Influence of nanostructure composition on its morphometric characterization by different techniques

Morphometric characterization of nanoparticles is crucial to determine their biological effects and to obtain a formulation pattern. Determining the best technique requires knowledge of the particles being analyzed, the intended application of the particles, and the limitations of the techniques being considered. The aim of this article was to present transmission (TEM) and scanning (SEM) electron microscopy protocols for the analysis of two different nanostructures, namely polymeric nanoemulsion and poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles, and to compare these results with conventional dynamic light scattering (DLS) measurements. The mean hydrodynamic diameter, the polydispersity index, and zeta potential of the nanostructures of polymeric nanoemulsion were 370.5 ± 0.8 nm, 0.133 ± 0.01, and ?36.1 ± 0.15 mV, respectively, and for PLGA nanoparticles were 246.79 ± 5.03 nm, 0.096 ± 0.025, and ?4.94 ± 0.86 mV, respectively. TEM analysis of polymeric nanoemulsion revealed a mean diameter of 374 ± 117 nm. SEM analysis showed a mean diameter of 368 ± 69 nm prior to gold coating and 448 ± 70 nm after gold coating. PLGA nanoparticles had a diameter of 131 ± 41.18 nm in TEM and 193 ± 101 nm in SEM. Morphologically, in TEM analysis, the polymeric nanoemulsions were spherical, with variable electron density, very few showing an electron‐dense core and others an electron‐dense surface. PLGA nanoparticles were round, with an electron‐lucent core and electron‐dense surface. In SEM, polymeric nanoemulsions were also spherical with a rough surface, and PLGA nanoparticles were round with a smooth surface. The results show that the “gold standards” for morphometric characterization of polymeric nanoemulsion and PLGA nanoparticles were, respectively, SEM without gold coating and TEM with negative staining. Microsc. Res. Tech. 77:691–696, 2014. © 2014 Wiley Periodicals, Inc.     

Transmission EBSD from 10 nm domains in a scanning electron microscope

The spatial resolution of electron diffraction within the scanning electron microscope (SEM) has progressed from channelling methods capable of measuring crystallographic characteristics from 10 μm regions to electron backscatter diffraction (EBSD) methods capable of measuring 120 nm particles. Here, we report a new form of low‐energy transmission Kikuchi diffraction, performed in the SEM. Transmission‐EBSD (t‐EBSD) makes use of an EBSD detector and software to capture and analyse the angular intensity variation in large‐angle forward scattering of electrons in transmission, without postspecimen coils. We collected t‐EBSD patterns from Fe–Co nanoparticles of diameter 10 nm and from 40 nm‐thick Ni films with in‐plane grain size 15 nm. The patterns exhibited contrast similar to that seen in EBSD, but are formed in transmission. Monte Carlo scattering simulations showed that in addition to the order of magnitude improvement in spatial resolution from isolated particles, the energy width of the scattered electrons in t‐EBSD is nearly two orders of magnitude narrower than that of conventional EBSD. This new low‐energy transmission diffraction approach builds upon recent progress in achieving unprecedented levels of imaging resolution for materials characterization in the SEM by adding high‐spatial‐resolution analytical capabilities.     

Radius dependence of solute concentration estimates of simulated ultrafine precipitates

Estimates of the radii and solute concentrations of simulated microstructures containing ultrafine spherical precipitates were determined from isoconcentration surfaces and proximity histograms. The accuracy of the estimates of the solute concentrations and the radii of precipitates was found to depend on the size of precipitates. Optimized parameters for analyzing 0.5‐ to 2‐nm‐radius precipitates are proposed. The solute content of 0.5‐nm‐radius precipitates was not estimated correctly by this method. The accuracy of the estimates of the solute concentration and the radius of precipitates were primarily influenced by the solute concentrations of the precipitates. The ranges of error of the solute concentration in the precipitates, which are associated with the analytical limitations of the ultrafine precipitates, were determined, and the results indicated a limitation of the estimates. Microsc. Res. Tech. 76:1196–1203, 2013. © 2013 Wiley Periodicals, Inc.     

