalities like retinal degeneration (Mun et al. 2009). Figure 3 shows the process of 3D reconstruction and the lamina structure in D. melanogaster retina as an example. In addition, Butcher et al. (2012) showed different classes of input and output neurons in the Drosophila mushroom body calyx through serial-section TEM. After development of the SBF-SEM system, as well as representative model systems like Caenorhabditis elegans and D. melanogaster, the detailed structure of the compound eye visual system in sea spider, Achelia langi, were studied through FIB-SEM based 3D reconstruction (Lehmann et al. 2014). These revolutionary serial section 3DEM techniques make it possible to construct 3D structures of large volumes
of tissue (hundreds of micrometers), and it is sure to overcome the limitations of structural studies using 2D images.
Single-particle analysis Three-dimensional structures are extremely important for understanding structural changes or the binding domain of proteins. X-ray crystallography, electron microscopy and nuclear magnetic resonance (NMR) have been used as traditional methods for creating 3D structures of proteins. Xray crystallography is limited because it can not analyze the structure of uncrystallized protein. On the contrary, electron microscopy can be used for studying large protein complexes and the interactions of different proteins, because it is not necessary to crystallize the proteins. Electron microscopy is known to have significantly lower resolution than X-ray crystallography, but its resolution has approached the atomic level following the development of software and devices. The 2D averaging technique obtains the mean value of images and classifies them according to shape, making it possible to reconstruct a 3D structure (Jung et al. 2008). In order to investigate detailed structure of protein, at least tens of thousands images of single molecules should be used during image processing. The software for automated data acquisition and image processing has been developed and
Figure 3 Serial section and 3D reconstruction through transmission electron microscopy (SSTEM). Sections are cut manually with an ultramicrotome, floated onto a water boat and then picked up onto one-hole grids. Conventional TEM imaging after serial section (thin sections with 60–70 nm thickness) or high-voltage electron microscopy serial imaging of thick sections (250 nm) can be applied for 3D TEM. Examples of 2D and 3D images are lamina structure in D. melanogaster.
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Entomological Research •• (2016) •• –•• © 2016 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
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Figure 4 3D reconstruction of protein through single-particle analysis. Transmission electron microscope images of a protein were boxed the same size and aligned for 3D reconstruction. A series of 2D projections were calculated from a 3D reference model. 3D reconstruction of a thin filament in D. melanogaster showed known binding sites between tropomyosin and F-actin in the filament.
commercialized to minimize the time needed for studying detailed structures of proteins (Egelman 2010). For studies on the binding site between two muscle proteins purified from D. melanogaster, proteins were imaged by TEM, and then 3D structure was reconstructed through single-particle analysis techniques including iterative helical real space reconstruction (IHRSR) (Fig. 4). These muscle proteins have almost the same structure and binding sites as mouse recombinant protein (Mun et al. 2014, 2016; Previs et al. 2015, 2016). A known X-ray structure can be fitted to the 3DEM structure to help to analyze which domain is the binding site between proteins (Mun et al. 2011). These analyses can show the change of binding site and the structural change of the protein itself relating to myopathy (Previs et al. 2015). Several groups have reported the applications of singleparticle analysis to myosin filaments from tarantula (Zoghbi et al. 2004; Zhao et al. 2008; Alamo et al. 2015, 2016) and insect flight muscle (Al-Khayat et al. 2004, 2009). In addition, troponin complex from Lethocerus indicus asynchronous flight muscle was studied by single-particle analysis (Wendt & Leonard 1999). These data revealed how the proteins control muscle contraction through structural change. Because detailed structural changes of proteins cause various functional changes, imaging analysis through 2D averaging with high-resolution and 3D reconstruction is necessary for the study of proteins. It will be a very useful imaging tool for proteins in insects.
Further directions There is a global trend of increase in demand for high-resolution 3D imaging techniques in biology and medicine. The investigation of cellular structural change and related functional study in entomology are necessary for analyzing biological phenomena or specific diseases. In addition, the study of Entomological Research •• (2016) •• –•• © 2016 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
molecular structures of proteins can directly be used for identifying the mechanism of how the proteins cause changes in cellular functions. Furthermore, visualization of the mechanism through 3DEM can help to develop and produce new medicines. Therefore, the rapid expansion of the 3DEM field coupled with tremendous technological developments has brought about large opportunities and challenges in structural biology. The resolution revolution through cryotechniques is still developing as a major technological breakthrough. Correlative light and electron microscopy (CLEM) made it possible for fluorescently labeled proteins localized by light microscopy to be visualized with subcellular structures by electron microscopy (Bykov et al. 2016). These techniques combined with 3DEM will certainly yield further structural information in entomology.
Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2015R1C1A1A02037153).
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