Preparation of biomolecule gel matrices for electron microscopy

We report a new sample preparation method that allows the direct transmission electron microscopy evaluation of the architectural characteristics of biomolecules entrapped in gel matrices. We demonstrate that this sample preparation technique can be used for the identification and ultrastructural characterization of liposomes, collagen I and collagen III embedded in gel matrices, and has the potential to be useful for transmission electron microscopy (TEM) characterization of other biomolecule-gel matrix systems.     

A novel "ghost"-free tomographic image reconstruction method applicable to rotary-shadowed replica specimens

Electron tomography by conventional filtered back-projection is often seriously impaired by anisotropic resolution due to unavoidable limitation in specimen tilt-angles. We propose a new approach to overcome the problem for thin film-like replica-type specimens in which internal density is supposed as homogenous and contiguously distributed, by imposing a reasonable constraint of density-existing region in the reconstruction procedure. The objects were approximated as a distribution of binary voxels and the intensity of the projected images being proportional to the thickness along the projection ray. The new reconstruction algorithm consists of initial determination of approximate constraint region by a topographic analysis by stereo-photogrammetry, followed by iterative computation to find the unique solution of simultaneous equations, so that all the intensity distribution in tilt-series images are included within pre-determined voxel arrangement. During a trial run with a new methodology, we realized its significantly advantageous feature that much less number of projection images than conventional back-projection is required to perform the reconstruction of almost equivalent quality. Here, we show the performance of this novel algorithm by 3-D reconstruction of quick-freeze deep-etch replica specimens without any trace of spurious ghosting caused by missing-wedge problems.     

Astigmatic intensity equation for electron microscopy based phase retrieval

Phase retrieval, in principle, can be performed in a transmission electron microscope (TEM) using arbitrary aberrations of electron waves; provided that the aberrations are well-characterised and known. For example, the transport of intensity equation (TIE) can be used to infer the phase from a through-focus series of images. In this work an "astigmatic intensity equation" (AIE) is considered, which relates phase gradients to intensity variations caused by TEM objective lens focus and astigmatism variations. Within the paraxial approximation, it is shown that an exact solution of the AIE for the phase can be obtained using efficient Fourier transform methods. Experimental requirements for using the AIE are the measurement of a through-focus derivative and another intensity derivative, which is taken with respect to objective lens astigmatism variation. Two quasi-experimental investigations are conducted to test the validity of the solution.     

A formula for the image intensity of phase objects in Zernike mode

I present an analytical expression for the image intensity of a phase object visualized in Zernike phase contrast mode. The formula is valid for periodic and non-periodic weak and strong objects, and accounts for the effects of finite illumination. The expression provided is intended as a generalization of the standard reference formula given in the Born and Wolf [Principles of Optics, sixth ed., Pergamon Press, New York, 1980, p. 427] textbook as well as of the formalism employed to evaluate imaging doses in Zernike mode [M. Malac, M. Beleggia, R. Egerton, Y. Zhu, Ultramicroscopy 108 (2008) 126]. I illustrate the usefulness of the improved expression by means of three examples: a sinusoidal phase grating, a Gaussian object, and a phase step. The optimal Zernike phase angle yielding maximum image contrast is found to be object-dependent and not necessarily equal to pi/2. Phase plate optimization criteria are derived and presented for two of the examples considered.     

Characterization of JEOL 2100F Lorentz-TEM for low-magnification electron holography and magnetic imaging

