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, [8] more effectively suppresses phase and diffraction contrast, providing image intensities that vary with the projected mass-thickness of samples up to micrometres thick for materials with low atomic number. ADF-STEM also acts as a low-pass filter, eliminating the edge-enhancing artifacts common in BF/HRTEM. Thus, provided that the features can be resolved, ADF-STEM tomography can yield a reliable reconstruction of the underlying specimen which is extremely important for its application in materials science. [9] For 3D imaging, the resolution is traditionally described by the Crowther criterion. In 2010, a 3D resolution of 0.5±0.1×0.5±0.1×0.7±0.2 nm was achieved with a single-axis ADF-STEM tomography. [10]
Atomic level resolution in 3D electron tomography reconstructions has been demonstrated. Reconstructions of crystal defects such as stacking faults, grain boundaries, dislocations, and twinning in structures have been achieved. [11] 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. [12] ADF-STEM tomography has recently been used to directly visualize the atomic structure of screw dislocations in nanoparticles. [13] [14] [15] [16] AET has also been used to find the 3D coordinates of 3,769 atoms in a tungsten needle with 19 pm precision [17] and 20,000 atoms in a multiply twinned palladium nanoparticle. [18] The combination of AET with electron energy loss spectroscopy (EELS) allows for investigation of electronic states in addition to 3D reconstruction. [19] 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. [20] 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 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. [21]
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, [8] more effectively suppresses phase and diffraction contrast, providing image intensities that vary with the projected mass-thickness of samples up to micrometres thick for materials with low atomic number. ADF-STEM also acts as a low-pass filter, eliminating the edge-enhancing artifacts common in BF/HRTEM. Thus, provided that the features can be resolved, ADF-STEM tomography can yield a reliable reconstruction of the underlying specimen which is extremely important for its application in materials science. [9] For 3D imaging, the resolution is traditionally described by the Crowther criterion. In 2010, a 3D resolution of 0.5±0.1×0.5±0.1×0.7±0.2 nm was achieved with a single-axis ADF-STEM tomography. [10]
Atomic level resolution in 3D electron tomography reconstructions has been demonstrated. Reconstructions of crystal defects such as stacking faults, grain boundaries, dislocations, and twinning in structures have been achieved. [11] 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. [12] ADF-STEM tomography has recently been used to directly visualize the atomic structure of screw dislocations in nanoparticles. [13] [14] [15] [16] AET has also been used to find the 3D coordinates of 3,769 atoms in a tungsten needle with 19 pm precision [17] and 20,000 atoms in a multiply twinned palladium nanoparticle. [18] The combination of AET with electron energy loss spectroscopy (EELS) allows for investigation of electronic states in addition to 3D reconstruction. [19] 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. [20] 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 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. [21]