In ADF-STEM electron tomography, a focused electron beam with sub-nanometre diameter is rastered across a sample of interest. Electrons scattered from the sample are recorded using an annular dark field detector, which generates a 2D projection image of the sample27,28.

The viewing angle is changed by rotating the specimen and a series of projection images from different angles is acquired. For the vast majority of electron microscopes, a single axis of rotation is permitted by the stage, although more complex tilt geometries have been demonstrated17,29,30. After each successive tilt during the experiment, the specimen moves relative to the electron beam and must be re-centred. The specimen can only be re-centred approximately at the time of acquisition and so the data is said to be ‘misaligned’. The end result of a tomographic experiment is a set of ‘misaligned’ STEM images corresponding to a specific specimen tilt.

Aligning the STEM images prior to reconstruction is vital to establish a common axis of rotation (i.e., tilt-axis) in the image series. Alignment methods include the use of fiducial markers31,32, cross correlation33, and centre of mass. Once aligned, a reconstruction algorithm is used to generate a 3D reconstruction of the sample.

The basic steps of the tomographic method are illustrated in Fig. 1.

Figure 1: Illustration of electron tomography data acquisition and reconstruction process. Series of 2D images acquired of object of unknown 3D structure at different viewing angles. Images shown are from tiltser_Co2P.tif. 2D images combined into image stack ordered by viewing angle i.e., a tilt series. Tilt series is aligned, and reconstruction algorithm is applied to produce 3D reconstruction of object. A 3D isosurface visualization of recon_Co2P.tif is shown as an example rendered using tomviz. Full size image

Tom_1: Tomography of hyperbranched Co 2 P nanoparticle

Sample preparation

Hyperbranched Co 2 P nanocrystal synthesis methods and scientific relevance are discussed in detail by Zhang et al.26 Samples were prepared for tomographic analysis by pipetting a drop of organic solution containing Co 2 P nanocrystals onto the surface of a copper TEM grid coated with an amorphous carbon film. Once the organic solution had dried, Co 2 P nanocrystals were dispersed over the grid. A drop of a solution of gold nanoparticles in water was then pipetted onto the grid, and allowed to dry. The gold nanoparticles were used as fiducial markers to align the tilt series.

Data acquisition

The tomographic tilt series of Co 2 P nanocrystals was acquired using an FEI Tecnai F20 scanning transmission electron microscope (STEM) at Cornell University. The microscope was operated at an accelerating voltage of 200 kV, with a convergence semi-angle of 9.6 mrad, and beam current of ~8–10 pA. This yields a nominal 2D resolution of up to 1.6 Å for STEM annular dark field (ADF) images. The tomographic tilt series was acquired over a 150° range at 2° intervals using a high angle annular dark-field (HAADF) detector. The scale in each image is ~0.71 nm per pixel.

Alignment and reconstruction

Each of the 76 projections in the tilt series, tiltser_Co2P.tif, has been aligned to a fiducial particle close to the Co 2 P nanocrystal using manual alignment techniques (except projections 50 and 51, which are blank to correct for 4° goniometer backlash during acquisition). Similarly, the tilt axis was determined by manually choosing the axis of rotation that minimized artifacts and maximized detail in the final reconstruction. We provide an example reconstruction, recon_Co2P.tif, produced from tiltser_Co2P.tif using the SIRT algorithm.

Tom_2: 180° Tomography of nanoparticles on nanofibre

Sample preparation

Graphitized nanofibres, loaded with platinum nanoparticles at 10 wt. % were dispersed in a methanol solution and dried onto the tip of a tungsten omniprobe needle. The needle was inserted into a Fischione 2050 On-Axis Rotation Tomography Holder for data acquisition.

Data acquisition

One 93° tilt series and one 95° tilt series were acquired with an offset of ~85.3° between the viewing angle of the first image of the first series, and the viewing angle of the first image of the second series. The two tilt series together therefore cover slightly more than the full 180° tilt range. The overlapping region between the two tilt series was used to align them together in post-processing. The angular increment for each tilt series was 1°. In order to reduce scan noise and thus improve signal to noise ratio, 16 images, each with a 1 μs per pixel dwell time, were recorded at each viewing angle and saved as an image stack to be aligned in post processing. Data was acquired using an FEI Tecnai F20 scanning transmission electron microscope (STEM) at Cornell University. The microscope was operated in low angle annular dark field (LAADF) mode an accelerating voltage of 200 kV with a probe current of approximately 5 pA. A convergence angle of ~6.9 mrad was used to optimize resolution over a large depth of field. Images were acquired with 1024×1024 pixels. Non-orthogonality in probe scan direction was observed and was corrected in all images by applying a 0.6 degree shear parallel to the tilt axis with linear interpolation. The field of view in each image was 363.52 nm.

