Showing papers in "Microscopy and Microanalysis in 2010"

Enhancing Serial Block-Face Scanning Electron Microscopy to Enable High Resolution 3-D Nanohistology of Cells and Tissues

Abstract: Serial block face scanning electron microscopy (SBFSEM) is a powerful technique originally introduced by Leighton [1], substantially improved by Denk [2] and subsequently commercialized (Gatan Inc., Pleasanton, CA.). SBFSEM allows for the automated image acquisition of relatively large volumes of tissue at near nanometer-scale resolution, using a dry cutting ultramicrotome fitted into an SEM. In an automated process, a low voltage backscatter electron (BSE) image is obtained from the surface of an epoxy embedded tissue block face. The ultramicrotome then removes an ultra-thin section of tissue with a specially designed oscillating diamond knife (Diatome AG, Switzerland), and a block face image from the corresponding region is again obtained. This sequence is repeated over and over until the desired volume of tissue has been imaged. Although SBFSEM overcomes many obstacles routinely encountered with serial section TEM reconstruction, until recently there was a significant limitation to the resolution obtainable by this method compared to conventional TEM. This was due primarily to difficulties encountered using BSE imaging at low accelerating voltages. To overcome this we have developed a protocol for vastly increasing the heavy metal staining of specimens to improve BSE yield. This is accomplished by combining a variety of preexisting heavy metal staining methodologies not normally used together, including ferrocyanide-reduced osmium tetroxide, thiocarbohydrazide-osmium tetroxide (OTO), prolonged uranyl acetate treatment and en bloc lead aspartate staining. Using this approach, we demonstrate a dramatic improvement in image contrast and resolution from existing methods in a variety of specimens (Fig. 1).

Colocalization analysis in fluorescence micrographs: verification of a more accurate calculation of pearson's correlation coefficient.

Abstract: One of the most routine uses of fluorescence microscopy is colocalization, i.e., the demonstration of a relationship between pairs of biological molecules. Frequently this is presented simplistically by the use of overlays of red and green images, with areas of yellow indicating colocalization of the molecules. Colocalization data are rarely quantified and can be misleading. Our results from both synthetic and biological datasets demonstrate that the generation of Pearson's correlation coefficient between pairs of images can overestimate positive correlation and fail to demonstrate negative correlation. We have demonstrated that the calculation of a thresholded Pearson's correlation coefficient using only intensity values over a determined threshold in both channels produces numerical values that more accurately describe both synthetic datasets and biological examples. Its use will bring clarity and accuracy to colocalization studies using fluorescent microscopy.

Spatial resolution in atom probe tomography

Abstract: 3 Science et Ingenierie des MAteriaux et Procedes (SIMaP) - UMR 5266 CNRS-Grenoble INP-UJF, Saint-Martin-d'Heres, France Abstract: This article addresses gaps in definitions and a lack of standard measurement techniques to assess the spatial resolution in atom probe tomography. This resolution is known to be anisotropic, being better in-depth than laterally. Generally the presence of atomic planes in the tomographic reconstruction is considered as being a sufficient proof of the quality of the spatial resolution of the instrument. Based on advanced spatial distribution maps, an analysis methodology that interrogates the local neighborhood of the atoms within the tomographic reconstruction, it is shown how both the in-depth and the lateral resolution can be quantified. The influences of the crystallography and the temperature are investigated, and models are proposed to explain the observed results. We demonstrate that the absolute value of resolution is specimen specific.

Microfluidic system for transmission electron microscopy.

Abstract: We present a microfluidic system that maintains liquid flow in a specimen chamber for scanning transmission electron microscope (STEM) imaging. The specimen chamber consists of two ultrathin silicon nitride windows supported by silicon microchips. They are placed in a specimen holder that seals the sample from the vacuum in the electron microscope and incorporates tubing to and from the sample connected to a syringe pump outside the microscope. Using results obtained from fluorescence microscopy of microspheres flowing through the system, an equation to characterize the liquid flow through the system was calibrated. Gold nanoparticles of diameters of 30 and 100 nm moving in liquid were imaged with a 200 kV STEM. It was concluded that despite strong influences from Brownian motion, and sensitivity to small changes in the depth of the bypass channel, the electron microscopy flow data matched the calculated flow speed within an order of magnitude. The system allows for rapid (within a minute) liquid exchange, which can potentially be used, for example, to investigate the response of specimens, e.g., eukaryotic or bacterial cells, to certain stimuli.

