Determination of UTW kXSi factors for seven elements from oxygen to iron

This study reports k factors for an ultra-thin window (UTW) Si(Li) energy dispersive (ED) X-ray detector which extends the limit for X-ray microanalysis in the analytical electron microscope (AEM) to elements below Na in the periodic table. The oxygen k factors reported for the UTW ED detector suggest that quantitative thin specimen X-ray analysis can be extended to oxygen.     

Beam induced mass loss in high resolution biological microanalysis

Electron beam induced loss of mass from the organic matrix and from higher Z constituents of biological samples was measured by monitoring bremsstrahlung and peak changes in EDS spectra. When any effects of contamination, extraneous X-rays, beam current drift, specimen drift, and specimen shrinkage were monitored and corrected for, the three types of samples gave consistent and similar results at 296 K. Bremsstrahlung losses averaged 45%, 46% and 50% respectively for muscle homogenate, salivary gland sections and albumin. Sulphur losses average 74%, 72% and 86% for the same three sample types. No other elements suffered significant losses. D_l/e for bremsstrahlung averaged 0·14 C/cm². Bremsstrahlung loss at 93 K began approximately one order of magnitude higher in dose, and the extent of loss varied. Sulphur losses, however, were greatly reduced at low temperatures.     

Solute-enriched surface layers and X-ray microanalysis of thin foils of a commercial aluminium alloy

Thin foils of the commercial Al alloy 7075 were subjected to X-ray microanalysis in a Jeol 100 CX TEMSCAN using a modified double-tilting stage. The ratio of the Cu K_x and Al K_x peaks, I_cu/I_Al, was found to decrease rapidly with increasing foil thickness, t, in regions thinner than about 200 nm. This was attributed to the formation of surface layers enriched in Cu during electropolishing, consistent with the findings of other investigators. An equation predicting the variation of I_B/I_A with t in an A-rich alloy in which B (or additional solute elements) is dilute is derived for a sample with surface layers enriched in B. The derivation predicts that I_B/I_A should vary linearly with t^?1, and that the intercept of such a plot should be positive. It is suggested that consistency with this prediction serves as a criterion for true surface enrichment. Our data on Cu obey these predictions, and further analysis indicates that the layers on both surfaces are enriched by a factor of about 5.6. The ratio of the intensities of the Zn K_x and Al K_x X-ray peaks was found to be nearly independent of foil thickness to t?400 nm. This result, combined with additional analysis, indicates that the apparent enrichment observed by other investigators is most likely due to the influence of instrumental factors.     

An extrapolation method for the determination of Cliff-Lorimer kAB factors at zero foil thickness

An extrapolation method is proposed to be useful for the determination of the Cliff-Lorimer k_AB factor at zero foil thickness. The method consists of measuring k_AB factors as a function of the measured foil thickness, t_M, and extrapolating the relationship toward t_M=0. The intersection between the extrapolated line and the ordinate of t_M=0 gives (k_AB)₀ which is free from the effect of absorption. The straight line extrapolation that can be achieved by a linear-least squares method is particularly developed to eliminate arbitrariness introduced in the extrapolation process. The extrapolation method is applied to data available in the literature. It is shown that the method yields the (k_AB)₀ factors compatible with those predicted from the theoretical calculation. It is also shown that this method can circumvent several problems which make it complex and difficult to determine accurate values of the absorption-free k_AB factors. Using the straight line extrapolatioin, it is possible to estimate the degree of the thickness overestimation which arises when the foil thickness is measured by the contamination spot separation (CSS) method. Validity of the straight line extrapolation is further discussed.     

