Specimen Drift Research Articles

High-resolution wave field reconstruction using a through-focus series of images taken under limited electron dose

The three-dimensional Fourier filtering method and Schiske's Wiener filtering method are compared with the aim of high-resolution wave field reconstruction of an unstained deoxyribonucleic acid (DNA) molecular fiber using a through-focus series of images taken under a limited electron dose. There were some definite differences between the two reconstructed images, although the two kinds of processing are essentially equivalent except for the dimension and the filter used for processing. Through theoretical analyses together with computer simulations, the differences were proved to be primarily due to specimen drift during the experiment. Although the observed structure of the DNA molecular fiber was heavily damaged by electron beam irradiation, reconstructed images by the three-dimensional Fourier filtering method provided higher resolution information on the molecular structure even when relatively large specimen drift was included in the through-focus series. In contrast, in Schiske's Wiener filtering method, the detailed information of the structure was lost because of the drift, although the reconstructed image showed a higher signal-to-noise ratio. The three dimensional Fourier filtering method seems to be more applicable for observing radiation-sensitive materials under an extremely low electron dose, because specimen drift cannot be completely avoided.

Reprint of “Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)” [J. Struct. Biol. 160 (2007) 35–48

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides 3-D distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2-D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L(2,3) edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell's nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.

Development of Stage-scanning System for Confocal Scanning Transmission Electron Microscopy

We developed a stage-scanning system for confocal scanning transmission electron microscopy (confocal STEM), which enables us to perform STEM under a fixed lens configuration without a beam-scanning system. A specimen holder with a piezo-driven stage was modified and controlled by a computer program for stage scanning. The signals of transmitted electrons detected by an STEM detector were displayed on a computer screen and were synchronized with the voltage supplied to the piezoelectric devices used for stage positioning, which consequently produced a stage-scanning STEM image. Furthermore, we evaluated the stability of the developed stage-scanning STEM system and the resolution of the obtained images. 0.2 nm lattice firings could be observed in Au particles, which indicated that this system could demonstrate atomic-resolution STEM imaging at desired z positions. This is attributed to the small specimen drift, stable stage scanning and the computer program developed for stage axis corrections. Finally, we applied this stage-scanning system to confocal imaging. [DOI: 10.1380/ejssnt.2008.111]

Applications of direct detection device in transmission electron microscopy

A prototype direct detection device (DDD) camera system has shown great promise in improving both the spatial resolution and the signal to noise ratio for electron microscopy at 120-400 keV beam energies (Xuong et al., 2007. Methods in Cell Biology, 79, 721-739). Without the need for a resolution-limiting scintillation screen as in the charge coupled device (CCD), the DDD camera can outperform CCD based systems in terms of spatial resolution, due to its small pixel size (5 microm). In this paper, the modulation transfer function (MTF) of the DDD prototype is measured and compared with the specifications of commercial scientific CCD camera systems. Combining the fast speed of the DDD with image mosaic techniques, fast wide-area imaging is now possible. In this paper, the first large area mosaic image and the first tomography dataset from the DDD camera are presented, along with an image processing algorithm to correct the specimen drift utilizing the fast readout of the DDD system.

Oxidation Mapping by Postpeak Technique

Department of Physics, University of Alberta, Edmonton T6G 2G7, CanadaSpatially resolved energy -loss near-edge structure (ELNES) reveals geometric and electronic structure of materials at high spatial resolution [1]. In scanning transmission ele ctron microscope (STEM), ELNES allows to study the valence state of atoms at interfaces of a nanocomposite system. The obtainable signal-to-noise ratio (SNR), set by specimen drift and radiation damage, is an obstacle in obtaining interpretable ELNES data at high spatial resolution. The white line ratio (WLR) is often used as a “fingerprinting” technique in identifying the oxidation of transition metals (TMs) [2], but hard to extract when the SNR is low. We recently found that a broad postpeak following the L-edge, observed in thin specimens where plural scattering is negligible, is characteristic of TMs in an oxidized state [3]. The postpeak was applied to identify surface oxidation of nanoparticles (NP) by examining average oxidation state of many NPs [4]. Here we show that the postpeak can be a convenient and sensitive indicator of oxidation of single iron NP by forming point analysis using a STEM with a small (0.5 nm) probe. The measurements were performed on isolated Fe NPs embedded in silica, shown in the high-angle annular dark-field (HAADF) image (Fig. 1a). The data were obtained using a JEOL 2200 FS (a 200 kV Schottky field emission instrument equipped with an W filter). Fig. 1b shows the ELNES of Fe L-edges recorded at different sites of a selected particle, as labelled in HAADF image in Fig. 1a. The signal comes mostly from the surface of the NP when the beam is focused at its edge, and mainly from the interior when the beam is focused at the center. A broad postpeak, about 40 eV above the onset of Fe L