Visualization of localization microscopy data.

Abstract: Localization microscopy techniques based on localizing single fluorophore molecules now routinely achieve accuracies better than 30 nm. Unlike conventional optical microscopies, localization microscopy experiments do not generate an image but a list of discrete coordinates of estimated fluorophore positions. Data display and analysis therefore generally require visualization methods that translate the position data into conventional images. Here we investigate the properties of several widely used visualization techniques and show that a commonly used algorithm based on rendering Gaussians may lead to a 1.44-fold loss of resolution. Existing methods typically do not explicitly take sampling considerations into account and thus may produce spurious structures. We present two additional visualization algorithms, an adaptive histogram method based on quad-trees and a Delaunay triangulation based visualization of point data that address some of these deficiencies. The new visualization methods are designed to suppress erroneous detail in poorly sampled image areas but avoid loss of resolution in well-sampled regions. A number of criteria for scoring visualization methods are developed as a guide for choosing among visualization methods and are used to qualitatively compare various algorithms.

Information transfer in a TEM corrected for spherical and chromatic aberration.

Abstract: For the transmission electron aberration-corrected microscope (TEAM) initiative of five U.S. Department of Energy laboratories in the United States, a correction system for the simultaneous compensation of the primary axial aberrations, the spherical aberration Cs, and the chromatic aberration Cc has been developed and successfully installed. The performance of the resulting Cc /Cs-corrected TEAM instrument has been investigated thoroughly. A significant improvement of the linear contrast transfer can be demonstrated. The information about the instrument one obtains using Young's fringe method is compared for uncorrected, Cs-corrected, and Cc /Cs-corrected instruments. The experimental results agree well with simulations. The conclusions might be useful to others in understanding the process of image formation in a Cc /Cs-corrected transmission electron microscope.

Nanoscale X-ray microscopic imaging of mammalian mineralized tissue.

Abstract: A novel hard transmission X-ray microscope (TXM) at the Stanford Synchrotron Radiation Lightsource operating from 5 to 15 keV X-ray energy with 14 to 30 microm2 field of view has been used for high-resolution (30-40 nm) imaging and density quantification of mineralized tissue. TXM is uniquely suited for imaging of internal cellular structures and networks in mammalian mineralized tissues using relatively thick (50 microm), untreated samples that preserve tissue micro- and nanostructure. To test this method we performed Zernike phase contrast and absorption contrast imaging of mouse cancellous bone prepared under different conditions of in vivo loading, fixation, and contrast agents. In addition, the three-dimensional structure was examined using tomography. Individual osteocytic lacunae were observed embedded within trabeculae in cancellous bone. Extensive canalicular networks were evident and included processes with diameters near the 30-40 nm instrument resolution that have not been reported previously. Trabecular density was quantified relative to rod-like crystalline apatite, and rod-like trabecular struts were found to have 51-54% of pure crystal density and plate-like areas had 44-53% of crystal density. The nanometer resolution of TXM enables future studies for visualization and quantification of ultrastructural changes in bone tissue resulting from osteoporosis, dental disease, and other pathologies.

Synchrotron UV fluorescence microscopy uncovers new probes in cells and tissues.

Abstract: Use of deep ultraviolet (DUV, below 350 nm) fluorescence opens up new possibilities in biology because it does not need external specific probes or labeling but instead allows use of the intrinsic fluorescence that exists for many biomolecules when excited in this wavelength range. Indeed, observation of label free biomolecules or active drugs ensures that the label will not modify the biolocalization or any of its properties. In the past, it has not been easy to accomplish DUV fluorescence imaging due to limited sources and to microscope optics. Two worlds were coexisting: the spectrofluorometric measurements with full spectrum information with DUV excitation, which lacked high-resolution localization, and the microscopic world with very good spatial resolution but poor spectral resolution for which the wavelength range was limited to 350 nm. To combine the advantages of both worlds, we have developed a DUV fluorescence microscope for cell biology coupled to a synchrotron beamline, providing fine tunable excitation from 180 to 600 nm and full spectrum acquired on each point of the image, to study DUV excited fluorescence emitted from nanovolumes directly inside live cells or tissue biopsies.

