Depth sectioning in scanning transmission electron microscopy based on core-loss spectroscopy

Recent and ongoing improvements in aberration correction have opened up the possibility of depth sectioning samples using the scanning transmission electron microscope in a fashion similar to the confocal scanning optical microscope. We explore questions of principle relating to image interpretability in the depth sectioning of samples using electron energy loss spectroscopy. We show that provided electron microscope probes are sufficiently fine and detector collection semi-angles are sufficiently large we can expect to locate dopant atoms inside a crystal. Furthermore, unlike high angle annular dark field imaging, electron energy loss spectroscopy can resolve dopants of smaller atomic mass than the supporting crystalline matrix.     

An expanded approach to noise reduction from high-resolution STEM images based on the maximum entropy method

An expanded use of the maximum entropy method (MEM) is suggested to reduce noise from an experimental high-angle annular dark-field (HAADF) scanning transmission electron microscope (STEM) image. The MEM is combined with an estimate of the standard deviation of noise from an experimental HAADF STEM image and low-pass filtering using the information limit for an incoherent STEM image. Consequently, the present method has just one parameter of a Lagrange multiplier. It is demonstrated that the present method can reduce noise efficiently in high-resolution HAADF STEM images.     

Electron diffraction analysis of individual single-walled carbon nanotubes

We present a detailed electron diffraction study of individual single-walled carbon nanotubes. A novel sample preparation procedure provides well-separated, long and straight individual single-shell nanotubes. Diffraction experiments are carried out at 60 kV, below the threshold for knock-on damage in carbon nanotubes. We describe experimental parameters that allow single-tube electron diffraction experiments with widely available thermal emission transmission electron microscopes. Further, we review the simulation of diffraction patterns for these objects.     

Effects of specimen tilt in ADF-STEM imaging of a-Si/c-Si interfaces

Annular dark field scanning transmission electron microscopy (ADF-STEM) imaging of a crystal depends strongly on specimen orientation, but for an amorphous sample it is insensitive to orientation changes. To fully investigate the effects of specimen tilt, an interface of amorphous Si (a-Si) and crystalline Si (c-Si) was rotated systematically off a zone axis in a STEM equipped with low-angle ADF (LAADF) and high-angle ADF (HAADF) detectors. The change of relative intensity across the interface shows very different trends in the LAADF and the HAADF images upon tilting. More importantly, it is found that the HAADF signal decreases much more rapidly when tilted off a zone axis than does the LAADF signal. The high-resolution lattice fringes also disappear much faster in the HAADF image than in the LAADF image. These trends reflect the fact that the channeling peaks that are responsible for scattering into the HAADF detector decrease more quickly upon tilting than the lower angle scattering to the LAADF detector does.     

Subtleties in ADF imaging and spatially resolved EELS: A case study of low-angle twist boundaries in SrTiO3

A screw dislocation network at the low-angle SrTiO3/Nb:SrTiO3 twist grain boundary has been analyzed by annular dark field (ADF) imaging and spatially resolved electron energy loss spectroscopy (EELS) in a scanning transmission electron microscope (STEM). The cores of one set of dislocations running parallel to the beam direction appear dark in the ADF STEM images. EELS on the dislocation core reveals a reduced Sr/Ti ratio compared to the bulk suggesting Sr-deficient cores. The second set of dislocations, orthogonal to the latter, is imaged by its strain field using low-angle annular dark field (LAADF) imaging. Multislice image simulations suggest channeling of the electron probe on the atomic columns for small tilts, theta < 1 degree, where the Sr columns act as beam guides. Only for larger tilts is the channeling effect strongly reduced and the fringe contrast approaches the value predicted by a purely incoherent imaging model. Ti-L(2,3) EELS across the dislocation core shows an asymmetry between the EELS and the ADF signal which cannot be explained by the geometry or beam broadening. This asymmetry might be explained by an effective nonlocal potential representing inelastic scattering in EELS.     

Effect of small crystal tilt on atomic-resolution high-angle annular dark field STEM imaging

Using a slightly tilted convergent electron beam, high-angle annular dark field scanning transmission electron microscopy observations have been performed of a [0 11]-oriented Si crystal. A small tilt of the crystal zone axis with respect to the coma-axis of the probe-forming lens causes a difference in intensity between bright spots of a Si dumbbell. The semiangle of the beam probe and the tilting angle with respect to the specimen hormal were determined by means of convergent beam micro-diffraction. The simulation using these parameters accounts for the image contrasts satisfactorily.     

