Quantitative EFTEM mapping of near physiological calcium concentrations in biological specimens

Although electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) provides high sensitivity for measuring the important element, calcium, in biological specimens, the technique has been difficult to apply routinely, because of long acquisition times required. Here we describe a refinement of the complementary analytical technique of energy-filtered transmission electron microscopy (EFTEM), which enables rapid imaging of large cellular regions and measurement of calcium concentrations approaching physiological levels. Extraction of precise quantitative information is possible by averaging large numbers of pixels that are contained in organelles of interest. We employ a modified two-window approach in which the behavior of the background signal in the EELS spectrum can be modeled as a function of specimen thickness t expressed in terms of the inelastic mean free path λ. By acquiring pairs of images, one above and one below the Ca L_2,3 edge, together with zero-loss and unfiltered images, which are used to determine a relative thickness (t/λ) map, it is possible to correct the Ca L_2,3 signal for plural scattering. We have evaluated the detection limits of this technique by considering several sources of systematic errors and applied this method to determine mitochondrial total calcium concentrations in freeze-dried cryosections of rapidly frozen stimulated neurons. By analyzing 0.1 μm² areas of specimen regions that do not contain calcium, it was found that the standard deviation in the measurement of Ca concentrations was about 20 mmol/kg dry weight, corresponding to a Ca:C atomic fraction of approximately 2×10⁻⁴. Calcium concentrations in peripheral mitochondria of recently depolarized, and therefore stimulated and Ca loaded, frog sympathetic neurons were in reasonable agreement with previous data.     

Surface finish prediction models for precision grinding of silicon

Conventional grinding of silicon substrates results in poor surface quality unless they are machined in ductile mode on expensive ultra-precision machine tools. However, precision grinding can be used to generate massive ductile surfaces on silicon so that the polishing time can be reduced immensely and surface quality improved. However, precision grinding has to be planned with reliability in advance and the process has to be performed with high rates of reproducibility. Therefore, this work reports the empirical models developed for surface parameters R _a, R _max, and R _t with precision grinding parameters, depths of cut, feed rates, and spindle speeds using conventional numerical control machine tools with Box–Behnken design. Second-order models are developed for the surface parameters in relation to the grinding parameters. Analysis of variance is used to show the parameters as well as their interactions that influence the roughness models. The models are capable of navigating the design space. Also, the results show large amounts of ductile streaks at depth of cut of 20?μm, feed rate of 6.25?mm/min, and spindle speed of 70,000?rpm with a 43-nm R _a. Optimization experiments by desirability function generate 37-nm R _a, 400-nm R _max, and 880-nm R _t with massive ductile surfaces.     

An experimental method for calibration of the plasmon mean free path

Transmission electron microscopy specimens in the form of elongated, conical needles were made using a dual‐beam focused ion beam system, allowing the specimen thickness to be geometrically determined for a range of thickness values. From the same samples electron energy loss maps were acquired and the plasmon mean free path (λ) for inelastic scattering was determined experimentally from the measured values of specimen thickness. To test the method λ was determined for Ni (174 ± 17 nm), α‐Al₂O₃ (143 ± 14 nm), Si (199 ± 20 nm) and amorphous SiO₂ (238 ± 12 nm), and compared both to experimental values of λ taken from the literature and to calculated values. The calculated values of λ significantly underestimate the true sample thickness for high accelerating voltages (300 kV) and large collection angles. A linear dependence of λ on thickness was confirmed for t/λ < 0.5–0.6, but this method also provides an approach for calibrating λ at sample thicknesses for which multiple scattering occurs, thus expanding the thickness range over which electron energy loss spectroscopy can be used to determine the absolute sample thickness (t/λ > 0.6). The experimental method proposed in this contribution offers a means to calibrate λ for any type of material or phase that can be milled using a focused ion beam system.     