Three-dimensional scanning transmission electron microscopy of biological specimens.

Abstract: A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2-3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset.

Three-dimensional analysis of carbon nanotube networks in interconnects by electron tomography without missing wedge artifacts.

Abstract: The three-dimensional ~3D! distribution of carbon nanotubes ~CNTs! grown inside semiconductor contact holes is studied by electron tomography. The use of a specialized tomography holder results in an angular tilt range of 6908, which means that the so-called "missing wedge" is absent. The transmission electron microscopy ~TEM! sample for this purpose consists of a micropillar that is prepared by a dedicated procedure using the focused ion beam ~FIB! but keeping the CNTs intact. The 3D results are combined with energy dispersive X-ray spectroscopy ~EDS! to study the relation between the CNTs and the catalyst particles used during their growth. The reconstruction, based on the full range of tilt angles, is compared with a reconstruc- tion where a missing wedge is present. This clearly illustates that the missing wedge will lead to an unreliable interpretation and will limit quantitative studies.

D-STEM: A Parallel Electron Diffraction Technique Applied to Nanomaterials

Abstract: An electron diffraction technique called D-STEM has been developed in a transmission electron microscopy/scanning transmission electron microscopy (TEM/STEM) instrument to obtain spot electron diffraction patterns from nanostructures, as small as ∼3 nm. The electron ray path achieved by configuring the pre- and postspecimen illumination lenses enables the formation of a 1-2 nm near-parallel probe, which is used to obtain bright-field/dark-field STEM images. Under these conditions, the beam can be controlled and accurately positioned on the STEM image, at the nanostructure of interest, while sharp spot diffraction patterns can be simultaneously recorded on the charge-coupled device camera. When integrated with softwares such as GatanTM STEM diffraction imaging and Automated Crystallography for TEM or DigistarTM, NanoMEGAS, the D-STEM technique is very powerful for obtaining automated orientation and phase maps based on diffraction information acquired on a pixel by pixel basis. The versatility of the D-STEM technique is demonstrated by applying this technique to nanoparticles, nanowires, and nano interconnect structures.

Cryomesh™: A New Substrate for Cryo-Electron Microscopy

Abstract: Here we evaluate a new grid substrate developed by ProtoChips Inc. (Raleigh, NC) for cryo-transmission electron microscopy. The new grids are fabricated from doped silicon carbide using processes adapted from the semiconductor industry. A major motivating purpose in the development of these grids was to increase the low-temperature conductivity of the substrate, a characteristic that is thought to affect the appearance of beam-induced movement (BIM) in transmission electron microscope (TEM) images of biological specimens. BIM degrades the quality of data and is especially severe when frozen biological specimens are tilted in the microscope. Our results show that this new substrate does indeed have a significant impact on reducing the appearance and severity of beam-induced movement in TEM images of tilted cryo-preserved samples. Furthermore, while we have not been able to ascertain the exact causes underlying the BIM phenomenon, we have evidence that the rigidity and flatness of these grids may play a major role in its reduction. This improvement in the reliability of imaging at tilt has a significant impact on using data collection methods such as random conical tilt or orthogonal tilt reconstruction with cryo-preserved samples. Reduction in BIM also has the potential for improving the resolution of three-dimensional cryo-reconstructions in general.

Electron probe microanalysis of thin films and multilayers using the computer program XFILM.

Abstract: XFILM is a computer program for determining the thickness and composition of thin films on substrates and multilayers by electron probe microanalysis. In this study, we describe the X-ray emission model implemented in the latest version of XFILM and assess its reliability by comparing measured and calculated k-ratios from thin-film samples available in the literature. We present and discuss examples of applications of XFILM that illustrate the capabilities of the program.

Streamlined embedding of cell monolayers on gridded glass-bottom imaging dishes for correlative light and electron microscopy.

Abstract: Correlative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.

