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Electron tomography (ET) is a tomography technique for obtaining detailed 3D structures[1] of sub-cellular, macro-molecular, or materials specimens. Electron tomography is an extension of traditional transmission electron microscopy and uses a transmission electron microscope to collect the data. In the process, a beam of electrons is passed through the sample at incremental degrees of rotation around the center of the target sample. This information is collected and used to assemble a three-dimensional image of the target. For biological applications, the typical resolution of ET systems[2] are in the 5–20 nm range, suitable for examining supra-molecular multi-protein structures, although not the secondary and tertiary structure of an individual protein or polypeptide.[3][4] Recently, atomic resolution in 3D electron tomography reconstructions has been demonstrated.[5][6]
In the field of biology, bright-field transmission electron microscopy (BF-TEM) and high-resolution TEM (HRTEM) are the primary imaging methods for tomography tilt series acquisition. However, there are two issues associated with BF-TEM and HRTEM. First, acquiring an interpretable 3-D tomogram requires that the projected image intensities vary monotonically with material thickness. This condition is difficult to guarantee in BF/HRTEM, where image intensities are dominated by phase-contrast with the potential for multiple contrast reversals with thickness, making it difficult to distinguish voids from high-density inclusions.[7] Second, the contrast transfer function of BF-TEM is essentially a high-pass filter – information at low spatial frequencies is significantly suppressed – resulting in an exaggeration of sharp features. However, the technique of annular dark-field scanning transmission electron microscopy (ADF-STEM), which is typically used on material specimens,Cite error: Closing </ref> missing for <ref> tag This method is relevant to the physical sciences, where cryo-EM techniques cannot always be used to locate the coordinates of individual atoms in disordered materials. AET reconstructions are achieved using the combination of an ADF-STEM tomographic tilt series and iterative algorithms for reconstruction. Currently, algorithms such as the real-space algebraic reconstruction technique (ART) and the fast Fourier transform equal slope tomography (EST) are used to address issues such as image noise, sample drift, and limited data.[8] ADF-STEM tomography has recently been used to directly visualize the atomic structure of screw dislocations in nanoparticles.Cite error: Closing </ref> missing for <ref> tag and 20,000 atoms in a multiply twinned palladium nanoparticle.[9] The combination of AET with electron energy loss spectroscopy (EELS) allows for investigation of electronic states in addition to 3D reconstruction.[10] Challenges to atomic level resolution from electron tomography include the need for better reconstruction algorithms and increased precision of tilt angle required to image defects in non-crystalline samples.
The most popular tilting methods are the single-axis and the dual-axis tilting methods. The geometry of most specimen holders and electron microscopes normally precludes tilting the specimen through a full 180° range, which can lead to artifacts in the 3D reconstruction of the target.[11] Standard single-tilt sample holders have a limited rotation of ±80°, leading to a missing wedge in the reconstruction. A solution is to use needle shaped-samples to allow for full rotation. By using dual-axis tilting, the reconstruction artifacts are reduced by a factor of [math]\displaystyle{ \sqrt{2} }[/math] compared to single-axis tilting. However, twice as many images need to be taken. Another method of obtaining a tilt-series is the so-called conical tomography method, in which the sample is tilted, and then rotated a complete turn.[12]
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Categories: [Electron microscopy] [Multidimensional signal processing] [Condensed matter physics]