Bandgap measurement of thin dielectric films using monochromated STEM-EELS

High-resolution electron energy-loss spectroscopy (HR-EELS), achieved by attaching electron monochromators to transmission electron microscopes (TEM), has proved to be a powerful tool for measuring bandgaps. However, the method itself is still uncertain, due to Cerenkov loss and surface effects that can potentially influence the quality of EELS data. In the present study, we achieved an energy resolution of about 0.13 eV at 0.1 s, with a spatial resolution of a few nanometers, using a monochromated STEM-EELS technique. We also assessed various methods of bandgap measurement for a-SiNx and SiO₂ thin dielectric films. It was found that the linear fit method was more reliable than the onset reading method in avoiding the effects of Cerenkov loss and specimen thickness. The bandgap of the SiO₂ was estimated to be 8.95 eV, and those of a-SiNx with N/Si ratios of 1.46, 1.20 and 0.92 were measured as 5.3, 4.1 and 2.9 eV, respectively. These bandgap-measurement results using monochromated STEM-EELS were compared with those using Auger electron spectroscopy (AES)-reflective EELS (REELS).     

Surface roughness measurement in turning carbon steel AISI 1045 using wiper inserts

Surface roughness of the workpiece is an important parameter in machining technology. Wiper inserts have emerged as a significantly class of cutting tools, which are increasingly being utilized in last years. This study considers the influence of the wiper inserts when compared with conventional inserts on the surface roughness obtained in turning. Experimental studies were carried out for the carbon steel AISI 1045 because of its great application in manufacturing industry. Surface roughness is represented by different amplitude parameters (R_a, R_zD, R_3z, R_q, R_t, R_a/R_q, R_q/R_t, R_a/R_t). With wiper inserts and high feed rate it is possible to obtain machined surfaces with R_a < 0.8 μm (micron). Consequently it is possible to get surface quality in workpiece of mechanics precision without cylindrical grinding operations.     

Investigation for optimal parametric combination for achieving better surface finish during turning of Al/SiC-MMC

This paper presents an experimental investigation of the influence of cutting conditions on surface finish during turning of Al/SiC-MMC. In this study, the Taguchi method, a powerful tool for experiment design,is used to optimise cutting parameters for effective turning of Al/SiC-MMC using a fixed rhombic tooling system. An orthogonal L₂₇(3¹³) array is used for 3³ factorial design and analysis of variance (ANOVA) is employed to investigate the influence of cutting speed, feed and depth of cut on the surface roughness height R _a and R _t respectively. The influence of the interaction of cutting speed/feed on the surface roughness height R _a and R _t and the effect of cutting speed on cutting speed/feed two factor cell total interaction for surface roughness height R _a and R _t are analysed through various graphical representations. Taking significant cutting parameters into consideration and using multiple linear-regression, mathematical models relating to surface roughness height R _a and R _t are established to investigate the influence of cutting parameters during turning of Al/SiC-MMC. Confirmation test results established the fact that the mathematical models are appropriate for effectively representing machining performance criteria, e.g. surface roughness heights during turning of Al/SiC-MMC.     

Electron beam damage of behenic acid films

The rate of fading of electron diffraction patterns of behenic acid monolayer crystals as well as multilayer crystals was measured at 100 kV at room temperature to investigate the dependence of beam damage on specimen thickness. The diffracted intensities for monolayers and double layers decreased nearly exponentially with electron exposure; however, the intensities for multilayers were unchanged during initial electron exposures, often increased temporarily and then decreased with electron exposure. The critical dose, D_e, defined as the dose at which the diffracted intensity falls to 1/e of its initial value, was 1.0 electrons/Ų for the monolayers, 1.8 electrons/Ų for the double layers and more for multilayers. These results lead to the conclusion that D_e for behenic acid increases nearly linearly with specimen thickness in the range of about 25–100 Å for dose rate of 0.1–2 electrons/Ų min.     

An integrated optimization of cutting parameters and tool path generation in ultraprecision raster milling

This paper provides a new methodology for the integrated optimization of cutting parameters and tool path generation (TPG) based on the development of prediction models for surface roughness and machining time in ultraprecision raster milling (UPRM). The proposed methodology simultaneously optimizes the cutting feed rate, the path interval, and the entry distance in the feed direction to achieve the best surface quality in a given machining time. Cutting tests are designed to verify the integrated optimization methodology. The experimental results show that, in the fabrication of plane surface, the changing of entry distance improves surface finish about 40 nm (R _a ) and 200 nm (R _t ) in vertical cutting and decreases about 8 nm (R _a ) and 35 nm (R _t ) in horizontal cutting with less than 2 s spending extra machining time. The optimal shift ratio decreases surface roughness about 7 nm (R _a ) and 26 nm (R _t ) in the fabrication of cylinder surfaces, while the total machining time only increases 2.5 s. This infers that the integrated optimization methodology contributes to improve surface quality without decreasing the machining efficiency in ultraprecision milling process.     