Clustering and local magnification effects in atom probe tomography: a statistical approach.

Abstract: Local magnification effects and trajectory overlaps related to the presence of a second phase (clusters) are key problems and still open issues in the assessment of quantitative composition data in three-dimensional atom probe tomography (APT) particularly for tiny solute-enriched clusters. A model based on the distribution of distance of first nearest neighbor atoms has been developed to exhibit the variations in the apparent atomic density in reconstructed volumes and to correct compositions that are biased by local magnification effects. This model was applied to both simulated APT reconstructions and real experimental data and shows an excellent agreement with the expected composition of clusters.

Development of a new quantitative X-ray microanalysis method for electron microscopy.

Abstract: Quantitative X-ray microanalysis of thick samples is usually performed by measuring the characteristic X-ray intensities of each element in a sample and in corresponding standards. The ratio of the measured intensities from the unknown material to that from the standard is related to the concentration using the ZAF or ϕ(ρz) equations. Under optimal conditions, accuracies approaching 1% are possible. However, all the experimental conditions must remain the same during the sample and standard measurements. This is not possible with cold field emission scanning electron microscopes (FE-SEMs) where beam current can fluctuate around 5% in its stable regime. Very little work has been done on variable beam current conditions (Griffin, B.J. & Nockolds, C.E., Scanning 13, 307-312, 1991), and none relating to cold FE-SEM applications. To address this issue, a new method was developed using a single spectral measurement. It is similar in approach to the Cliff-Lorimer method developed for the analytical transmission electron microscope. However, corrections are made for X rays generated from thick specimens using the ratio of the characteristic X-ray intensities of two elements in the same material. The proposed method utilizes the ratio of the intensity of a characteristic X-ray normalized by the sum of X-ray intensities of all the elements measured for the sample, which should also reduce the amplitude of error propagation. Uncertainties in the physical parameters of X-ray generation are corrected using a calibration factor that must be previously acquired or calculated. As an example, when this method was applied to the calculation of the composition of Au-Cu National Institute of Standards and Technology standards measured with a cold field emission source SEM, relative accuracies better than 5% were obtained.

Simulating STEM Imaging of Nanoparticles in Micrometers-Thick Substrates

Abstract: Scanning transmission electron microscope (STEM) images of three-dimensional (3D) samples were simulated. The samples consisted of a micrometer(s)-thick substrate and gold nanoparticles at various vertical positions. The atomic number (Z) contrast as obtained via the annular dark-field detector was generated. The simulations were carried out using the Monte Carlo method in the CASINO software (freeware). The software was adapted to include the STEM imaging modality, including the noise characteristics of the electron source, the conical shape of the beam, and 3D scanning. Simulated STEM images of nanoparticles on a carbon substrate revealed the influence of the electron dose on the visibility of the nanoparticles. The 3D datasets obtained by simulating focal series showed the effect of beam broadening on the spatial resolution and on the signal-to-noise ratio. Monte Carlo simulations of STEM imaging of nanoparticles on a thick water layer were compared with experimental data by programming the exact sample geometry. The simulated image corresponded to the experimental image, and the signal-to-noise levels were similar. The Monte Carlo simulation strategy described here can be used to calculate STEM images of objects of an arbitrary geometry and amorphous sample composition. This information can then be used, for example, to optimize the microscope settings for imaging sessions where a low electron dose is crucial for the design of equipment, or for the analysis of the composition of a certain specimen.

The Formation and Utility of Sub-Angstrom to Nanometer-Sized Electron Probes in the Aberration-Corrected Transmission Electron Microscope at the University of Illinois

Abstract: We evaluate the probe forming capability of a JEOL 2200FS transmission electron microscope equipped with a spherical aberration (Cs) probe corrector. The achievement of a real space sub-Angstrom (0.1 nm) probe for scanning transmission electron microscopy (STEM) imaging is demonstrated by acquisition and modeling of high-angle annular dark-field STEM images. We show that by optimizing the illumination system, large probe currents and large collection angles for electron energy loss spectroscopy (EELS) can be combined to yield EELS fine structure data spatially resolved to the atomic scale. We demonstrate the probe forming flexibility provided by the additional lenses in the probe corrector in several ways, including the formation of nanometer-sized parallel beams for nanoarea electron diffraction, and the formation of focused probes for convergent beam electron diffraction with a range of convergence angles. The different probes that can be formed using the probe corrected STEM opens up new applications for electron microscopy and diffraction.

