Mass thickness determination by electron energy loss for quantitative X-ray microanalysis in biology

As is well known, electron energy loss spectroscopy can be used to determine the relative sample thickness in the electron microscope. This paper considers how such measurements can be applied to biological samples in order to obtain the mass thickness for quantitative X-ray microanalysis. The important quantity in estimating the mass thickness from an unknown sample is the total inelastic cross section per unit mass. Models for the cross section suggest that this quantity is constant to within ±20% for most biological compounds. This is comparable with the approximation made in the continuum method for measuring mass thickness. The linearity of the energy loss technique is established by some measurements on evaporated films and quantitation is demonstrated by measurements on thin calcium standards. A significant advantage of the method is that the energy loss spectrum can be recorded at very low dose, so that mass thickness determination can be made before even the most sensitive samples suffer damage resulting in mass loss. The energy loss measurements avoid the necessity to correct the continuum measurement for stray radiation produced in the vicinity of the sample holder. Unlike the continuum method the energy loss technique requires uniform mass thickness across the probe area, but this is not usually a problem when small probes (<100 nm diameter) are used.     

Glassy cholesteric structure: thickness variation induced by electron radiation in transmission electron microscopy investigated by atomic force microscopy.

During the observation of glassy cholesteric liquid crystals in transmission electron microscopy (TEM), a new contrast is created or enhanced by electron radiation which has a direct relationship with the periodic microstructure of the specimen. In this paper, we investigate the variations of the sample thickness and mass density as possible causes of this irradiation contrast. By means of observations in atomic force microscopy (AFM) coupled to TEM, we compared the surface corrugations of non-irradiated and irradiated specimens. It is shown that the final contrast is the result of several processes. including fracture during ultramicrotomy and mass loss during irradiation. Mass loss acts as an etching, and hence results in a decrease of the sample thickness. The etching depends on the initial molecular orientation, thus evidencing the latent structure. An electron channelling mechanism is suggested to explain this behaviour.     

Dose-rate dependence of electron-induced mass loss from organic specimens

Electron energy-loss measurements on thin films of collodion at low temperatures (90 K) show that the characteristic electron dose D_e for mass loss first increases and then decreases with increasing dose rate (current density). This behaviour is explained in terms of the limited diffusion rates at low specimen temperature and the heating effect of the electron beam, and can be approximately modelled using a simple computer program. For a small-diameter electron probe, the increase in D_e can be several orders of magnitude, suggesting a substantial advantage of STEM (in comparison to fixed-beam TEM) for examining beam-sensitive specimens.     

Specimen thickness dependence of hydrogen evolution during cryo‐transmission electron microscopy of hydrated soft materials

The evolution of hydrogen from many hydrated cryo‐preserved soft materials under electron irradiation in the transmission electron microscope can be observed at doses of the order of 1000 e nm^?2 and above. Such hydrogen causes artefacts in conventional transmission electron microscope or scanning transmission electron microscopy (STEM) imaging as well as in analyses by electron energy‐loss spectroscopy. Here we show that the evolution of hydrogen depends on specimen thickness. Using wedge‐shaped specimens of frozen‐hydrated Nafion, a perfluorinated ionomer, saturated with the organic solvent DMMP together with both thin and thick sections of frozen‐hydrated porcine skin, we show that there is a thickness below which hydrogen evolution is not detected either by bubble observation in transmission electron microscope image mode or by spectroscopic analysis in STEM electron energy‐loss spectroscopy mode. We suggest that this effect is due to the diffusion of hydrogen, whose diffusivity remains significant even at liquid nitrogen temperature over the length scales and time scales relevant to transmission electron microscopy analysis of thin specimens. In short, we speculate that sufficient hydrogen can diffuse to the specimen surface in thin sections so that concentrations are too low for bubbling or for spectroscopic detection. Significantly, this finding indicates that higher electron doses can be used during the imaging of radiation‐sensitive hydrated soft materials and, consequently, higher spatial resolution can be achieved, if sufficiently thin specimens are used in order to avoid the evolution of hydrogen‐based artefacts.     

Radiation damage in dry and frozen hydrated organic material

Electron-induced radiation damage can cause errors in interpreting electron micrographs. Radiation damage is distinguished from contamination (polymerization of hydrocarbons) and etching (radiolysis in the presence of water), both of which can be controlled by a proper specimen environment in the microscope. While temperature has little effect on the primary interactions of fast electrons with matter, most secondary radiation-damage processes are temperature dependent. Because damage mechanisms differ so greatly among materials, there is no simple factor by which specimen stability is improved as a function of temperature (some cases improve fivefold, others improve 100-fold). While some specimens are stable to almost arbitrarily high doses, some tests reveal damage at 1 e/nm². This paper surveys damage rates and temperature dependencies of various materials as a guide for future electron microscopic studies of organic specimens.     