EELS log-ratio technique for specimen-thickness measurement in the TEM

We discuss measurement of the local thickness t of a transmission microscope specimen from the log-ratio formula t = λ In (I_t/I₀) where I_t and I₀ are the total and zero-loss areas under the electron-energy loss spectrum. We have measured the total inelastic mean free path λ in 11 materials of varying atomic number Z and have parameterized the results in the form λ = 106F (E₀/E_m)/ln (2βE₀/E_m) where F = (1 + E₀/1,022)/(1 + E₀/511)², the incident energy E₀ is in keV, the spectrum collection semiangle β is in mrad, and E_m = 7.6Z^0.36. This formulation should allow absolute thickness to be determined to an accuracy of ±20% in most inorganic specimens.     

Simulation of surface generated during abrasive flow finishing of Al/SiCp-MMC using neural networks

Abrasive flow machining (AFM) is a multivariable finishing process which finds its use in difficult to finish surfaces on difficult to finish materials. Near accurate prediction of generated surface by this process could be very useful for the practicing engineers. Conventionally, regression models are used for such prediction. This paper presents the use of artificial neural networks (ANN) for modeling and simulation of response characteristics during AFM process in finishing of Al/SiC_p metal matrix composites (MMCs) components. A generalized back-propagation neural network with five inputs, four outputs, and one hidden layer is designed. Based upon the experimental data of the effects of AFM process parameters, e.g., abrasive mesh size, number of finishing cycles, extrusion pressure, percentage of abrasive concentration, and media viscosity grade, on performance characteristics, e.g., arithmetic mean value of surface roughness (R _a, micrometers), maximum peak–valley surface roughness height (R _t, micrometers), improvement in R _a (i.e., ΔR _a), and improvement in R _t (i.e., ΔR _t), the networks are trained for finishing of Al/SiC_p-MMC cylindrical components. ANN models are compared with multivariable regression analysis models, and their prediction accuracy is experimentally validated.     

Electron-beam-induced reduction of Fe in iron phosphate dihydrate,ferrihydrite, haemosiderin and ferritin as revealed by electron energy-loss spectroscopy

The effect of high-energy electron irradiation on ferritin/haemosiderin cores (in an iron-overloaded human liver biopsy), its mineral analogue; six-line ferrihydrite (6LFh), and iron phosphate dihydrate (which has similar octahedral ferric iron to oxygen coordination to that in ferrihydrite and ferritin/haemosiderin cores) has been investigated using electron energy-loss spectroscopy (EELS). Fe L_2,3-ionisation edges were recorded on two types of electron microscope: a 200 keV transmission electron microscope (TEM) and a 100 keV scanning transmission electron microscope (STEM), in order to investigate the damage mechanisms in operation and to establish a methodology for minimum specimen alteration during analytical electron microscopic characterisation. A specimen damage mechanism dominated by radiolysis that results in the preferential loss of iron co-ordinating ligands (O, OH and H₂O) is discussed. The net result of irradiation is structural re-organisation and reduction of iron within the iron hydroxides. At sufficiently low electron fluence and particularly in the lower incident energy, finer probe diameter STEM, the alteration is shown to be minimal. All the materials examined exhibit damage which as a function of cumulative fluence is best fitted by an inverse power-law, implying that several chemical and structural changes occur in response to the electron beam and we suggest that these are governed by secondary processes arising from the primary ionisation event. This work affirms that electron fluence and current density should be considered when measuring mixed valence ratios with EELS.     

Quantitative and qualitative characterization of aquatic iron oxyhydroxide particles by EF-TEM

Electron energy-loss spectroscopy (EELS) and elemental imaging under the energy-filtered transmission electron microscope are powerful tools for the characterization of iron-rich particles present in natural waters. Features present in EEL spectra (Fe-M_2,3 Fe-L_2,3 and O-K ionization edges) of goethite (α-FeOOH) have been studied with an energy filter operated at 80 keV to determine optimal quantification and elemental imaging of Fe-rich natural aquatic particles in the 30–200 nm range of thickness. For quantitative aims, the Fe-M_2,3 ionization edge cannot be used easily, but the Fe-L_2,3 edge provides more accurate results owing to a better background extrapolation. The partial cross-section of the Fe(III) M shell has been determined for iron oxide. The use of two-windows (jump-ratio) and three-windows (background stripping) imaging methods is discussed in relation to the specimen thickness.     