Imaging, core-loss, and low-loss electron-energy-loss spectroscopy mapping in aberration-corrected STEM.

Abstract: High-angle annular dark-field and annular bright-field imaging experiments were carried out on an aberration-corrected transmission electron microscope. These techniques have been demonstrated on thin films of complex oxides Ba3.25La0.75Ti3O12 and on LaB6. The results show good agreement between theory and experiments, and for the case of LaB6 they demonstrate the detection of contrast from the B atoms in the annular bright-field images. Elemental mapping with electron-energy-loss spectroscopy has been used to deduce the distribution of Cr and Fe in a thin film of the complex oxide Bi2(Fe1/2Cr3/2)O6 at the unit cell level and the changes in the near-edge structure within the inequivalent regions in the crystalline unit cell. Energy-filtered images in the low-loss region of the energy-loss spectrum show contrast and resolution consistent with the modulation of the signals from elastic scattering. High-resolution contrast, mediated by phonon scattering, is observed for interband transitions. The limitations in terms of detection and signal are discussed.

Three-dimensional imaging in aberration-corrected electron microscopes.

Abstract: This article focuses on the development of a transparent and uniform understanding of possibilities for three-dimensional (3D) imaging in scanning transmission and confocal electron microscopes (STEMs and SCEMs), with an emphasis on the annular dark-field STEM (ADF-STEM), bright-field SCEM (BF-SCEM), and ADF-SCEM configurations. The incoherent imaging approximation and a 3D linear imaging model for ADF-STEM are reviewed. A 3D phase contrast model for coherent-SCEM as well as a pictorial way to find boundaries of information transfer in reciprocal space are reviewed and applied to both BF- and ADF-SCEM to study their 3D point spread functions and contrast transfer functions (CTFs). ADF-STEM is capable of detecting the depths of dopant atoms in amorphous materials but can fail for crystalline materials when channeling substantially modifies the electron propagation. For the imaging of extended (i.e., nonpointlike) features, ADF-STEM and BF-SCEM exhibit strong elongation artifacts due to the missing cone of information. ADF-SCEM shows an improvement over ADF-STEM/BF-SCEM due to its differential phase contrast eliminating slowly varying backgrounds, an effect that partially suppresses the elongation artifacts. However, the 3D CTF still has a cone of missing information that will result in some residual feature elongation as has been observed in A. Hashimoto et al., J Appl Phys 160(8), 086101 (2009).

Contributions of electron microscopy to understand secretion of immune mediators by human eosinophils.

Abstract: Mechanisms governing secretion of proteins underlie the biologic activities and functions of human eosinophils, leukocytes of the innate immune system, involved in allergic, inflammatory, and immunoregulatory responses. In response to varied stimuli, eosinophils are recruited from the circulation into inflammatory foci, where they modulate immune responses through the release of granule-derived products. Transmission electron microscopy (TEM) is the only technique that can clearly identify and distinguish between different modes of cell secretion. In this review, we highlight the advances in understanding mechanisms of eosinophil secretion, based on TEM findings, that have been made over the past years and that have provided unprecedented insights into the functional capabilities of these cells.

New electrostatic phase plate for phase-contrast transmission electron microscopy and its application for wave-function reconstruction.

Abstract: A promising novel type of electrostatic phase plate for transmission electron microscopy (TEM) is presented. The phase plate consists of a single microcoaxial cable-like rod with its electrode exposed to the undiffracted electrons. The emerging field is used to shift the phase of the undiffracted electrons with respect to diffracted electrons. The design overcomes the drawback of the spatial frequency-blocking ring electrode of the Boersch phase plate. First, experimental phase-contrast images are presented for PbSe and Pt nanoparticles with clearly varying phase contrast, which depends on the applied voltage and resulting phase shift of the unscattered electrons. With the new phase-plate design, we show for the first time the reconstruction of an object wave function based on a series of only three experimental phase-contrast TEM images obtained with an electrostatic phase plate.