A study of factors influencing the accuracy of quantitative X-ray analysis of thin foil specimens

Needle-shaped specimens were used to determine experimental values of X-ray intensity ratios for L/K and M/L lines. These ratios can be used to determine k_AB factors for L and M lines. They also provide necessary data for absorption correction with the X-ray intensity ratio method. Recent theoretical calculations of k_AB factors are evaluated and new constants in the parametrization of ionization cross-sections for L and M lines are presented. The average X-ray production per unit thickness has been measured as a function of mass thickness for a stainless steel specimen using acceleration voltages of 100 and 200 kV. An increase in X-ray production by more than 20% was found at 100 kV for mass thicknesses up to 1 mg/cm² while at 200 kV the increase was about 5%. The contamination spot method for thickness measurements has also been investigated using needle-shaped specimens. The reported overestimate of thickness using this method can be explained by the formation of broad contamination layers around the deposited spots.     

Quantitative analysis of backscattered-electron contrast in scanning electron microscopy

Backscattered-electron scanning electron microscopy (BSE-SEM) imaging is a valuable technique for materials characterisation because it provides information about the homogeneity of the material in the analysed specimen and is therefore an important technique in modern electron microscopy. However, the information contained in BSE-SEM images is up to now rarely quantitatively evaluated. The main challenge of quantitative BSE-SEM imaging is to relate the measured BSE intensity to the backscattering coefficient η and the (average) atomic number Z to derive chemical information from the BSE-SEM image. We propose a quantitative BSE-SEM method, which is based on the comparison of Monte–Carlo (MC) simulated and measured BSE intensities acquired from wedge-shaped electron-transparent specimens with known thickness profile. The new method also includes measures to improve and validate the agreement of the MC simulations with experimental data. Two different challenging samples (ZnS/Zn(O_xS_1–x)/ZnO/Si-multilayer and PTB7/PC₇₁BM-multilayer systems) are quantitatively analysed, which demonstrates the validity of the proposed method and emphasises the importance of realistic MC simulations for quantitative BSE-SEM analysis. Moreover, MC simulations can be used to optimise the imaging parameters (electron energy, detection-angle range) in advance to avoid tedious experimental trial and error optimisation. Under optimised imaging conditions pre-determined by MC simulations, the BSE-SEM technique is capable of distinguishing materials with small composition differences.     

Entwicklung einer verbesserten sondenstromstabilisierung für rasterelektronenmikroskope development of an improved probe current stabilizing unit for SEMs

A probe current stabilizing unit is important in quantitative XRMA with long counting times. The uncorrected long-term drift in a Cambridge Mk II A SEM is of the order of 40% during the first 3 h after switch-on and is caused by

• a) an increase in the emission current by more than 20%, probably due to thermal expansion of the long metal tube containing the filament mounting which reduces the distance to the Wehnelt electrode, and
• b) a decrease in probe current due to a horizontal shift of the filament tip and a misalignment of the electron beam.

Summarizing the literature the following stabilizing methods can be determined:

• 1. A motor-driven readjustment of the filament (Balk et al 1975),
• 2. control of the Wehnelt bias (Cleaver and Smith 1972, 1973),
• 3. regulation of the filament current (Chapman 1972, Close and Yarwood 1972, Wellenstein and Ensman 1973, Reed 1968, Nakagawa and Yanaka 1975, Baines et al 1975).