Machining with tool-chip contact on the tool secondary rake face—Part I: a new slip-line model

Given the growing number of applications of groove-type chip breaker tools in modern machining, it is becoming increasingly important to study the tool-chip contact on the tool secondary rake face. This type of tool-chip contact significantly changes not only the state of stresses in the plastic deformation region, but also changes the distribution of forces and temperatures over the tool rake face. A new slip-line model accounting for the tool-chip contact on the tool secondary rake face is proposed in this paper. The model also takes into account chip curl and incorporates seven slip-line models developed for machining during the last six decades as special cases. Dewhurst and Collins's matrix technique for numerically solving slip-line problems and Powell's algorithm of nonlinear optimization are employed in the mathematical formulation of the model. The inputs of the model include (a) the tool primary rake angle γ₁, (b) the tool secondary rake angle γ₂, (c) the tool land length h, (d) the undeformed chip thickness t₁, (e) the ratio of hydrostatic pressure P_A to the material shear flow stress k, (f) the ratio of frictional shear stress τ₁ on the tool primary rake face to the material shear flow stress k, and (g) the ratio of frictional shear stress τ₂ on the tool secondary rake face to the material shear flow stress k. The outputs of the model include (a) the cutting force F_c/kt₁w and the thrust force F_t/kt₁w, (b) the chip up-curl radius R_u, (c) the chip thickness t₂, and (d) the natural tool-chip contact length l_n.     

Growth behavior of short surface fatigue cracks in 2 1/4 Cr-1 Mo steel

Fatigue tests by axial loading (R-0.05) were carried out to investigate short fatigue crack growth behavior in 2 1/4 Cr-1 Mo steel at room temperature using smooth and a small notched flat specimen. All the data of the fatigue crack growth rate in the present tests were analyzed as a function of the stress intensity factor equation in conjunction with crack closure behavior. Analysis was performed accounting for the relation of surface effective stress range,Ua and depth effective stress range,Ub. In the case of isotropic crack growth properties,Ub=(ΔK_ta/ΔK_tb) ·Ua. By use ofUb obtained from the analysis, crack growth rates to surface direction coincide with those of depth direction.     

Electron energy-loss spectroscopy study of thin film hafnium aluminates for novel gate dielectrics

We have used conventional high‐resolution transmission electron microscopy and electron energy‐loss spectroscopy (EELS) in scanning transmission electron microscopy to investigate the microstructure and electronic structure of hafnia‐based thin films doped with small amounts (6.8 at.%) of Al grown on (001) Si. The as‐deposited film is amorphous with a very thin (～0.5 nm) interfacial SiO_x layer. The film partially crystallizes after annealing at 700 °C and the interfacial SiO₂‐like layer increases in thickness by oxygen diffusion through the Hf‐aluminate layer and oxidation of the silicon substrate. Oxygen K‐edge EELS fine‐structures are analysed for both films and interpreted in the context of the films’ microstructure. We also discuss valence electron energy‐loss spectra of these ultrathin films.     

A multivariate surface roughness modeling and optimization under conditions of uncertainty

Correlated responses can be written in terms of principal component scores, but the uncertainty in the original responses will be transferred and will influence the behavior of the regression function. This paper presents a model building strategy that consider the multivariate uncertainty as weighting matrix for the principal components. The main objective is to increase the value of R² predicted to improve model’s explanation and optimization results. A case study of AISI 52100 hardened steel turning with Wiper tools was performed in a Central Composite Design with three-factors (cutting speed, feed rate and depth of cut) for a set of five correlated metrics (R_a, R_y, R_z, R_q and R_t). Results indicate that different modeling methods conduct approximately to the same predicted responses, nevertheless the response surface to Weighted Principal Component – case b – (WPC1^b) presented the highest predictability.     