Suberized cell walls of cork from cork oak differ from other species.

Abstract: Plants have suberized cells that act as protective interfaces with the environment or between different plant tissues. A lamellar structure of alternating dark and light bands has been found upon transmission electron microscopy (TEM) observation of cork cells and considered a typical feature of the suberized secondary wall. We observed cork cells from periderms of Quercus suber, Quercus cerris, Solanum tuberosum, and Calotropis procera by TEM after uranyl acetate and lead citrate staining. A lamellated structure was observed in S. tuberosum and C. procera but not in Q. suber and Q. cerris where the suberized cell wall showed a predominantly hyaline aspect with only a dark dotted staining. Removal of suberin from Q. suber cells left a thinner secondary wall that lost the translucent aspect. We hypothesize that the species' specific chemical composition of suberin will result in different three-dimensional macromolecular development and in a different spatial location of lignin and other aromatics. A lamellated ultrastructure is therefore not a general feature of suberized cells.

Transmission electron microscopy of lipid vesicles for drug delivery: comparison between positive and negative staining.

Abstract: Lipid-containing nanostructures, in the form of solid lipid nanoparticles or iron oxide nanoparticles (NPs) coated with a lipid shell, were used as case studies for assessing and optimizing staining for transmission electron microscopy structural and compositional characterization. These systems are of paramount importance as drug delivery systems or as bio-compatible contrast agents. In particular, we have treated the systems with a negative (phospshotungstic acid) or with a positive (osmium tetroxide) staining agent. For iron-oxide NPs coated with the lipid shell, negative staining was more efficient with respect to the positive one. Nevertheless, in particular cases the combination of the two staining procedures provided more complete morphological and compositional characterization of the particles.

A Comparison between Bright Field and Phase-Contrast Image Analysis Techniques in Activated Sludge Morphological Characterization

Abstract: Different approaches using microscopy image analysis procedures were employed for characterization of activated sludge systems. The approaches varied mainly on the type of visualization and acquisition method used for collection of data. In this context, this study focused on the comparison of the two most common acquisition methods: bright field and phase-contrast microscopy. Images were acquired from seven different wastewater treatment plants for a combined period of two years. Advantages and disadvantages of each acquisition technique and the results are discussed. Bright field microscopy proved to be more simple and inexpensive and provided the best overall results.

Comparison of the Detection Limits of EDS and EELS in S/TEM

Abstract: The detection limit for analytical techniques has been the subject of many publications. [1] The most general approach [2] is to define the detection limit at 99% confidence limit of detecting a minor element as Ip-Ib > 3*SQRT(2*Ib) where Ip is the total intensity in an energy window for a given element and Ib is the background intensity under the same window. The ionization cross-section of a given element determines the number of innershell energy loss events in both EDS and EELS, but there are 3 major differences: 1. the X-ray production for an inner-shell transition competes with the generation of Auger electrons, giving rise to a Z-dependent fluorescence yield factor (ω<1) 2. the detection efficiency of EELS systems is generally much higher (up to 80% at low count rates) than for EDS systems (< 1%) 3. the peak-to-background ratios (P/B=Ip-Ib/Ib)) for EELS are generally much lower for EELS than EDS, especially near the detection limit. These differences make EELS a much more sensitive technique for light elements and EDS for heavy elements. However, with the advent of new detector technology it has been possible to improve the EDS detection efficiency by an order of magnitude [3]. This paper re-examines the detection limits of the two techniques under identical conditions in an FEI 200kV S/TEM equipped with a Super-X EDS detector with 0.9 sr collection angle and an EELS spectrometer. The detection efficiency (as a %age of 4π) of the Super-X is compared with Si(Li) detectors in Fig.1; the high efficiency for low X-ray energy (light elements) is due to the absence of a window in front of the detector. The dead time is also much lower for Super-X at the same energy resolution (Fig.1 right) Fig.2 shows simultaneous spectra acquired on a titanium alloy (0.5%Fe, 3%Cr, 5%V, 5%Mo by wt.) of medium specimen thickness, containing Fe-rich precipitates. All the minor elements in the alloy are visible in the EDS spectra, but not in EELS. The P/B ratio of Ti is 35 in EDS compared to ~3 in EELS; for Fe in the precipitates the P/B ratios are 3 for EDS and ~0.01 for EELS. In Fig.3 a line scan from the edge of the specimen shows the specimen thickness t varying from about 0.1 to 0.8 times the inelastic mean free path MFP (λ). In the thin regions (t<0.3 λ) both the EDS and the EELS spectra show iron signals in the precipitates, but the P/B ratio is very low (P/B <0.01) in the EELS case, making accurate quantifications impossible. For a relatively thick (t~0.8λ) region the Fe peak is not visible in EELS signals due to multiple scattering, but is strong in the EDS spectra. The detection limit for EDS is therefore better than for EELS at all specimen thicknesses for all the elements in this alloy. Further examples will be given for a wide range of Z numbers, for example on a chromium doped TiB2 WB alloy, for which the detection limits have been determined by both techniques. 1312 doi:10.1017/S1431927610058940 Microsc. Microanal. 16 (Suppl 2), 2010 © Microscopy Society of America 2010