A novel method for focus assessment in atomic resolution STEM HAADF experiments

Analysis of the Fourier components of through-focal images in scanning transmission electron microscopy with a high angle annular dark field detector is used to assess illumination defocus values. The method is based on a least squares fitting of the peculiar dependence of Fourier components of the high angle annular dark field image on defocus. The validity of the method has been checked against simulations and experiments obtaining a good level of accuracy on the defocus measurement (δf=2 nm) for simulated specimen thickness up to 40 nm. The difference between simulated and experimental Fourier coefficients for large defoci can be used to estimate the specimen thickness at least up to 30 nm but with decreasing precision for larger thickness.     

Comparison of intensity distributions in tomograms from BF TEM,ADF STEM,HAADF STEM,and calculated tilt series

The three-dimensional (3D) morphology of a nanometer-sized object can be obtained using electron tomography. Variations in composition or density of the object cause variations in the reconstructed intensity. When imaging homogeneous objects, variations in reconstructed intensity are caused by the imaging technique, imaging conditions, and reconstruction. In this paper, we describe data acquisition, image processing, and 3D reconstruction to obtain and compare tomograms of magnetite crystals from bright field (BF) transmission electron microscopy (TEM), annular dark-field (ADF) scanning transmission electron microscopy (STEM), and high-angle annular dark field (HAADF) STEM tilt series. We use histograms, which plot the number of volume elements (voxels) at a given intensity vs. the intensity, to measure and quantitatively compare intensity distributions among different tomograms. In combination with numerical simulations, we determine the influence of maximum tilt angle, tilt increment, contrast changes with tilt (diffraction contrast), and the signal-to-noise ratio (SNR) as well as the choice of the reconstruction approach (weighted backprojection (WB) and sequential iterative reconstruction technique (SIRT)) on the histogram. We conclude that because ADF and HAADF STEM techniques are less affected by diffraction, and because they have a higher SNR than BF TEM, they are better suited for tomography of nanometer-sized crystals.     

Comparison of intensity distributions in tomograms from BF TEM, ADF STEM, HAADF STEM, and calculated tilt series

The three-dimensional (3D) morphology of a nanometer-sized object can be obtained using electron tomography. Variations in composition or density of the object cause variations in the reconstructed intensity. When imaging homogeneous objects, variations in reconstructed intensity are caused by the imaging technique, imaging conditions, and reconstruction. In this paper, we describe data acquisition, image processing, and 3D reconstruction to obtain and compare tomograms of magnetite crystals from bright field (BF) transmission electron microscopy (TEM), annular dark-field (ADF) scanning transmission electron microscopy (STEM), and high-angle annular dark field (HAADF) STEM tilt series. We use histograms, which plot the number of volume elements (voxels) at a given intensity vs. the intensity, to measure and quantitatively compare intensity distributions among different tomograms. In combination with numerical simulations, we determine the influence of maximum tilt angle, tilt increment, contrast changes with tilt (diffraction contrast), and the signal-to-noise ratio (SNR) as well as the choice of the reconstruction approach (weighted backprojection (WB) and sequential iterative reconstruction technique (SIRT)) on the histogram. We conclude that because ADF and HAADF STEM techniques are less affected by diffraction, and because they have a higher SNR than BF TEM, they are better suited for tomography of nanometer-sized crystals.     

Contributions to the contrast in experimental high-angle annular dark-field images

This paper reports on a study of the contributions to the image contrast of high-angle annular dark field (HAADF) images acquired in scanning transmission electron microscopy. Experimental HAADF images were obtained from a model system consisting of an epitaxial perovskite PbTiO3 film grown on a SrTiO3 single crystal. This sample allowed for the study of the intensities of a wide range of atomic numbers. The main objective of the paper was to quantify the influence of TEM foil thickness on the image contrast, but the effects of the annular detector inner angle and the probe forming lens focus were also studied. Sample thicknesses ranging from approximately 10 nm to more than 400 nm were investigated. The image contrast was relatively insensitive to changes in inner angle. The main impact of sample thickness was a rapid increase in a background intensity that contributed equally to the intensities of the atomic columns and the channels between them. The background intensity and its increase with thickness reflected the average atomic number of the crystal. Subtraction of the background intensity allowed for a quantitative interpretation of image contrast in terms of atomic numbers and comparison with multislice image simulations. The consequences for the analysis of interfaces in terms of atom column occupancies are discussed.     