Alignment and reconstruction

Each of the 1 μs per pixel image stacks of 16 images was aligned by cross correlation. Each stack was then summed to form a single image. The single images from both tilt series were then combined into a single 180° image stack, tiltser_180.tif, for reconstruction, with duplicate viewing angles discarded. The tilt series was aligned using a centre of mass method. A 3D reconstruction of the data, recon_180.tif, was produced using a weighted back projection algorithm. Data illustrated in Fig. 2a.

Figure 2: Illustrations of tilt series and sample reconstructions. (a) Sample image from tiltser_180.tif. Mixed 3D volume/isosurface visualizations of recon_180.tif show exterior of fibre, with nanoparticles visible on exterior, and hollow interior of nanofibre, containing nanoparticles. (b) Sample image from tiltser_PtNP.tif. Mixed 3D volume/isosurface visualization of recon_PtNP.tif and volume visualization of 3D Fourier transform of recon_PtNP.tif, showing platinum reciprocal lattice spots. (c) Sample image from tiltser_W.tif. Mixed 3D volume and isosurface visualization of recon_W.tif and of the 3D Fourier transform (cropped) of recon_W.tif, showing tungsten reciprocal lattice spots. All 3D visualizations produced using tomviz. Full size image

Tom_3: Atomic resolution tomography of platinum nanoparticle

Sample preparation

Platinum nanoparticles were deposited onto a grid consisting of a 5-nm-thick silicon nitride membrane with dimensions of 100 μm×1500 μm, supported on a 100 μm-thick silicon frame designed for loading into a TEM (TEMwindows.com). High temperature coating of a 1–2 nm carbon layer was applied to mitigate charging effects due to the electron beam. The grid was then loaded onto a Fischione 2020 tomographic sample holder for data acquisition in the TEM.

Data acquisition

The tomographic tilt series of platinum nanoparticles was acquired using an uncorrected FEI Titan STEM at the University of California, Los Angeles. The microscope was operated with a beam energy of 200 keV, a 100 pA probe current, and a 10.7 mrad convergence semi-angle. A tilt series of 104 projections was acquired from a platinum nanoparticle with equal-slope increments and a tilt range of ±72.6°.

Alignment and reconstruction

The images in the tilt series, tiltser_PtNP.tif, were aligned using a centre of mass (CM) alignment method after background subtraction and removal15. We present a reconstruction of this data, recon_PtNP.tif, produced using the equal slope tomography (EST) iterative algorithm, a method described by Miao et al.23 No Fourier filters were applied to the final reconstruction. Data illustrated in Fig. 2b.

Tom_4: Atomic resolution tomography of tungsten needle

Sample preparation

A 99.95% pure tungsten wire was annealed under tension, creating a large crystalline domain with the [011] crystallographic axis aligned along the wire axis. The wire was electrochemically etched in a NaOH solution to form a sharp tip with a <10 nm diameter. The wire was plasma cleaned in an Ar/O 2 gas mixture and then heated to 1,000 °C under vacuum (~10−5 Pa) to remove the oxide layer generated by the plasma cleaning. The wire was mounted in a 1 mm sample puck compatible with the TEAM microscope stage.

Data acquisition

Tomographic data was acquired using the TEAM I at the National Center for Electron Microscopy. The microscope was operated at 300 kV beam voltage in ADF-STEM mode with a convergence semi-angle of ~ 30 mrad and a ~ 70 pA beam current. The tomography rotation axis was aligned to the wire axis [011]. An equally sloped tomographic tilt series of 62 images, covering the complete angular range of ±90° was acquired from the tungsten needle sample. Two images of 1024×1024 pixels each with 6 μs per pixel dwell time and 0.405 Å pixel resolution were acquired at each angle in order to correct for drift. The TEAM stage, which is a tilt-rotate design with full 360° rotation about both axes, enabled rotation around the [011] crystalline axis.

Alignment and reconstruction

Raw experimental data can be found in tiltser_W.zip, which contains tif stacks of the two images acquired at each viewing angle, as described above. In addition, we provide an aligned tilt series, tiltser_W.tif. In the raw data, the tilt axis has a different in-plane orientation at each viewing angle, and in order to obtain an aligned tilt series, this was corrected by using Fourier methods to align the tilt direction along the image horizontal in every image. Both sample drift and scan distortion were corrected for in all images in the tilt series using Fourier techniques34. The tilt series was then aligned using a centre of mass method, with a mask applied to remove background noise. The tilt series was cropped in order to only feature the tip of the needle, which remained within the depth of focus throughout data acquisition. We present a reconstruction of the data, recon_W.tif, produced from tiltser_W.tif using the equal slope tomography iterative algorithm. The alignment and reconstruction process is explained in detail by Xu et al.16 Data illustrated in Fig. 2c.