Diffraction imaging in a He+ ion beam scanning transmission microscope

Abstract: The scanning helium ion microscope has been used in transmission mode to investigate both the feasibility of this approach and the utility of the signal content and the image information available. Operating at 40 keV the penetration of the ion beam, and the imaging resolution achieved, in MgO crystals was found to be in good agreement with values predicted by Monte Carlo modeling. The bright-field and annular dark-field signals displayed the anticipated contrasts associated with beam absorption and scattering. In addition, the diffraction of the He ion beam within the sample gave rise to crystallographic contrast effects in the form of thickness fringes and dislocation images. Scanning transmission He ion microscopy thus achieves useful sample penetration and provides nanometer scale resolution, high contrast images of crystalline materials and crystal defects even at modest beam energies.

Using DTSA-II to simulate and interpret energy dispersive spectra from particles.

Abstract: A high quality X-ray spectrum image of a 33 mum diameter sphere of K411 glass resting on a copper substrate was collected at 25 keV The same sample configuration was modeled using the NISTMonte Monte Carlo simulation of electron and X-ray transport as is integrated into the quantitative X-ray microanalysis software package DTSA-II The distribution of measured and simulated X-ray intensity compare favorably for all the major lines present in the spectra The simulation is further examined to investigate the influence of angle-of-incidence, sample thickness, and sample diameter on the generated and measured X-ray intensity The distribution of generated X-rays is seen to deviate significantly from a naive model which assumes that the distribution of generated X-rays is similar to bulk within the volume they share in common It is demonstrated that the angle at which the electron beam strikes the sample has nonnegligible consequences It is also demonstrated that within the volume that the bulk and particle share in common that electrons, which have exited and later reentered the particle volume, generate a significant fraction of the X-rays Any general model of X-ray generation in particles must take into account the lateral spread of the scattered electron beam

Experimental Evaluation of the Depth Resolution of a Raman Microscope

Abstract: Raman microscopy has been attractive because of its ability to characterize materials on a spatial scale commensurate with optical microscopy. Typically the lateral spatial resolution is quoted as determined by the Airy disc[1] which is 1.22λ/NA where λ is the wavelength of the illuminating light, and NA is the numerical aperture which is equal to nsinθ, where n is the index of refraction of the medium (1.0 in the case of air) and is the angle subtended by the optics. However, the Airy disc description cannot be correct for a Raman microscope. The Airy disc assumes uniform illumination of the focusing optic, and the laser profile is anything but. In addition, in some instruments the Gaussian laser profile is not well matched to the aperture of the focusing objective. At any rate, this article is going to concentrate on the depth resolution of the Raman microscope. Optical calculations for depth resolution of an optical microscope state that the it is proportional to λ/(NA). The essential point to recognize is that the spatial resolution of any Raman microscope depends on the detection optics as well as the focusing optics. How effectively does the optical system collect the Raman signal excited in the laser focal spot, and reject the signal from the surrounding volume that is illuminated by the laser but not in focus?