STEM tomography for thick biological specimens

Scanning transmission electron microscopy (STEM) tomography was applied to biological specimens such as yeast cells, HEK293 cells and primary culture neurons. These cells, which were embedded in a resin, were cut into 1-microm-thick sections. STEM tomography offers several important advantages including: (1) it is effective even for thick specimens, (2) 'dynamic focusing', (3) ease of using an annular dark field (ADF) mode and (4) linear contrasts. It has become evident that STEM tomography offers significant advantages for the observation of thick specimens. By employing STEM tomography, even a 1-microm-thick specimen (which is difficult to observe by conventional transmission electron microscopy (TEM)) was successfully analyzed in three dimensions. The specimen was tilted up to 73 degrees during data acquisition. At a large tilt angle, the specimen thicknesses increase dramatically. In order to observe such thick specimens, we introduced a special small condenser aperture that reduces the collection angle of the STEM probe. The specimen damage caused by the convergent electron beam was expected to be the most serious problem; however, the damage in STEM was actually smaller than that in TEM. In this study, the irradiation damage caused by TEM- and STEM-tomography in biological specimens was quantitatively compared.     

Contrast enhancement of negatively stained macromolecules and biomembranes by single sideband phase contrast interference.

A straightforward procedure is described for the production of contrast enhancement of negatively stained macromolecules and biological membranes by single sideband phase contrast interference (electron optical shadowing). The instrumental adjustment required to produce this type of phase contrast illumination is readily achieved by beam deflection from the strioscopic (dark field) mode. Part of the hollow cone of electrons from the annular condenser aperture that are unscattered by the specimen are permitted to pass through the objective aperture and interfere with the scattered beam. The electron optical shadowing effect is produced because only one side of the unscattered beam is used. Careful adjustment of the beam tilt control, with the ability to tilt in any azimuth, allows optimal illumination conditions to be achieved. The results presented show the increased image contrast obtained using as specimens the purified cylindrical macromolecule from human erythrocyte membranes, purified nuclear envelopes and collagen fibres.     

Backscattered electron imaging and scanning transmission electron microscopy imaging of multi-layers

Experimental and theoretical results on image contrast of semiconductor multi-layers in scanning electron microscopy investigation are reported. Two imaging modes have been considered: backscattered electron imaging of bulk specimen and scanning transmission imaging of thinned specimens. The following main results have been reached. The image resolution of the multi-layers is, in both cases, defined by the probe size. The contrast, governed by density and atomic number differences, is affected by the size of the interaction volume in backscattered electron imaging and by the beam broadening in scanning transmission. Operating in the scanning transmission mode, the contrast of bright field images can be easily related to local variation in atomic number and density of the specimen while the dark field image contrast is strongly affected by electron beam energy, detector collection angles and specimen thickness. All these factors are able to produce contrast reversals that are difficult to explain without the support of a suitable simulation code.     

Spatially resolved diffractometry with atomic-column resolution

We demonstrate spatially resolved diffractometry in which diffraction patterns are acquired at two-dimensional positions on a specimen using scanning transmission electron microscopy (STEM), resulting in four-dimensional data acquisition. A high spatial resolution of about 0.1 nm is achieved using a stabilized STEM instrument, a spherical aberration corrector and various post-acquisition data processings. We have found a few novel results in the radial and the azimuthal scattering angle dependences of atomic-column contrast in STEM images. Atomic columns are clearly observed in dark field images obtained using the excess Kikuchi band intensity even in small solid-angle detection. We also find that atomic-column contrasts in dark field images are shifted in the order of a few tens of picometers on changing the azimuthal scattering angle. This experimental result is approximately interpretable on the basis of the impact parameter in Rutherford scattering. Spatially resolved diffractometry provides fundamental knowledge related to various STEM techniques, such as annular dark field (ADF) and annular bright field (ABF) imaging, and it is expected to become an analytical platform for advanced STEM imaging.     

Historical perspective and current trends in emission microscopy, mirror electron microscopy and low-energy electron microscopy. An introduction to the proceedings of the Second International Symposium and Workshop on Emission microscopy and Related Techniques

Emission microscopes and related instruments comprise a specialized class of electron microscopes that have in common an acceleration field in combination with the first stage of imaging (i.e., an immersion objective lens, also called a cathode lens or emission lens). These imaging techniques include photoelectron emission microscopy (PEEM or PEM), electron emission induced by heat, ions, or neutral particles, mirror electron microscopy (MEM), and low-energy electron microscopy (LEEM), among others. In these instruments the specimen is placed on a flat cathode or is the cathode itself. The low-energy electrons that are emitted, reflected, or backscattered from the specimen are first accelerated and then imaged by means of an electron lens system resembling that of a transmission electron microscope. The image is formed in a parallel mode in all of the above instruments, in contrast to the image in scanning electron microscopes, where the information is collected sequentially by scanning the specimen. A brief history and introduction to emission microscopy, MEM, and LEEM is presented as a background for the Proceedings of the Second International Symposium and Workshop on this subject, held in Seattle, Washington, August 16-17, 1990. Current trends in this field gleaned from the presentations at that meeting are discussed.     

