Compressed sensing electron tomography of needle-shaped biological specimens--Potential for improved reconstruction fidelity with reduced dose.

Electron tomography is an invaluable method for 3D cellular imaging. The technique is, however, limited by the specimen geometry, with a loss of resolution due to a restricted tilt range, an increase in specimen thickness with tilt, and a resultant need for subjective and time-consuming manual segmentation. Here we show that 3D reconstructions of needle-shaped biological samples exhibit isotropic resolution, facilitating improved automated segmentation and feature detection. By using scanning transmission electron tomography, with small probe convergence angles, high spatial resolution is maintained over large depths of field and across the tilt range. Moreover, the application of compressed sensing methods to the needle data demonstrates how high fidelity reconstructions may be achieved with far fewer images (and thus greatly reduced dose) than needed by conventional methods. These findings open the door to high fidelity electron tomography over critically relevant length-scales, filling an important gap between existing 3D cellular imaging techniques.

Choice of operating voltage for a transmission electron microscope.

An accelerating voltage of 100-300kV remains a good choice for the majority of TEM or STEM specimens, avoiding the expense of high-voltage microscopy but providing the possibility of atomic resolution even in the absence of lens-aberration correction. For specimens thicker than a few tens of nm, the image intensity and scattering contrast are likely to be higher than at lower voltage, as is the visibility of ionization edges below 1000eV (as required for EELS elemental analysis). In thick (>100nm) specimens, higher voltage ensures less beam broadening and better spatial resolution for STEM imaging and EDX spectroscopy. Low-voltage (e.g. 30kV) TEM or STEM is attractive for a very thin (e.g. 10nm) specimen, as it provides higher scattering contrast and fewer problems for valence-excitation EELS. Specimens that are immune to radiolysis suffer knock-on damage at high current densities, and this form of radiation damage can be reduced or avoided by choosing a low accelerating voltage. Low-voltage STEM with an aberration-corrected objective lens (together with a high-angle dark-field detector and/or EELS) offers atomic resolution and elemental identification from very thin specimens. Conventional TEM can provide atomic resolution in low-voltage phase-contrast images but requires correction of chromatic aberration and preferably an electron-beam monochromator. Many non-conducting (e.g. organic) specimens damage easily by radiolysis and radiation damage then determines the TEM image resolution. For bright-field scattering contrast, low kV can provide slightly better dose-limited resolution if the specimen is very thin (a few nm) but considerably better resolution is possible from a thicker specimen, for which higher kV is required. Use of a phase plate in a conventional TEM offers the most dose-efficient way of achieving atomic resolution from beam-sensitive specimens.

Atomic structure from large-area, low-dose exposures of materials: a new route to circumvent radiation damage.

Beam-induced structural modifications are a major nuisance in the study of materials by high-resolution electron microscopy. Here, we introduce a new approach to circumvent the radiation damage problem by a statistical treatment of large, noisy, low-dose data sets of non-periodic configurations (e.g. defects) in the material. We distribute the dose over a mixture of different defect structures at random positions and with random orientations, and recover representative model images via a maximum likelihood search. We demonstrate reconstructions from simulated images at such low doses that the location of individual entities is not possible. The approach may open a route to study currently inaccessible beam-sensitive configurations.

Z dependence of electron scattering by single atoms into annular dark-field detectors.

A simple parameterization is presented for the elastic electron scattering cross sections from single atoms into the annular dark-field (ADF) detector of a scanning transmission electron microscope (STEM). The dependence on atomic number, Z, and inner reciprocal radius of the annular detector, q(0), of the cross section σ(Z,q(0)) is expressed by the empirical relation [see formula in text] where A(q(0)) is the cross section for hydrogen (Z = 1), and the detector is assumed to have a large outer reciprocal radius. Using electron elastic scattering factors determined from relativistic Hartree-Fock simulations of the atomic electron charge density, values of the exponent n(Z,q(0)) are tabulated as a function of Z and q(0), for STEM probe sizes of 1.0 and 2.0 Å. Comparison with recently published experimental data for single-atom scattering [Krivanek et al. (2010). Nature 464, 571-574] suggests that experimentally measured exponent values are systematically lower than the values predicted for elastic scattering from low-Z atoms. It is proposed that this discrepancy arises from the inelastic scattering contribution to the ADF signal. A simple expression is proposed that corrects the exponent n(Z,q(0)) for inelastic scattering into the annular detector.

Mass mapping of large globin complexes by scanning transmission electron microscopy.

Scanning transmission electron microscopy (STEM) of unstained, freeze-dried biological macromolecules in the dark-field mode provides an image based on the number of electrons elastically scattered by the constituent atoms of the macromolecule. The image of each isolated particle provides information about the projected structure of the latter, and its integrated intensity is directly related to the mass of the selected particle. Particle images can be sorted by shape, providing independent histograms of mass to study assembly/disassembly intermediates. STEM is optimized for low-dose imaging and is suitable for accurate measurement of particle masses over the range from about 30 kDa to 1,000 MDa. This article describes the details of the method developed at the Brookhaven National Laboratory STEM facility and illustrates its application to the mass mapping of large globin complexes.

Quantitative amplitude and phase contrast imaging in a scanning transmission X-ray microscope.

Phase contrast in X-ray imaging provides lower radiation dose, and dramatically higher contrast at multi-keV photon energies when compared with absorption contrast. We describe here the use of a segmented detector in a scanning transmission X-ray microscope to collect partially coherent bright field images. We have adapted a Fourier filter reconstruction technique developed by McCallum, Landauer and Rodenburg to retrieve separate, quantitative maps of specimen phase shift and absorption. This is demonstrated in the imaging of a germanium test pattern using 525eV soft X-rays.

Determinación del sellado marginal en incrustaciones cerámicas MOD / Marginal seal determination in MOD ceramic inlays

Se determina mediante MEB el comportamiento de la resina compuesta de cementación, in vitro en restauraciones cerámicas grabadas. Los resultados indican que las microporosidades fueron penetradas por las resinas de cementación. Se obtuvo un espesor promedio de la línea de ementación de 93,7 µm, presentándose mayor desajuste en las zonas marginales. En las 12 piezas estudiadas la resina de cementación mostró un contorno armónico respecto de la restauración cerámica y la pieza dentaria