Characterization of precipitates size distribution: validation of low‐voltage STEM

The size distribution of second phase precipitates is frequently determined using conventional transmission electron microscopy (CTEM). However, other techniques, which present different advantages, can also be used for this purpose. In this paper, we focus on high angle annular dark field (HAADF) in TEM and scanning TEM (STEM) in scanning electron microscopy (SEM) imaging modes. The mentioned techniques will be first described, then compared to more conventional ones for the measurement of carbides size distribution in two FeCV and FeCVNb model alloys. This comparative study shows that STEM in SEM, a technique much easier to undertake compared to TEM, is perfectly adapted for size distribution measurements of second phase particles, with sizes ranging between 5 and 200 nm in these systems.     

High resolution SEM imaging of gold nanoparticles in cells and tissues

The growing demand of gold nanoparticles in medical applications increases the need for simple and efficient characterization methods of the interaction between the nanoparticles and biological systems. Due to its nanometre resolution, modern scanning electron microscopy (SEM) offers straightforward visualization of metallic nanoparticles down to a few nanometre size, almost without any special preparation step. However, visualization of biological materials in SEM requires complicated preparation procedure, which is typically finished by metal coating needed to decrease charging artefacts and quick radiation damage of biomaterials in the course of SEM imaging. The finest conductive metal coating available is usually composed of a few nanometre size clusters, which are almost identical to the metal nanoparticles employed in medical applications. Therefore, SEM monitoring of metal nanoparticles within cells and tissues is incompatible with the conventional preparation methods. In this work, we show that charging artefacts related to non‐conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive substrate. By growing the cells on glass pre‐coated with a chromium layer, we were able to observe the uptake of 10 nm gold nanoparticles inside uncoated and unstained macrophages and keratinocytes cells. Imaging in back scattered electrons allowed observation of gold nanoparticles located inside the cells, while imaging in secondary electron gave information on gold nanoparticles located on the surface of the cells. By mounting a skin cross‐section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals.     

SEM characterization of silicon nanostructures: can we meet the challenge?

The current semiconductor technology road map for device scaling champions a 4.5 nm gate length in production by 2022. The scanning electron microscope (SEM) as applied to critical dimensions (CD) metrology and associated characterization modes such as electron beam-induced current and cathodoluminescence (CL) has proved to be a workhorse for the semiconductor industry during the microelectronics era. We review some of the challenges facing these techniques in light of the silicon nanotechnology road map. We present some new results using voltage contrast imaging and CL spectroscopy of top-down fabricated silicon nanopillar/nanowires (<100 nm diameter), which highlight the visualization challenge. However, both techniques offer the promise of providing process characterization on the 10-20 nm scale with existing technology. Visualization at the 1 nm scale with these techniques may have to wait for aberration-corrected SEM to become more widely available. Basic secondary electron imaging and CD applications may be separately addressed by the He-ion microscope.     

Argon broad ion beam tomography in a cryogenic scanning electron microscope: a novel tool for the investigation of representative microstructures in sedimentary rocks containing pore fluid

The contribution describes the implementation of a broad ion beam (BIB) polisher into a scanning electron microscope (SEM) functioning at cryogenic temperature (cryo). The whole system (BIB‐cryo‐SEM) provides a first generation of a novel multibeam electron microscope that combines broad ion beam with cryogenic facilities in a conventional SEM to produce large, high‐quality cross‐sections (up to 2 mm²) at cryogenic temperature to be imaged at the state‐of‐the‐art SEM resolution. Cryogenic method allows detecting fluids in their natural environment and preserves samples against desiccation and dehydration, which may damage natural microstructures. The investigation of microstructures in the third dimension is enabled by serial cross‐sectioning, providing broad ion beam tomography with slices down to 350 nm thick. The functionalities of the BIB‐cryo‐SEM are demonstrated by the investigation of rock salts (synthetic coarse‐grained sodium chloride synthesized from halite‐brine mush cold pressed at 150 MPa and 4.5 GPa, and natural rock salt mylonite from a salt glacier at Qom Kuh, central Iran). In addition, results from BIB‐cryo‐SEM on a gas shale and Boom Clay are also presented to show that the instrument is suitable for a large range of sedimentary rocks. For the first time, pore and grain fabrics of preserved host and reservoir rocks can be investigated at nm‐scale range over a representative elementary area. In comparison with the complementary and overlapping performances of the BIB‐SEM method with focused ion beam‐SEM and X‐ray tomography methods, the BIB cross‐sectioning enables detailed insights about morphologies of pores at greater resolution than X‐ray tomography and allows the production of large representative surfaces suitable for FIB‐SEM investigations of a specific representative site within the BIB cross‐section.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