Effect of nozzle geometry on a standard cavitation erosion test using a cavitating jet

In order to accurately and reliably evaluate the cavitation erosion resistance of materials using cavitating jet apparatus according to ASTM G134, the effect of various types of nozzle geometries on the erosion rate was investigated. As the erosion rate depends on the erosion time and the distance from the nozzle to the specimen, i.e., the standoff distance, the mass loss as a function of erosion time at the optimum standoff distance was measured. It was shown that the erosion rate depended on the nozzle geometry. In fact, the aggressive intensity of the cavitating jet I_J depends on the nozzle geometry. When a cavitating jet of low I_J was used in the erosion test, it took some time to reach the maximum cumulative erosion rate E_Rmax, which is recommended in ASTM G134 as a parameter for determining the cavitation erosion resistance of materials. In the present experiment, the difference in E_Rmax was more than 600%, and the time required to reach E_Rmax was also scattered over 600%, for the different nozzles used. It was also revealed that E_Rmax could be obtained from the product of I_J and the reciprocal of the relative cavitation erosion resistance of the material, R_ER.     

Electron energy loss analysis of near-trace-element concentrations of calcium

The quantitation of near-trace-element concentrations of calcium (25 ppm atomic fraction) with electron energy loss spectroscopy (EELS) is demonstrated. The data collection, with an energy-stabilized parallel recording spectrometer, subsequent signal processing, and quantitation procedures are described. The quantitative results obtained with EELS, in the biologically relevant range of 1 to 100 mmol/kg, are directly compared with simultaneously collected and previously validated energy-dispersive X-ray spectroscopy (EPMA). The experimentally determined sensitivity of EELS for Ca detection is five-fold better than for EPMA, and the theoretically attainable sensitivity of EELS is ten-fold better than for EPMA. However, the attainment of this sensitivity with EELS is technically more difficult and limited by specimen thickness. The sensitivity of EELS experimentally demonstrated in this study permits the detection of three calcium atoms in a 10 nm diameter spot of an organic matrix, with a field-emission-gun-equipped scanning transmission electron microscope.     

Grinding-aided electrochemical discharge machining of particulate reinforced metal matrix composites

A grinding-aided electrochemical discharge machining (G-ECDM) process has been developed to improve the performance of the conventional ECDM process in machining particulate reinforced metal matrix composites (MMCs). The G-ECDM process functions under a combined action of electrochemical dissolution, spark erosion, and direct mechanical grinding. The tool electrode has a coating containing a hard reinforcement phase of diamond particles. The MMC employed in this study was Al₂O₃ particulate reinforced aluminum 6061 alloy. The material removal mechanism of this hybrid process has been analyzed. The results showed that the grinding action can effectively remove re-cast material deposited on the machining surface. The surface roughness (R _a) measured for the G-ECDM specimen was ten times smaller than that of the specimen machined without grinding aid (i.e., ECDM alone). Moreover, the material removal rate (MRR) of G-ECDM was about three times higher than that of ECDM under the experimental conditions of this study. The voltage waveform and crater distribution were also analyzed, and the experimental results showed that the G-ECDM process operates in a stable condition. The relative importance of the various processing parameters on MRR was established using orthogonal analysis. The results showed that MRR is influenced by the machining parameters in the order of duty cycle?>?current?>?electrolyte concentration. This study showed that the G-ECDM process is superior to the ECDM process for machining particulate reinforced MMCs, where a higher machining efficiency and a better surface quality can be obtained.     

Aspects of scanning microdensitometry: II. Spot size,focus and resolution

In scanning microdensitometry increasing the size of the measuring spot, or throwing the specimen out of focus, decreases the apparent integrated absorbance A_pE_p of a discrete specimen. Both experimental observations and elementary geometrical theory (i.e. ignoring diffraction effects) show that with moderate spot sizes the relative error in A_pE_p is greater with small objects or objects of high absorbance, and that with a given object the absolute error is approximately proportional to the spot diameter (round spot) or spot width (square spot). From the observed apparent integrated absorbances AE₁ and AE₂ obtained using measuring spots of width s₁ and s₂ respectively, the true integrated absorbance AE₀ corresponding to zero spot size can therefore be calculated from the approximate expression which reduces to With very large spot sizes, or with the specimen grossly out of focus, the apparent integrated absorbance of a specimen of radius R and transmittance I_t tends theoretically to a limit: A_pE_p = 0.4343πR² (I-I_t). Provided the true absorbance of the specimen does not exceed about 0.5 at any point, the true integrated absorbance could in principle be estimated with less than 3% error from the expression but in practice A_pE_p can probably not be measured with sufficient accuracy. In scanning measurements of apparent specimen area, the results depend both on the threshold absorbance used and the size of the flying spot. With low thresholds the apparent area of the specimen at first increases and later decreases, as the spot size is increased or as the focus is changed.