We present results that characterize the performance and capabilities of the JEOL 2100F-LM electron microscope to carry out holography and quantitative magnetic imaging. We find the microscope is well-suited for studies of magnetic materials, or for semi-conductor dopant profiling, where a large hologram width ( approximately 1 microm) and fine fringe spacing ( approximately 1.5 nm) are obtained with good contrast ( approximately 20%). We present, as well, measurements of the spherical aberration coefficient Cs=(108.7+/-9.6)mm and minimum achievable focal step delta f=(87.6+/-1.4)nm for the specially designed long-focal-length objective lens of this microscope. Further, we detail experiments to accurately measure the optical parameters of the imaging system typical of conventional holography setup in a transmission electron microscope. The role played by astigmatic illumination in the hologram formation is also assessed with a wave-optical model, which we present and discuss. The measurements obtained for our microscope are used to simulate realistic holograms, which we compare directly to experimental holograms finding good agreement. These results indicate the usefulness of measuring these optical parameters to guide the optimization of the experimental setup for a given microscope, and to provide an additional degree of practical experimental possibility.     

Correlation of the orientation of stacked aragonite platelets in nacre and their connection via mineral bridges

In this work, we studied the correlation of the orientation of stacked aragonite platelets of Haliotis laevigata nacre, using selected area diffraction (SAD) in transmission electron microscopy (TEM). From the position of the center of Laue circle (COLC) within the diffraction patterns the tilt angles of the investigated platelets relatively to a reference platelet (oriented in zone axis) are determined. The strong correlation of the platelets supports the existence of mineral bridges, which connect the stacked platelets and enable a transfer of the platelet orientation during growth. Electron tomography and subsequent reconstruction of the obtained data yield information about the shape of the mineral bridges. The crystalline structure of the material within the mineral bridges was investigated by high resolution TEM (HRTEM).     

On the interpretation of the forbidden spots observed in the electron diffraction patterns of flat Au triangular nanoparticles

In many cases nanostructures present forbidden spots in their electron diffraction patterns when they are observed by transmission electron microscopy (TEM). To interpret their TEM and high resolution transmission electron microscopy (HRTEM) images properly, an understanding of the origin of these spots is necessary. In this work we comment on the origin of the forbidden spots observed in the [111] and [112] electron diffraction patterns of flat gold triangular nanoparticles. The forbidden spots were successfully indexed as corresponding to the first laue Zone (FOLZ) and the HRTEM images presented a contrast produced by the interference of the zero-order Laue zone (ZOLZ) and FOLZ spots. We discuss the use of the forbidden spots in the study of the structure of nanoparticles and show that they are related to the shape and incompleteness of layers in the very thin particles.     

Limits to the spatial, energy and momentum resolution of electron energy-loss spectroscopy

We discuss various factors that determine the performance of electron energy-loss spectroscopy (EELS) and energy-filtered (EFTEM) imaging in a transmission electron microscope. Some of these factors are instrumental and have undergone substantial improvement in recent years, including the development of electron monochromators and aberration correctors. Others, such as radiation damage, delocalization of inelastic scattering and beam broadening in the specimen, derive from basic physics and are likely to remain as limitations. To aid the experimentalist, analytical expressions are given for beam broadening, delocalization length, energy broadening due to core-hole and excited-electron lifetimes, and for the momentum resolution in angle-resolved EELS.     

Maximum diameter of the rod-shaped specimen for transmission electron microtomography without the "missing wedge"

In our recent study, the complete rotation of a rod-shaped specimen during transmission electron microscopy (TEM) has been successfully carried out, yielding a truly quantitative three-dimensional (3D) structure of a ZrO(2)/polymer nano-composite. This result allows the further development of transmission electron microtomography (TEMT) for materials science. The diameter of the rod-shaped specimen was about 150 nm, which may not be statistically large enough to evaluate structural parameters, e.g., volume fraction of Zr nano-particles. Thus, it is preferable to image rods with larger diameters in 3D. In this study, several rod-shaped specimens whose diameters ranged from 150 to 530 nm were subjected to the "distortion-free TEMT". The maximum diameters, l, observable under 200 and 300 kV-TEMTs were, respectively, 460-470 and 600-670 nm (corresponding the maximum relative diameters, l/lambda (lambda: mean free path), were ca. 2.2 and 2.7-3.0).     