TEM and HREM study of Al3Zr precipitates in an Al-Mg-Si-Zr alloy

The structure of Al₃Zr precipitates in Al‐1.0Mg‐0.6Si‐0.5Zr (in wt.%) alloy was investigated using conventional transmission electron microscopy (TEM) and high‐resolution TEM (HREM). After annealing of the alloy in the temperature range 450–540 °C, spherical precipitates of metastable L1₂‐Al₃Zr phase appeared nearly homogeneously within the matrix, and elongated particles were found at grain boundaries. L1₂‐structured Al₃Zr were about 20–30 nm in diameter and coherent with the matrix. Inside some of them, planar faults parallel to {100} planes were revealed by use of HREM. Most probably, these faults are an indication of the transition stage of transformation to the stable D0₂₃‐type Al₃Zr phase. The elongated precipitates (about 100 nm) were identified as D0₂₂‐type Al₃Zr. Energy‐dispersive X‐ray analysis showed that they contain, apart from Al, mainly Zr with small amounts of Si. The substitution of Al by Si increased the stability of the D0₂₂‐Al₃Zr as compared with D0₂₃‐Al₃Zr.     

A Brief Discussion About Image Quality and SEM Methods for Quantitative Fractography of Polymer Composites

The methodology for fracture analysis of polymeric composites with scanning electron microscopes (SEM) is still under discussion. Many authors prefer to use sputter coating with a conductive material instead of applying low‐voltage (LV) or variable‐pressure (VP) methods, which preserves the original surfaces. The present work examines the effects of sputter coating with 25 nm of gold on the topography of carbon‐epoxy composites fracture surfaces, using an atomic force microscope. Also, the influence of SEM imaging parameters on fractal measurements is evaluated for the VP‐SEM and LV‐SEM methods. It was observed that topographic measurements were not significantly affected by the gold coating at tested scale. Moreover, changes on SEM setup leads to nonlinear outcome on texture parameters, such as fractal dimension and entropy values. For VP‐SEM or LV‐SEM, fractal dimension and entropy values did not present any evident relation with image quality parameters, but the resolution must be optimized with imaging setup, accompanied by charge neutralization. SCANNING 35: 196‐204, 2013. © 2012 Wiley Periodicals, Inc.     

Microstructural characterization of Ti‐6Al‐4V alloy subjected to the duplex SMAT/plasma nitriding

In this study, microstructural characterization of Ti‐6Al‐4V alloy, subjected to the duplex surface mechanical attrition treatment (SMAT)/nitriding treatment, leading to improve its mechanical properties, was carried out through novel and original samples preparation methods. Instead of acid etching which is limited for morphological characterization by scanning electron microscopy (SEM), an original ion polishing method was developed. Moreover, for structural characterization by transmission electron microscopy (TEM), an ion milling method based with the use of two ions guns was also carried out for cross‐section preparation. To demonstrate the efficiency of the two developed methods, morphological investigations were done by traditional SEM and field emission gun SEM. This was followed by structural investigations through selected area electron diffraction (SAED) coupled with TEM and X‐ray diffraction techniques. The results demonstrated that ionic polishing allowed to reveal a variation of the microstructure according to the surface treatment that could not be observed by acid etching preparation. TEM associated to SAED and X‐ray diffraction provided information regarding the nanostructure compositional changes induced by the duplex SMAT/nitriding process. Microsc. Res. Tech. 76:897–903, 2013. © 2013 Wiley Periodicals, Inc.     

MRT Letter: High resolution SEM imaging of nano‐architecture of cured urea‐formaldehyde resin using plasma coating of osmium

Nanoarchitecture of cured urea‐formaldehyde (UF) resins was examined with a field‐emission scanning electron microscope (FE‐SEM) after coating samples with osmium, which is considered to produce particles of considerably smaller size compared to other metal coatings used in SEM studies. This method enabled comparison of the nanoarchitecture of UF resins of low (1.0) and high (1.6) formaldehyde/urea (F/U) mole ratios to be made, based on imaging of extremely small size particles as part of UF resin architecture, not described before. Imaging revealed presence of relatively large globular particles (148.084–703.983 nm size range) as well as smaller substructures (28.004–39.604 nm size range) as part of the architecture of 1.0‐mole UF resin. Globular particles were also present in 1.6 mole UF resin, but of considerably smaller size (14.760–50.269 nm). The work presented demonstrates usefulness of osmium coating in unraveling the intricacies of the nanostructural organization of cured UF resins, prompting wider application of this immensely useful but grossly underutilized metal coating type in high resolution SEM examination of biological and materials samples. Microsc. Res. Tech. 76:1108–1111, 2013. © 2013 Wiley Periodicals, Inc.     