Deconvolution processing of HAADF STEM images

A deconvolution processing of high-resolution high-angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) images, combined with maximum entropy method, is applied to two experimental [0 11]-Si images; one having unresolved dumbbells and the other having resolved dumbbells and artificial bright spots. The deconvoluted images for these images show bright spots corresponding to the projected atomic columns and no artificial bright spots. Thus, the deconvolution processing provides almost a real projected atomic structure by eliminating effects of the probe function from HAADF STEM images.     

Local crystal structure analysis with several picometer precision using scanning transmission electron microscopy

We report a local crystal structure analysis with a high precision of several picometers on the basis of scanning transmission electron microscopy (STEM). Advanced annular dark-field (ADF) imaging has been demonstrated using software-based experimental and data-processing techniques, such as the improvement of signal-to-noise ratio, the reduction of image distortion, the quantification of experimental parameters (e.g., thickness and defocus) and the resolution enhancement by maximum-entropy deconvolution. The accuracy in the atom position measurement depends on the validity of the incoherent imaging approximation, in which an ADF image is described as the convolution between the incident probe profile and scattering objects. Although the qualitative interpretation of ADF image contrast is possible for a wide range of specimen thicknesses, the direct observation of a crystal structure with deep-sub-angstrom accuracy requires a thin specimen (e.g., 10 nm), as well as observation of the structure image by conventional high-resolution transmission electron microscopy.     

A new approach for 3D reconstruction from bright field TEM imaging: beam precession assisted electron tomography

The successful combination of electron beam precession and bright field electron tomography for 3D reconstruction is reported. Beam precession is demonstrated to be a powerful technique to reduce the contrast artifacts due to diffraction and curvature in thin foils. Taking advantage of these benefits, Precession assisted electron tomography has been applied to reconstruct the morphology of Sn precipitates embedded in an Al matrix, from a tilt series acquired in a range from +49° to -61° at intervals of 2° and with a precession angle of 0.6° in bright field mode. The combination of electron tomography and beam precession in conventional TEM mode is proposed as an alternative procedure to obtain 3D reconstructions of nano-objects without a scanning system or a high angle annular dark field detector.     

An approach to the reduction of hydrocarbon contamination in the scanning electron microscope

The deposition of electron beam-induced specimen contamination in both the transmission (TEM) and scanning electron microscopes (SEM) has remained a problem since the beginning of these forms of microscopy. Generally, sources of SEM contamination can be attributed to one or a combination of three major contributors: (1) the pumping system; (2) outgassing of other internal SEM component parts (i.e., specimen stage, stage lubricants, O-rings, etc.), or (3) the sample (including its preparation and handling). Generally, because of the nature of SEM, specimen contamination can be minimized but is difficult to avoid fully. This work outlines three approaches taken with instruments at NIST to reduce the deposition of contamination in high-resolution cold-field emission SEMs. With some modification these techniques could be applied to any SEM. These approaches have been in successful operation for several years, resulting in a reduction in electron beam-induced hydrocarbon contamination.     

Morphology visualization of irregular shape bacteria by electron holography and tomography

In the current work, irregular morphology of Staphylococcus aureus bacteria has been visualized by phase retrieval employing off‐axis electron holography (EH) and 3D reconstruction electron tomography using high‐angle annular dark field scanning transmission electron microscopy (HAADF‐STEM). Bacteria interacting with gold nanoparticles (AuNP) acquired a shrunken or irregular shape due to air dehydration processing. STEM imaging shows the attachment of AuNP on the surface of cells and suggests an irregular 3D morphology of the specimen. The phase reconstruction demonstrates that off‐axis electron holography can reveal with a single hologram the morphology of the specimen and the distribution of the functionalized AuNPs. In addition, EH reduces significantly the acquisition time and the cumulative radiation damage (in three orders of magnitude) over biological samples in comparison with multiple tilted electron expositions intrinsic to electron tomography, as well as the processing time and the reconstruction artifacts that may arise during tomogram reconstruction.