Title: Cryo-Electron Tomography: 3D Cell-Imaging at Molecular Resolution
Author: Ryan Gomoto
Abstract: Cryo-electron tomography is an emerging 3D cell-imaging technique, which can resolve subcellular structures down to a few nanometers. The goal of cryo-ET is to create a 3D computerized reconstruction of a cell. Cryo-ET involves first cryogenically freezing a cell so rapidly that its water forms vitreous ice. Then a transmission electron microscope (TEM) images the frozen cell in small-angle increments. In the final step of cryo-ET, computer programs integrate the series of projection images into a 3D representation of the cell, called a tomogram.
In recent research, cryo-ET has revealed such structures as the synaptic vesicles in a neuron. If it can achieve atomic resolution, cryo-ET may one day let scientists and doctors peer into our very own cells.
Keywords: cryo-electron tomography, cryo-ET, electron microscopy, tomography, cell, imaging

What if we could see into our own cells? Imagine viewing life at the molecular level in high- definition. Imagine doctors diagnosing diseases by looking directly at the molecules in a cell.
Currently the best options for visualizing molecules are X-ray crystallography and nuclear magnetic resonance (NMR). But although these techniques can routinely reveal protein structures with atomic detail, we can only use them to study one protein at a time. Since the spatial distribution of proteins is an important factor in cell function, scientists need a technology that not only can show atomic details but can also report the positions of proteins in an intact cell.
Cryo-electron tomography (cryo-ET) has the potential to do both. Cryo-ET is a 3D cell-imaging technique, which can image subcellular structures down to a few nanometers. The goal of cryo-
ET is to create a 3D computerized reconstruction of a cell. Cryo-ET uses a transmission electron microscope (Fig. 1) to image a cryogenically frozen cell from multiple angles. Then computer programs reconstruct a model of the cell based on the series of 2D images. Cryo-ET has the potential to reveal both structural and positional details of every protein inside a cell. In recent research, cryo-ET has revealed such structures as the synaptic vesicles in a neuron. If it can achieve atomic resolution, cryo-ET may one day let scientists and doctors peer into our very own cells.
Figure 1. Titan Krios™ Transmission Electron Microscope, FEI Company [1].
At its simplest, a cell is an enclosed water droplet packed with moving molecules. Before we can image a cell, we must cryogenically freeze it, so that its molecules stop moving. Since a cell is mostly water, to understand how to freeze a cell, we must understand that water can freeze in different ways. Usually water molecules settle into a crystal lattice, forming ice. However, if they are frozen quickly enough, water molecules can stop moving before they can form an ordered array. This disordered structure of frozen water molecules is known as vitreous ice. In

vitreous ice, water molecules stop almost instantaneously, such that their arrangement resembles that of liquid water molecules frozen in time. Thus, any dissolved protein molecule is hydrated as it would be in its native state [2]. A cryogenically frozen cell is a snapshot of a live cell at an instant in time.
In electron tomography, a transmission electron microscope images an object in small-angle increments. Then a special software package integrates the series of projection images into a 3D representation of the object. A projection image is akin to a shadow on a wall. The idea behind electron tomography is that with enough shadows, we can reconstruct the shape of the object creating the shadows.
Transmission Electron Microscopy
A transmission electron microscope (TEM) creates a projection image of a cell by shining an electron beam through it. Then the TEM rotates the sample and acquires another image. Over a hundred projection images may be recorded per sample. A TEM is analogous to a common light microscope (summarized in Table 1). They are similar because both electrons and light
(photons) travel in waves. Like a ray of light, a beam of electrons can be focused and magnified to form an image. In transmission electron microscopy, we use electrons to "see" objects.
Table 1. Light microscope vs. transmission electron microscope. Adapted from [3].
LIGHT MICROSCOPE TRANSMISSION ELECTRON
MICROSCOPE
Source of illumination Ambient light Electron gun
Lens
Sample platform
Viewing the sample
Glass lenses
Glass slide
Eyepiece
Electromagnetic lenses
Electron microscopy grid
Digital camera
Use of vacuum No vacuum Entire electron path from gun to camera is under vacuum.
A basic TEM consists of an electron source, electron lenses, a specimen platform, and a detector
(Fig. 2). The electron source is typically an electron gun, which generates electrons and accelerates them using high voltages. Emitted electrons are monochromatic (all the same wavelength). The condenser lens focuses the monochromatic electrons into a narrow beam.
Since electrons are charged particles, focusing or magnifying an electron beam requires an electromagnetic lens (Fig. 3). This type of lens produces a magnetic field in order to deflect electrons that are far from the central axis inward, while leaving electrons that are on the center of the axis untouched. A narrow electron beam leaves the condenser lens and passes through the specimen, which is fixed to an electron microscopy (EM) grid. The EM grid is analogous to the glass slide in light microscopy. The grid is a metal mesh structure lined with carbon and is a few millimeters in diameter. Cell and tissue samples that are mounted onto the EM grid must be made thin, because the electron beam must be transmitted through it. After the electrons pass through the sample, the objective lens, also an electromagnetic lens, focuses them into a real image. Additional lenses (not shown) create a magnified image, which is captured by a detector.