Our first method of stabilizing the total emission current (Arndt et al 1978) is working well, but does not solve the problem of constant probe current. In the present experiments, we insulated the three spray apertures (Fig. 1) and recorded the emission current and the specimen current during the first 3 h (Fig. 2). The increasing current at aperture 1 confirms the effect (a) mentioned above. The approximately equal decrease in the current at aperture 3 and at the specimen, initiated experiments aimed at stabilizing the probe current using the current from spray aperture 3. However, this was not an appropriate method because overcompensation effects were observed. Therefore, we finally insulated the whole final aperture holder containing the three limiting apertures below the final lens. The voltage drop on a resistor of a few MΩ is amplified by an FET, and the difference voltage U compared to the compensation voltage is used to make a feed-back loop to the power supply unit of condenser lens 1. Fig. 3 shows the dependence of the probe current on the current I_C1 of this lens. The normal operating point is at I_C1 = 0.55 A. We limit the current of this lens to between values of I_C1 = 0.4–0.7A to avoid the cut-off region below 0.4A. The compensation voltage can be changed by a potentiometer “zero” (Fig. 12) to calibrate I_C1 to 0.55A with a current meter on the panel, because the electron emission of the filament does not only change with time but also from filament to filament. Fig. 4 shows the variations in the feedback voltage and I_C1 with a low and high sensitivity of the feed-back loop. Fig. 5 shows the dependence of the probe current on I_C2- Increasing values of I_C2 result in an equivalent decrease in I_C1. In order to change the probe current using condenser lens 2 a compensation voltage is generated by a logarithmic amplification of a voltage drop at a resistor in series with the lens current meter. Fig. 6 shows the difference between the actual voltage obtained by this method and the voltage required for ideal compensation. This difference causes a small shift of I_C1 if I_C2 is changed (Fig. 7). The time constant was of the order of 4s to avoid oscillations, because the magnetic lenses are slow to react to fast current changes. The result obtained with the stabilizing unit is presented in Fig. 8. The preexisting decrease of 47% in 3 h was reduced to approximately 1.6% (for comparison see Fig. 2). Figs. 9 and 10 demonstrate how the stabilizing unit regulates the current using high and low sensitivity when the filament current is decreased from 3.0 to 2.1 A. The decrease in emission current is actually compensated for over a broad range. The overall efficiency of the stabilizing unit can be seen from Fig. 11. No defocusing could be seen at low and intermediate magnifications but a small shift of 1 μm was observed at M = 20000 × when the full range of stabilizing was used. It normally takes several hours to reach the limits of the stabilizing range and this unwanted shift can therefore be ignored.     

Some stereological and statistical consequences derived from Cartier's formula

Let X and Y be two random variables with distribution functions F_x and F_Y, respectively. If Cartier's formula E[Y|X]=X holds, then F_x is necessarily less dispersed than F_Y. In this paper, the main consequences of Cartier's formula are derived. Some examples where such a formula holds are also given, namely in stereology (comparison of various estimators of Minkowsky's functionals) and in geostatistics (the change of support problem).     

Theory of confocal fluorescence imaging in the programmable array microscope (PAM)

The programmable array microscope (PAM) uses a spatial light modulator (SLM) to generate an arbitrary pattern of conjugate illumination and detection elements. The SLM dissects the fluorescent light imaged by the objective into a focal conjugate image, I_c, formed by the ‘in-focus’ light, and a nonconjugate image, I_nc, formed by the ‘out-of-focus’ light. We discuss two different schemes for confocal imaging using the PAM. In the first, a grid of points is shifted to scan the complete image. The second, faster approach, uses a short tiled pseudorandom sequence of two-dimensional patterns. In the first case, I_c is analogous to a confocal image and I_nc to a conventional image minus I_c. In the second case I_c and I_nc are the sum and the difference, respectively, of a conventional and a confocal image. The pseudorandom sequence approach requires post-processing to retrieve the confocal part, but generates significantly higher signal levels for an equivalent integration time.     

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.     

Detection limits of impurities in a light filler in an X-ray fluorescent arrangement with a secondary target

The application of the arrangement with a secondary target for the X-ray fluorescent analysis of microimpurities in water solutions without sample destructions and preliminary sample preparations is considered. The limiting contrast values of fluorescence spectra are estimated, the limits of detecting impurities by the arrangement are theoretically calculated and experimentally measured (C _min is smaller than 0.5 ppm). The range of chemical elements, which are efficiently excited by the secondary target, is determined by the C _min criterion.     

Surface microanalysis by reflection electron energy-loss spectroscopy

Several basic physical concepts of applying eq. I_k = IσN^xt to surface microanalysis by reflection electron energy-loss spectroscopy (REELS) are clarified. Here I^k and I are the integrated intensities of the core ionization edge and the low loss part, σ is the scattering cross section of element x with atomic concentration N^x, and t is the specimen thickness. The reflected inelastic electrons are found to be distributed almost symmetrically around the Bragg sports and can be reasonably described by a Lorentzian function. EELS microanalysis can be performed by using the diffracted sports. The ω correction, arising from the angular contributions of the neighbouring spots into the spectrometer collecting aperture, is required to be considered.     