Mixed lagrange time delay estimation autoregressive Wiener filter application for real‐time SEM image enhancement

A new filter is developed for the enhancement of scanning electron microscope (SEM) images. A mixed Lagrange time delay estimation auto-regression (MLTDEAR)-based interpolator is used to provide an estimate of noise variance to a standard Wiener filter. A variety of images are captured and the performance of the filter is shown to surpass the conventional noise filters. As all the information required for processing is extracted from a single image, this method is not constrained by image registration requirements and thus can be applied in real-time in cases where specimen drift is presented in the SEM image.

Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides 3-D distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2-D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L2,3 edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell’s nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7nm-sized voxel.

Development of dedicated STEM with high stability

We developed a dedicated scanning transmission electron microscope with high-stability. The mechanical and electronic stabilities of the microscope were substantially improved, e.g. the specimen drift rate was found to be <0.2 nm min(-1). The Fourier transform of an ADF image showed spots of 0.105 nm at an acceleration voltage of 200 kV without spherical aberration corrector. The stabilized STEM instrument allows us to acquire distortion-free STEM images and high-signal to noise ratio analyses. We have shown the outline of the instrument and preliminary results.

Single channel blind image deconvolution from radially symmetric blur kernels

The multichannel exact blind image deconvolution theory tells us that exact recovery of unknown blur kernels is possible from multiple measurements of an identical scene through distinct blur channels. However, in many biological applications, there often exist technical difficulties in obtaining multiple distinct blur measurements, since the image content may vary for various reasons, including specimen drift between snapshots, specimen damage due to prolonged exposure, or physiological changes in live cell imaging. The main contribution of this paper is a new non-iterative single channel blind deconvolution method that eliminates the need of multiple blur measurements, but still guarantees an accurate estimation of the blurring kernel. The basic idea behind this novel method is to exploit the radial symmetry of a certain class of PSFs. This approach simplifies the PSF estimation to a 1-D channel identification problem with multiple excitations, which can be solved using a standard subspace method. Since radially symmetric PSFs are quite often encountered in many practical applications, such as optical imaging systems and electron microscopy, our theory may have great influence on many practical imaging applications. Simulation results as well as real experimental results using optical and electron microscopy confirm our theory.

Quantitative imaging of cells with multi-isotope imaging mass spectrometry (MIMS)—Nanoautography with stable isotope tracers

We describe some technical aspects of the application of multi-isotope imaging mass spectrometry (MIMS) to biological research, particularly the use of isotopic tags to localize and measure their incorporation into intracellular compartments. We touch on sample preparation, on image formation, on drift correction and on extraction of quantitative data from isotope ratio imaging. We insist on the wide variety of sample types that can be used, ranging from whole cells prepared directly on Si supports, to thin sections of cells and tissues on Si supports, to ultrathin TEM sections on carbon-coated grid. We attempt to dispel the myth of difficulties in sample preparation, which we view as a needless deterrent to the application of MIMS to the general biological community. We present protocols for the extraction of isotope ratio data from mass images. We illustrate the benefits of using sequential image plane acquisition followed by the application of an autocorrelation algorithm (nanotracking) to remove the effects of specimen drift. We insist on the advantages to display the isotope ratios as hue saturation intensity images.