In modern TEMs, the detector is a digital CCD (charge-coupled device) camera, which transmits the measured signals to a computer. The TEM's software compiles the signals into a projection image.
For electrons to behave most closely to light, the inside of a TEM must be under vacuum. Since electrons have mass, gas particles lingering in the path of the electron beam would distort the beam and thus the final image. Therefore the entire path of the electron beam, from electron gun to detector, must be evacuated of gas particles to ensure that electrons are focused, transmitted, and magnified accurately.
Figure 2. Transmission electron microscope diagram. Available: http://www.mete.metu.edu.tr/pages/tem/TEMtext/TEMtext.html
Figure 3. Electromagnetic electron lens. The size of the arrows represent the size of the electron beam before and after passing through the lens. Available: http://www.microscopy.ethz.ch/lens.htm
3D Reconstruction
During reconstruction, the final step in cryo-ET, computer programs analyze the "shadows" on the 2D projection images and create a 3D model or "tomogram" of the cell. Every projection image is a picture of how the electron beam navigates through the cell (Fig 4a). Therefore, we can qualitatively understand reconstruction with the concept back-projection (Fig 4b). Backprojection is the inverse process of image formation. As shown in Figure 4b, we can compare back-projection to shining a light through each image in the reverse direction. Regions in space where the light rays are intense represent dense areas in the cell. If the 2D images are back-

projected at the proper angles, they should intersect in a way that recreates the electron density pattern of the cell.
Another way to think about reconstruction is to imagine a cell divided into extremely tiny cubes, where each cube shapes the electron beam as it passes through the cell. Since electrons repel other electrons, the cubes with high electron density will block and scatter the electron beam, while the cubes with low electron density will transmit the beam. The goal of reconstruction, then, is to solve for the average electron density of each cube. All of the necessary information lies in the set of projection images. One projection image alone is not enough, because it conveys the cumulative effect of many cubes. However, by viewing the same set of cubes from many different angles, we can isolate the effect of each cube. In other words, we can find each cube's electron density. In the tomogram, a voxel (3D pixel) and its grayscale shade represent each cube and its electron density. 3D graphics software can render the tomogram into a colorful
3D image (Fig. 5).
Figure 4. 3D Reconstruction. a) 2D projection images. b) 3D reconstruction by back-projection. Available: http://www.nature.com/nature/journal/v422/n6928/fig_tab/nature01513_F6.html

Figure 5. Cryo-electron tomography of presynaptic vesicles (beige) connected by synaptic vesicle tethers (red) in a rat hippocampal neuron. Available: http://www.mpg.de/print/11952
Capabilities
Research groups around the world are using cryo-electron tomography to visualize subcellular structures. Baumeister and colleagues at the Max Planck Institute of Biochemistry in
Martinsried, Germany have used cryo-ET to study synaptic vesicles in a rat hippocampal neuron
(Fig. 5). Their research has provided insight into the spatial distribution of vesicles, synaptic vesicle size, the tethering of synaptic vesicles to the membrane, and the interconnectivity of vesicles [4]. Nearby, at the European Molecular Biology Laboratory (EMBL) in Heidelberg,
Germany, Beck and colleagues use cryo-ET to study the nuclear pore complex, which is a large protein complex regulating transport across the nuclear membrane (Fig. 6). They have combined cryo-ET with computational simulations in order to study dynamic events at the nuclear pores
[5]. Meanwhile in America, Jensen and colleagues at the California Institute of Technology in
Pasadena, California use cryo-ET to study the structure of bacterial cells. For a video discussing their work and the cryo-ET process in general, see [6]. In their research, they have reconstructed the motor-like structure that turns the flagellum in bacteria.
Figure 6. Cryo-ET tomogram of a nuclear pore complex. Available: http://www.embl.de/research/units/scb/beck/beck_2l.jpg

Barrier to atomic resolution
In the studies mentioned above, the cryo-ET tomograms have resolutions no better than 2 nm [4].
However, electrons should theoretically be able to resolve structures down to the atomic level
[7]. Cryo-ET cannot currently offer atomic resolution because the TEM electrons gradually damage the cell by breaking chemical bonds and forming free radicals [8]. The effects of high electron doses include molecular damage, cell shrinkage, and bubble formation [9]. Since these effects shift the contents of the cell during imaging, they cause blurred, low-resolution tomograms.
If these negative effects did not occur, the highest dose of electrons would be preferred, because it would maximize signal-to-noise ratio in the projection image and ultimately in the tomogram.
But because they do occur, it is clear that electron dose must be optimized. There is a tradeoff between preserving the sample and having a strong, clear signal. Although some researchers are starting to develop low-dose tomography strategies [9], electron damage in cryo-ET remains a key obstacle to atomic resolution.
If cryo-ET ever reaches atomic resolution, it would revolutionize science and medicine.
Scientific research would shift from reconstructing only large vesicles and large protein complexes, such as the nuclear pore complex and the flagellar motor, to visualizing every size of protein. We would be able to document every protein in a cell by simply inspecting a 3D image.
In medicine, doctors may even be able to diagnose a patient by analyzing a cryo-ET tomogram of the patient's diseased or cancerous cell. But even if cryo-ET never reaches atomic resolution, it is still a powerful imaging technique that can depict fundamental building blocks of life in a single image.
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