A study of chlorophyll-like and phycobilin pigments in the C endosymbiont of the apple- snail <i>Pomacea canaliculata</i>

Pigments present in the brown-greenish C morph of an intracellular endosymbiont of Pomacea canaliculata were investigated. Acetone extracts of the endosymbiotic corpuscles showed an absorption spectrum similar to that of chlorophylls. Three fractions obtained from silica gel column chromatography of the acetone extracts (C_I , C_II and C_III ), were studied by positive ion fast atom bombardment-mass spectrometry (FAB–MS) and hydrogen-nuclear magnetic resonance (H-NMR). Results indicated the presence of (1) a sterol in the yellow colored C_I fraction; (2) a mixture of pheophorbides a and b in the major green fraction, C_II; and (3) a modified pheophorbide a in the smaller green fraction, C_III . Aqueous extracts of the C endosymbiont did not show evidence of the occurrence of C-phycocyanin, allophycocyanin or phycoerithrin (light absorption, fluorescence emission, and electrophoresis of the protein moieties) while cyanobacterial cells (Nostoc sp.) showed evidence of C-phycocyanin and allophycocyanin. The possible phylogenetic and functional significance of the pigments present in the C endosymbiont is discussed.     

Recent developments and new strategies in scanning electron microscopy*

In addition to improvements in lateral resolution in scanning electron microscopy, recent developments of interest here concern extension of the incident beam energy, E₀, over two decades, from ≈ 20 keV to ≈ 0.1–0.5 keV and the possibility of changing the take-off emission, α, of detected secondary electrons. These two degrees of freedom for image acquisition permit a series of images of the same field of view of a specimen to be obtained, each image of the series differing from the others in some aspect. The origins of these differences are explored in detail and they are tentatively interpreted in terms of the change in the secondary electron emission yield δ vs. E₀, δ = f(E₀), and also of the change in δ vs. α, ∂δ/∂α. Various origins for the chemical contrast and topographic contrast have been identified. Illustrated by correlating a secondary electron image and a backscattered electron image, use of the scatter diagram technique facilitates image comparison. The difference between the lateral resolution and the size of the minimum detectable detail is outlined to avoid possible errors in nanometrology. Some aspects related to charging are also considered and possible causes of contrast reversal are suggested. Finally, the suggested strategy consists of the acquisition of various images of a given specimen by changing one parameter: primary beam energy and take-off angle for conductive specimens; working distance or beam intensity for high-resolution experiments; scanning frequency for insulating specimens.     

A spectroscopic study of some high-temperature superconductors using a low-temperature STM

We used a scanning tunnelling microscope (STM) to measure both the tunnel current, I, and the dynamic conductance, dI/dV, at 4·2 K for a number of high-transition temperature oxide superconductors. Large spatial variations in the tunnelling characteristics are observed. At low tunnel resistances, all samples show evidence of single electron tunnelling and incremental charging. Results on BiSrCaCu₂O_x show the coexistence of charging with Josephson coupling between grains within the sample. Results on both the Bi sample and a single crystal of YBa₂Cu₃O_6·5+x reveal possible energy gap (2A) values of 17 and 20 meV, respectively. A very sharp 5 meV gap, observed in ceramic samples of YBa₂Cu₃O_6·5+x and Y_0·5Al_0·05Ba₂Cu₃O_6·5+x, may indicate the presence of a lower temperature phase in these samples.     

Quantitative FRET measurement using emission‐spectral unmixing with independent excitation crosstalk correction

Quantification of fluorescence resonance energy transfer (FRET) needs at least two external samples, an acceptor‐only reference and a linked FRET reference, to calibrate fluorescence signal. Furthermore, all measurements for references and FRET samples must be performed under the same instrumental conditions. Based on a novel notion to predetermine the molar extinction coefficient ratio (R_C) of acceptor‐to‐donor for the correction of acceptor excitation crosstalk, we present here a robust and independent emission‐spectral unmixing FRET methodology, Iem‐spFRET, which can simultaneously measure the E and R_C of FRET sample without any external references, such that Iem‐spFRET circumvents the rigorous restriction of keeping the same imaging conditions for all FRET experiments and thus can be used for the direct measurement of FRET sample. We validate Iem‐spFRET by measuring the absolute E and R_C values of standard constructs with different acceptor‐to‐donor stoichiometry expressed in living cells. Our results demonstrate that Iem‐spFRET is a simple and powerful tool for real‐time monitoring the dynamic intermolecular interaction within single living cells.     