Transmission electron microtomography without the "missing wedge" for quantitative structural analysis

A three-dimensional (3D) visualization and structural analysis of a rod-shaped specimen of a zirconia/polymer nanocomposite material were carried out by transmission electron microtomography (TEMT) with particular emphasis on complete rotation of the specimen (tilt angular range: +/-90 degrees ). In order to achieve such an ideal experimental condition for the TEMT, improvements in the specimen as well as the sample holder were made. A rod-shaped specimen was necessary in order to obtain a high transmission of the specimen upon tilting to large angles. The image resolution of the reconstructed tomogram was isotropic, in sharp contrast to the anisotropic image resolution of the conventional TEMT with a limited angular range (the "missing wedge" problem). A volume fraction of zirconia, phi, evaluated from the 3D reconstruction was in quantitative agreement with the known composition of the nanocomposite. A series of 3D reconstructions was made from the tilt series with complete rotation by limiting the maximum tilt angle, alpha, from which a couple of structural parameters, the volume fraction and surface area per unit volume, Sigma, of the zirconia, were evaluated as a function of alpha. It was confirmed from actual experimental data that both phi and Sigma slightly decreased with the increasing alpha and reached constant values at around alpha=80 degrees , suggesting that the specimen may have to be tilted to +/-80 degrees for truly quantitative measurements.     

Quantitative TEM-based phase retrieval of MgO nano-cubes using the transport of intensity equation

Through focus series of images are collected from MgO nano-cube crystals in the transmission electron microscope (TEM). The experimental data is used to solve the transport of intensity equation (TIE) to retrieve phase maps, which portray the morphology of the cubes and are quantified by the mean inner potential V(0). Particular attention is given to the practical difficulties associated with TIE phase retrieval of non-conducting polyhedron particles.     

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.     

Effect of SP-C on surface potential distribution in pulmonary surfactant: Atomic force microscopy and Kelvin probe force microscopy study

The air–lung interface is covered by a molecular film of pulmonary surfactant (PS). The major function of the film is to reduce the surface tension of the lung's air–liquid interface, providing stability to the alveolar structure and reducing the work of breathing. Earlier we have shown that function of bovine lipid extract surfactant (BLES) is related to the specific molecular architecture of surfactant films. Defined molecular arrangement of the lipids and proteins of the surfactant film also give rise to a local highly variable electrical surface potential of the interface. In this work we investigated a simple model of artificial lung surfactant consisting of DPPC, eggPG, and surfactant protein C (SP-C).     

Prospects for analyzing the electronic properties in nanoscale systems by VEELS

Valence electron energy-loss spectroscopy in the scanning transmission electron microscope can provide detailed information on the electronic structure of individual nanostructures. By employing the latest advances in electron optical devices, such as a probe aberration corrector and an electron monochromator, the probe size, spectroscopic resolution, probe current and primary electron energy can be varied over a large range. This flexibility is particularly important for nanostructures where each of these variables needs to be carefully counterbalanced in order to collect spectroscopic data without altering the integrity of the sample. Here the implementation of valence electron energy-loss spectroscopy to the study of nanostructures is discussed, with particular mention to the theoretical understanding of each of the contributions to the overall spectrum.     

The impact of surface and retardation losses on valence electron energy-loss spectroscopy

The inelastic scattering of fast electrons transmitting thin foils of silicon (Si), silicon nitride (Si(3)N(4)), gallium arsenide (GaAs), gallium nitride (GaN) and cadmium selenide (CdSe) was analyzed using dielectric theory. In particular, the impact of surface and bulk retardation losses on valence electron energy-loss spectroscopy (VEELS) was studied as a function of the foil thickness. It is shown that for the materials analyzed, surface and retardation losses can cause a systematic, thickness-dependent modulation of the dielectric volume losses, which can hamper the determination of the bulk dielectric data as well as the identification of band-gap and interband transition energies by VEELS. For Si and GaAs, where the dielectric function is strongly peaked with high absolute values, retardation losses lead to additional intensity maxima in the spectrum. For thin films of these materials (below approximately 100 nm), the additional intensity maxima are related to retardation effects due to the finite size of the sample leading to the excitation of guided light modes. For thicker films, exceeding about 200 nm, the intensity maxima are caused by bulk retardation losses, i.e., Cerenkov losses. Although thickness-dependent modulations were observed for Si(3)N(4), GaN and CdSe, the form of the dielectric functions and their lower maxima, means that for TEM samples < 100 nm thick, the band-gap energies of these materials can be accurately identified by VEELS. Guidelines are given that allow for forecasting the impact of surface and retardation losses on VEELS.     