Tom_5: Through-focal tomography of Pt-Cu catalyst

Sample preparation

The through focal tilt series was acquired on PtCu nanoparticles on a 3D Vulcan carbon support. The synthesis methods and scientific relevance of the nanoparticles as a fuel-cell electrocatalyst are discussed in detail by Wang et al.35 To prepare for observation in the electron microscope, the particles were suspended in ethanol and pipetted onto a copper TEM grid with an ultra-thin, holey carbon support film.

Data acquisition

The through-focal tomographic tilt series of de-alloyed PtCu nanoparticles on an extended 3D carbon support was acquired using TEAM I at the National Center for Electron Microscopy; a tool that provides attributes to best demonstrate the advantages of this technique. Its large convergence angle provides high lateral resolution (<0.78 Å) and a small depth-of-field (~6 nm) at 300 kV accelerating voltage. Shadowing from the TEM grid limited tilts from −68° to +71° along our chosen axis of rotation.

The tomographic data was acquired over a 138° tilt range using a high angle annular dark field (HAADF) detector. The 30 mrad convergence angle provided a continuum of information in the through-focal CTF that spanned a ±1.72° wedge at low and medium frequencies. A 3° tilt increment was chosen to match the convergence angle. The PtCu nanoparticles decorate a 3D Vulcan carbon support with an extended structure that far exceeds the microscope’s depth of field—making it impossible to image multiple particles in-focus within a single field of view. At every tilt a 26 image through-focal series was taken over ±250 nm defocus with 20 nm focal steps in order to ensure all objects were imaged in focus. The microscope defocus steps are calibrated from a through-focal stack (Fig. 3). Each image had a 0.38 nm per pixel lateral resolution.

Figure 3: Illustration of raw data and sample reconstruction for Tom_5. A through-focal image series must be acquired at each viewing angle in through-focal tomography. Files 018.tif, 072.tif, and 120.tif are shown as examples. Through-focal tomography allows objects from an extended field of view to be reconstructed at high resolution in an aberration corrected STEM. A 3D isosurface visualization of the full view of PtCu nanoparticles on an extended carbon support in recon_ThroughFocal.tif is shown, along with high resolution 3D visualizations of individual PtCu particles in the reconstruction. All visualizations produced using tomviz. Full size image

Alignment and reconstruction

A five-dimensional alignment of the raw data in tiltser_ThroughFocal.zip was required: transverse x-y alignment, focal z-alignment, tilt axis rotation and shift. A fiduciary particle was used to align each through-focal stack in their respective x-y direction. The focal z-alignment for each focal stack was determined by identification of the best focus image to a fiduciary particle. Within each focal stack a cross-correlation alignment was used to reduce the small amounts of drift during the acquisition. After alignment, the data was reweighted in Fourier space by dividing with the microscope’s contrast transfer function (CTF) approximated by a 300 keV 30 mrad aberration-free probe plus a Wiener constant of 5 times the max CTF value. After this light deconvolution, each through-focal stack was mapped onto a universal Fourier space by bilinear extrapolation. This extrapolation distributes the complex value of an input point to its four nearest neighbors on the output Cartesian grid with a weighted average of points from all the through-focal stacks. A direct inverse 3D Fourier transform provided the final reconstruction, recon_ThroughFocal.tif. This method is described by Hovden et al.13. It should be noted that the alignment of each through-focal stack generated excess blank images for reference. Thus in the raw data provided, there are 43 images per stack; 26 images of the PtCu nanoparticles, and 17 blank reference images.

Code availability

Code equivalent to that used to reconstruct the data in Tom_1 and Tom_2 is available as part of the open source Tomviz software package at www.tomviz.org. Code used to reconstruct the data in Tom_3 is freely available online at http://www.physics.ucla.edu/research/imaging/EST/. Code used to reconstruct the data in Tom_4 is freely available online at http://www.physics.ucla.edu/research/imaging/3Datoms.

Code used to reconstruct the data in Tom_5 is available in the Supplementary Information to this paper (Supplementary File 1). Alignment tools are available as part of the open source Tomviz software package at www.tomviz.org, and the open source IMOD software package at http://bio3d.colorado.edu/imod/.