Elasticity solution for an axially compressed and encased cylindrical specimen

We present the solution of the linear elasticity equations governing the deformation of an elastic cylinder encased in a tube and subjected to uniform compression on the flat ends. The solutions for the stresses, strains, and displacements in the encased and compressed cylinder are all systematically determined from the basic solution of Lamé's classical elasticity problem of the long tube subjected to internal and external pressures. We first derive the general elastostatic analysis for an encased hollow cylinder, stress-free at the cavity, and later particularize the solution to a solid cylindrical specimen. The effective modulus E_eff of the encased sample is found to be a function of the bulk modulus k and Poisson's ratio ν of the material. E_eff differs from k except for nearly incompressible materials, where E_eff approaches the bulk modulus value. In the incompressible case, we also show how a load applied on the cylinder's flat ends is equivalent to, and can be replaced by, the same load acting on the curved surface. For compressible materials, a more general expression for E_eff is found that also accounts for the case deformation. These results explain the deformation of an axially compressed and encased cylindrical specimen tested in compressibility measuring devices such as those described by Matsuoka and Maxwell [Response of linear high polymers to hydrostatic pressure. Journal of Polymer Science 1958; 32:131–59]. The present analysis thus contributes to a better understanding of how this device works and to the interpretation of measurements taken with it.     

Excitation under the scanning electron microscope of DNA-associated fluorescence from chicken erythrocyte nuclei,polytene chromosomes and adenovirus 2 virions

Biological structures not seen by conventional light microscopy, such as longitudinal striations in polytene chromosomes, and, at the limit of sensitivity, virions of adenovirus 2, have been detected via DNA-associated fluorescence excited under the scanning electron microscope. The maximum sensitivity realized, about 1 detected photon per 700 base pairs, falls short by about an order of magnitude of that required to achieve, in unreplicated specimens, the 2 nm intrinsic resolution of the method. A combination of D₂O-H₂O substitution with freeze-drying provides the best unquenching procedure found for in situ DNA. DNA-associated fluorescence for light microscopy can be created by moderate exposure of the specimen in the electron microscope.     

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.     

An intelligent sensor system approach for reliable tool flank wear recognition

An intelligent sensor system approach for reliable flank wear monitoring in turning is described. Based on acoustic emission and force sensing, an intelligent sensor system integrates multiple sensing, advanced feature extraction and information fusion methodology. Spectral, statistical and dynamic analysis have been used to determine primary features from the sensor signals. A secondary feature refinement is further applied to the primary features in order to obtain a more correlated feature vector for the tool flank wear process. An unsupervised ART2 neural network is used for the fusion of AE and force information and decision-making of the tool flank wear state. The experimental results confirm that the developed intelligent sensor system can be reliably used to recognise the tool flank wear state over a range of cutting conditions.Notation mean - ² variance - k _n end condition factor of the cantilever beam - E Young's modulus of tool holder - I moment of inertia of tool holder at cross section - m mass of tool holder per unit length - L length of tool overhang - l the size of the moving window - fm, pm, sm, km the mean values of the four primary features (the tangential force component, the frequency band power, the skew, and the kurtosis) - fs, ps, ss, ks the standard deviation values of the four primary features - F= resultant feature vector ART2 neural network parameters I _i element of input vector - Y _i output node - W _i ,X _i ,U _i ,V _i ,P _i ,Q _i parameters inF ₁ layer - R _i orienting parameter - vigilance parameter - b _ij ,t _ji bottom-to-top and top-to-bottom weights - a, b, c network parameters - f() thresholding function