Defects in GaSe grown by Bridgman method

Optical quality GaSe crystals have been grown by vertical Bridgman method. The structural properties and micromorphology of a cleaved GaSe(001) surface have been evaluated by RHEED, SEM and AFM. The cleaved GaSe(001) is atomically flat with as low roughness as ～0.06 nm excepting local hillock type defects. The hillock‐type formations are round‐shaped with a bottom diameter of ～200 nm and a height of ～20–35 nm. The drastic depletion of the hillock material by gallium has been indicated by EDX measurements.     

New application of variable‐pressure/environmental microscopy to semiconductor inspection and metrology

Variable‐pressure/environmental scanning electron microscopy has been used for successful investigation binary and phase‐shifting chromium on quartz optical photomasks. This methodology was also applied to patterned 193 nm photoresist structures. The application of this methodology to semiconductor metrology is new because of the recent availability of variable‐pressure scanning electron microscopy (SEM) instrumentation equipped with high‐resolution, high‐signal, thermally assisted field emission technology in conjunction with large chamber and sample transfer capabilities. The variable‐pressure SEM methodology employs a gaseous environment around the sample to help diminish the charge build‐up that occurs under irradiation with the electron beam. Although very desirable for the charge reduction in many biological, pharmaceutical, and food applications, this methodology has not been employed for semiconductor photomask or wafer metrology until now. This is a new application of this technology to this area, and it shows great promise in inspection, imaging, and metrology in a charge‐free operational mode. For accurate metrology, variable‐pressure SEM methodology also affords a path that minimizes, if not eliminates, the need for charge modeling. This paper presents some of the early results in the variable‐pressure SEM metrology of photomask and photoresist structures.     

Achieving sub-nanometre particle mapping with energy-filtered TEM

A combination of state-of-the-art instrumentation and optimized data processing has enabled for the first time the chemical mapping of sub-nanometre particles using energy-filtered transmission electron microscopy (EFTEM). Multivariate statistical analysis (MSA) generated reconstructed datasets where the signal from particles smaller than 1 nm in diameter was successfully isolated from the original noisy background. The technique has been applied to the characterization of oxide dispersion strengthened (ODS) reduced activation FeCr alloys, due to their relevance as structural materials for future fusion reactors. Results revealed that most nanometer-sized particles had a core–shell structure, with an Yttrium–Chromium–Oxygen-rich core and a nano-scaled Chromium–Oxygen-rich shell. This segregation to the nanoparticles caused a decrease of the Chromium dissolved in the matrix, compromising the corrosion resistance of the alloy.     

Characterization of defects in semiconductors by combined application of SEM(EBIC) and SDLTS*

Besides the characterization of the geometrical structure of defects in semiconductors by TEM the estimation of their electrical activity is of importance. SEM(EBIC) and SDLTS (scanning deep level transient spectroscopy) are especially suitable for this purpose; they allow the inspection of electronic properties with a spatial resolution in the micron-range. On the one hand, SEM(EBIC) yields information on the recombination efficiency of defects in the crystal volume adjacent to a pn junction or a Schottky barrier; on the other hand, SDLTS enables the detection to be carried out of the distribution and the energetic levels of deep level defects lying in the space charge region. Accordingly, the combined application of these techniques is very promising for investigating physical processes implying an inhomogeneous incorporation of deep level defects in semiconductor crystals. In comparison to the widely used SEM(EBIC) technique SDLTS has only rarely been applied, a fact that is due to the high detection sensitivity necessary for measuring capacity transients. The application of a highly sensitive (10^—6 pF) micro-computer-controlled SDLTS system in combination with a conventional EBIC system allows a reliable inspection of semiconductor materials and devices, based on A₃B₅ compounds and on silicon. A typical application of the above technique is the investigation of the impurity distribution around extended crystal defects, like dislocations and precipitates, to study their gettering activity.     