Specimen temperature conditioning and drift before an S4 pipe fracture test

The resistance of plastic (primarily polyethylene) pipe to rapid axial crack propagation under pressure is specified using the ISO 13477 ‘S4’ (small-scale steady state) test method. Pipe performance in this test may be very sensitive to temperature under standard test conditions. There has recently been concern that the temperature conditioning of pipe specimens required by ISO 13477 may not be sufficiently precise. In order to compensate for an over-estimated increase in surface temperature between removal from the conditioning fluid and testing, some laboratories condition specimens well below the test temperature. Here, infra-red imaging data are used to calibrate a simple and conservative model for the evolution of surface temperature during this interval. The results broadly support the present specification for testing at 0 °C, and provide a more secure basis for conditioning of specimens to be tested at lower temperatures.

A specimen-drift-free EDX mapping system in a STEM for observing two-dimensional profiles of low dose elements in fine semiconductor devices

We developed a specimen-drift-free energy-dispersive X-ray (EDX) mapping system in a scanning transmission electron microscope (STEM) to improve the sensitivity and spatial resolution of EDX elemental mapping images. The amount of specimen drift was analysed from two STEM images before and after specimen drift by using the phase-correlation method, and was compensated for with an image-shift deflector of the STEM by the displacement of the scanning electron beam. We applied this system to observe the two-dimensional distribution of low dose arsenic in silicon semiconductor devices. The sensitivity of the elemental mapping was improved to several tenths atomic % for arsenic atoms while maintaining a spatial resolution of 2 nm.

Automated analysis of submicron particles by computer-controlled scanning electron microscopy.

Automated analysis of submicron particles by computer-controlled scanning electron microscopy is generally possible. The minimum diameter of the detectable particles is dependent on the mean atomic number of the particles and the operating parameters of the scanning microscope. The main limitation with regard to particle size is set by the quality of the particle detection system, which generally is the backscatter electron detector. The accuracy of the results of the x-ray analyses is very often strongly affected by specimen damage, omnipresent especially for environmental particles even at low electron energies and probe currents. With the exception for light elements, the detection limit is approximately 1 wt%. Device-related limitations to automated analysis may be specimen drift and an unreliable autofocus function.

A new method for the determination of the wave aberration function for high resolution TEM: 1. Measurement of the symmetric aberrations

A new method for the accurate determination of the symmetric coefficients of the wave aberration function has been developed. The relative defoci and displacements of images in a focus series are determined from an analysis of the phase correlation function between pairs of images, allowing the restoration of an image wave even when focus and specimen drift are present. Subsequently, the absolute coefficients of both defocus and 2-fold astigmatism are determined with a phase contrast index function. Overall this method allows a very accurate automated aberration determination even for largely crystalline samples with little amorphous contamination. Using experimental images of the complex oxide Nb 16W 18O 94 we have demonstrated the new method and critically compared it with existing diffractogram based aberration determinations. A series of protocols for practical implementation is also given together with a detailed analysis of the accuracy achieved. Finally a focal series restoration of Nb 16W 18O 94 with symmetric aberrations determined automatically using this method is presented.

Quantitative HREM: Reliable Structure Determination of Grain Boundaries in SrTiO3

High resolution transmission electron microscopy (HREM) is an excellent experimental method to image grain boundary structures with atomic resolution. The advantage of the method is the short exposure time of only about one second that is needed to record an image. Other methods like Z-contrast imaging require much longer exposure times and are therefore much more prone to specimen drift during recording. However there is the remaining difficulty to HREM that the evaluation of experimental images is not straightforward and a thorough analysis of the images is necessary in order to deduce quantitative information with small error bars of only a few pm (10-15m). A second inherent difficulty common to all atomic resolution imaging techniques is that the information is retrieved from a very small area of a specimen. The question arising from that is: can we nevertheless be sure to obtain a representative answer to a “real world” material science problem? A positive answer to this question is given by the investigations presented here.