In situ measurements and transmission electron microscopy of carbon nanotube field-effect transistors

We present the design and operation of a transmission electron microscopy (TEM)-compatible carbon nanotube (CNT) field-effect transistor (FET). The device is configured with microfabricated slits, which allows direct observation of CNTs in a FET using TEM and measurement of electrical transport while inside the TEM. As demonstrations of the device architecture, two examples are presented. The first example is an in situ electrical transport measurement of a bundle of carbon nanotubes. The second example is a study of electron beam radiation effect on CNT bundles using a 200 keV electron beam. In situ electrical transport measurement during the beam irradiation shows a signature of wall- or tube-breakdown. Stepwise current drops were observed when a high intensity electron beam was used to cut individual CNT bundles in a device with multiple bundles.     

A Bloch wave analysis of optical sectioning in aberration-corrected STEM

The reduction in the focal depth of field that occurs through the use of larger apertures in aberration-corrected STEM allows three-dimensional information to be retrieved by optical depth sectioning. This paper explores depth sectioning in zone-axis crystals using Bloch wave calculations. By decomposing the calculation into the contribution from individual states and from individual partial plane waves in the convergent cone of illumination, we explain the form of the electron intensity in the crystal as a function of depth. Two separate effects are found that can cause the intensity maximum to deviate from that of the expected defocus value. Firstly it is found that the unbound, high angle excited states give rise to a behaviour similar to that of the probe focusing in the vacuum, but with a prefocusing effect due to the lensing effect of the potential of the atomic column. Superimposed upon this prefocused peak is an oscillation due to interference between the channelling 1s state and the rest of the wavefunction. This oscillation can actually prevent an intensity maximum being formed at certain depths in the crystal, and will complicate the interpretation of optical sectioning data.     

A practical method to detect and correct for lens distortion in the TEM

A practical, offline method for experimental detection and correction for projector lens distortion in the transmission electron microscope (TEM) operating in high-resolution (HR) and selected area electron diffraction (SAED) modes is described. Typical TEM works show that, in the simplest case, the distortion transforms on the recording device, which would be a circle into an ellipse. The first goal of the procedure described here is to determine the elongation and orientation of the ellipse. The second goal is to correct for the distortion using an ordinary graphic program. The same experimental data set may also be used to determine the actual microscope magnification and the rotation between SAED patterns and HR images. The procedure may be helpful in several quantitative applications of electron diffraction and HR imaging, for instance while performing accurate lattice parameter determination, or while determining possible metrical deviations (cell edges and angles) from a given symmetry.     

Formation of pentacene wetting layer on the SiO2 surface and charge trap in the wetting layer

We studied the early-stage growth of vacuum-evaporated pentacene film on a native SiO(2) surface using atomic force microscopy and in-situ spectroscopic ellipsometry. Pentacene deposition prompted an immediate change in the ellipsometry spectra, but atomic force microscopy images of the early stage films did not show a pentacene-related morphology other than the decrease in the surface roughness. This suggested that a thin pentacene wetting layer was formed by pentacene molecules lying on the surface before the crystalline islands nucleated. Growth simulation based on the in situ spectroscopic ellipsometry spectra supported this conclusion. Scanning capacitance microscopy measurement indicated the existence of trapped charges in the SiO(2) and pentacene wetting layer.