Estimation of mass thickness response of embedded aggregated silica nanospheres from high angle annular dark‐field scanning transmission electron micrographs

In this study, we investigate the functional behaviour of the intensity in high‐angle annular dark field scanning transmission electron micrograph images. The model material is a silica particle (20 nm) gel at 5 wt%. By assuming that the intensity response is monotonically increasing with increasing mass thickness of silica, an estimate of the functional form is calculated using a maximum likelihood approach. We conclude that a linear functional form of the intensity provides a fair estimate but that a power function is significantly better for estimating the amount of silica in the z‐direction. The work adds to the development of quantifying material properties from electron micrographs, especially in the field of tomography methods and three‐dimensional quantitative structural characterization from a scanning transmission electron micrograph. It also provides means for direct three‐dimensional quantitative structural characterization from a scanning transmission electron micrograph.     

Improving the analysis of small precipitates in HSLA steels using a plasma cleaner and ELNES

The change from producing high strength low alloy (HSLA) steel sheet by conventional thick slab casting to producing it by direct charged thin slab casting causes a major change in the evolution of the precipitation. A key area of interest is the composition of the sub-10nm precipitates used to produce dispersion hardening. Carbon extraction replicas are frequently used to study precipitates in steels and other metals. When used with annular dark field imaging, this technique gives high contrast images of the precipitates while the thin carbon film adds little background or additional characteristic signals to either electron energy loss spectra or energy dispersive X-ray spectra. The method has the additional major advantage of removing the ferromagnetic matrix when studying HSLA steels. However, when the precipitates contain carbon, the C K-edge is dominated by the contribution from the amorphous carbon film. A plasma cleaner can be used to thin this carbon film to approximately 0.5 nm or less and then the contribution from the carbon in the precipitate can be separated from that in the carbon film using the electron energy loss near edge structure. A similar approach can be taken to separate the oxygen content of the precipitate from that of oxides formed from low-level impurities in the amorphous carbon during the plasma thinning process. In most cases, the precipitate studied here contained little or no oxygen even for the smallest sizes examined (approximately 4 nm). The precipitates contain mainly nitrogen with little carbon. For some compositions, the precipitates are clearly sub-stoichiometric.     

Analytical TEM study of annealed nanocrystalline cobalt–phosphorous electrodeposits

Investigation of thermal stability of two nanocrystalline Co–P alloys shows that P atoms segregate to the grain boundaries upon annealing until precipitation of Co₂P and CoP precipitates takes place. The P-rich precipitates formed have been investigated by analytical transmission electron microscopy to obtain statistical results of precipitate size, volume fraction and spatial distribution. Electron spectroscopic imaging maps show that the P-rich precipitates are 33 ± 9 nm in Co–1.1at.%P and 33 ± 12 nm in Co–3.2at.%P. The main differences between the alloys are the precipitate size distribution (Co–3.2at.%P having broader distribution) and precipitate volume number density (Co–3.2at.%P has 1.8 times more precipitates than Co–1.1at.%P). The volume fraction of precipitates is 3.0% in Co–1.1at.%P and 4.4% in Co–1.1at.%P. Most of the precipitates are of nearly spherical or slightly elongated shape, and only a few have a platelet-like shape as expected from previous tomographic atom probe measurements. Due to the truncation and projection effects, the composition of the precipitates could not be determined.     

Influence of structural parameters on magnetoresistive properties of CuFeNi melt spun ribbons

The microstructure of Cu₈₀Fe₁₀Ni₁₀ (at%) granular ribbon was investigated by means of atom probe tomography (APT). A granular system is composed of magnetic precipitates embedded in a non-magnetic matrix. In this ribbon, the magnetic precipitates have a diameter smaller than 5 nm in the as-spun state, and their crystallographic structure is very similar to the one of the matrix, which makes it difficult to characterize them using conventional techniques. Those data are of great importance to understand the magnetic and the transport behaviour of these ribbons. Using atom probe tomography, a 3D reconstruction of the microstructure of the as-spun and annealed ribbons was achieved and a precise characterization of the compositions of the two phases and of the composition profile at interfaces was carried out. In the as-spun state the composition of the matrix is Cu₈₉Fe₃Ni₈, the one of the precipitates is Cu₃₀Fe₄₀Ni₃₀. Upon annealing, the precipitates get enriched in iron. After annealing at 600 °C for 24 h, the measured compositions are close to the one predicted by Thermocalc, with Cu₉₄Fe₁Ni₅ for the matrix and Cu₅Fe₆₄Ni₃₁ for the precipitates.