Analysis of nanoscale multilayers by EDXS and EELS in the STEM

After a short description of a model for calculation of element specific linescan intensity profiles measured in an analytical transmission electron microscope on cross-sections of nanoscale multilayers by EDXS and EELS in the STEM mode the essential influences to the results are discussed. Particularly, the signal-to-noise ratio is considered. To confirm the model measured and calculated profiles are compared. Investigations on nanoscale multilayers require a field emission gun and a specimen stage with high stability. Specimen drift can lead to completely confused intensity profiles and has to be avoided or corrected during the measurement.

Accuracy of Spatial Measurements at Elevated Temperatures in an Environmental Scanning Electron Microscope

One of the unique abilities of the environmental scanning electron microscope is that it permits realtime dynamic observations of materials under controlled temperature/atmosphere conditions [1]. The need for accurate and precise measurements of dimensional changes at elevated temperatures arise in many material areas including determining the longitudinal and transverse coefficient of thermal expansion of fibers, sintering studies, hydration and dehydration of absorbent polymers, and strain measurement of materials under an applied load. The purpose of this research is to evaluate the capabilities of an ElectroScan 2020 environmental scanning electron microscope (ESEM™) in performing precise in situ spatial measurements at elevated temperatures. Identification and proposed solution and/or compensation of imaging difficulties such as specimen drift, sample stability, and thermal strain of a reference grid are addressed.In general, the accuracy of spatial measurements in a scanning electron microscope (SEM) depends on several factors including calibration of the scanned area, the effects of lens hysteresis and reproducibility in the working distance and column alignment.

Mapping Phosphorus In Macromolecular Assemblies At Near Single Atom Sensitivity By Stem-Eels

In principle, electron energy loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) should be able to detect single atoms of phosphorus in biological macromolecules. Such a capability would provide a direct method for measuring the degree of phosphorylation or nucleotide binding which are important in determining the functional states of protein assemblies. Although it has already been possible to map heavy elements, e.g., uranium, at near single atom sensitivity, this has not yet been achievable for lighter atoms like phosphorus. One difficulty in imaging light atoms in a biological specimen is that there is insufficient contrast to visualize the atoms directly so, in a stationary probe analysis, it is not known where to place the probe. Identifying single phosphorus atoms therefore necessarily involves compositional mapping in which EELS information is collected pixel by pixel, a technique known as spectrum-imaging. Recently, improvements have been made in the acquisition of such images, especially the ability to correct for specimen drift in a robust fashion.

A Method for Site Specific Characterization Using a Dedicated FIB System Combined With an Analyitical TEM

A method for site specific characterization of the materials using a dedicated focused ion beam(FIB) system and an analytical transmission electron microscope (TEM) was developed. Needless to say, in TEM specimen preparation using FIB system, stability of a specimen is quite important. The specimen stage employed in the developed FIB system is the one designed for high resolution TEM, and the specimen drift rate of the stage is less than lnm/min. In addition, FIB-TEM compatible specimen holder which allows milling of a specimen with the FIB system and observation of the specimen with the TEM without re-loading was developed. To obtain thin specimen from the area to be characterized correctly, confirmation of the area before final milling is needed. However, observation of cross sectional view in a FIB system is recommended because it causes damage by Ga ion irradiation. To solve this problem, we used a STEM unit as a viewer of FIB milled specimen.

Towards single atom analysis of biological structures

Mapping single atoms in biological structures is now becoming within the reach of analytical electron microscopy. Electron energy-loss spectroscopy (EELS) in the field-emission scanning transmission electron microscope (STEM) provides a particularly high sensitivity for detecting the biologically important element, phosphorus. Imaging can be performed at low dose with dark-field STEM prior to analysis at high dose, so that structures of macromolecular assemblies can be correlated with the numbers of specific atoms that they contain. Measurements confirm theoretical predictions that single atom detection requires a nanometer-sized probe. Although phosphorus atoms may have moved several nanometers from their original positions by beam-induced structural degradation at the high required dose of ∼10 9 e/nm 2, damaged molecules are nevertheless stable enough to be analyzed at 1 or 2 nm resolution. Such analyses can only be achieved by means of spectrum-imaging with correction for specimen drift. Optimal strategies for mapping small numbers of phosphorus atoms have been investigated using well-characterized specimens of DNA plasmids and tobacco mosaic virus.