Quantitative X-ray microanalysis of biological specimens

Qualitative X-ray microanalysis of biological specimens requires an approach that is somewhat different from that used in the materials sciences. The first step is deconvolution and background subtraction on the obtained spectrum. The further treatment depends on the type of specimen: thin, thick, or semithick. For thin sections, the continuum method of quantitation is most often used, but it should be combined with an accurate correction for extraneous background. However, alternative methods to determine local mass should also be considered. In the analysis of biological bulk specimens, the ZAF-correction method appears to be less useful, primarily because of the uneven surface of biological specimens. The peak-to-local background model may be a more adequate method for thick specimens that are not mounted on a thick substrate. Quantitative X-ray microanalysis of biological specimens generally requires the use of standards that preferably should resemble the specimen in chemical and physical properties. Special problems in biological microanalysis include low count rates, specimen instability and mass loss, extraneous contributions to the spectrum, and preparative artifacts affecting quantitation. A relatively recent development in X-ray microanalysis of biological specimens is the quantitative determination of local water content.     

Chemical etching in processing cortical bone specimens for scanning electron microscopy

Transverse and longitudinal sectioning of undecalcified cortical bone is a commonly employed technique for investigating the lamellar structure of the osteons. Since a flat surface is required, the specimen has to be grinded and then polished. Whereas the smear of debris and inorganic/organic deposits left by these treatments cannot be removed by ultrasonication alone, a chemical treatment of the specimen surface with either a basic or an acid etching solution is currently employed. A further effect of the latter can be the enhancement of the lamellar bone pattern. The kind of etching solution, its pH, the concentration of etchants, and the contact time significantly affect the sectioned surface when it is observed with scanning electron microscopy (SEM). The etching procedures can severely influence the obtained images. Homogeneous cortical bone specimens were sampled from the first metatarsal of two fresh human subjects. One or two cut surfaces were exposed to different acid and basic solutions in bonded conditions. Considering the type of chemical agents, the solution pH, and the exposure time of the specimens, the effects of several etching media have been investigated and compared. Strong etching, either acid or basic produced surface decalcification and severe damage of the collagen matrix, compromising any morphological or morphometric analysis. Weak acid etching (for example citric and acetic acid), even though causing distinctive alteration of the sample, enhanced the visibility of the lamellar pattern, while the polyphosphate treatment of the surface decalcified a thin layer matrix, ensuring a good visibility of fibrils and avoiding rough distortions. Microsc. Res. Tech. 77:653–660, 2014. © 2014 Wiley Periodicals, Inc.     

Interfacial energies and surface-tension forces involved in the preparation of thin,flat crystals of biological macromolecules for high-resolution electron microscopy

It is generally agreed that surface-tension forces and the direct interaction between the specimen and either the air-water interface or the water-substrate interface can influence significantly the preparation of biological materials for electron microscopy. Even so, there is relatively little systematic information available that would make it possible to control surface-tension forces and interfacial energies in a quantitative fashion. The main objective in undertaking the present work has been to understand somewhat better the factors that influence the degree of specimen flatness of large, monolayer crystals of biological macromolecules. However, the data obtained in our work should be useful in understanding the preparation of specimens of biological macromolecules in general. Data collection by electron diffraction and electron microscopy at high resolution and high tilt angles requires thin crystals of biological macromolecules that are flat to at least 1°, and perhaps less than 0·2°, over areas as large as 1 μm² or more. In addition to determining empirically by electron diffraction experiments whether sufficiently flat specimens can be prepared on various types of modified or unmodified carbon support films, we have begun to use other techniques to characterize both the surfaces involved and the interaction of our specimen with these surfaces. In the specific case of large, monolayer crystals of bacteriorhodopsin prepared as glucose-embedded specimens on hydrophobic carbon films, it was concluded that the initial interfacial interaction involves adsorption of the specimen to the air-water interface rather than adsorption of the specimen to the substrate. Surface-tension forces at the air-water interface and an apparently repulsive interaction between the specimen and the hydrophobic carbon seem to be major factors influencing the specimen flatness in this case. In the more general case it seems likely that interfacial interactions with either the substrate or the air-water interface can be variously manipulated in the search to find desirable conditions of specimen preparation.     

Freeze-fracturing: a review of methods and results

Freeze-fracturing may be accomplished either under vacuum, or at atmospheric pressure. The devices available for freeze-cleaving are discussed in relation to the vacuum and temperature conditions prevailing during cleaving, etching and replication. It is concluded that most specimens can be satisfactorily cleaved (even at 4 K), and processed using simple cleavage devices and systems that provide ample cold-trapping protection for the specimen. Only for special purposes are microtome assemblies or ultra-high vacuum units essential. The fracturing process may produce artefacts by plastic deformation. Such artefacts have been noted in both non-biological and biological polymers cleaved at temperatures as low as 4 K. Contamination of the frozen surface need not be a problem provided that the specimen is transferred under ‘safe’ conditions, and is protected by well-designed cold traps. Freeze-cleavage and freeze-sectioning are compared. It is considered that there will be a temperature range for most heterogeneous specimens within which both cleavage and fracturing may occur, depending upon the nature of the molecules in the cleavage/sectioning plane. Local heating during freeze-sectioning will produce deformation artifacts as in freeze-cleavage, and may also lead to more general surface ‘flow’. The relationships between sectioning and fracturing biological specimens at low temperature require further clarification.     

Aspects of the performance of a femtosecond laser-pulsed 3-dimensional atom probe

Specimen heating is shown to occur in the laser-pulsed 3-dimensional atom probe (3DAP), even in the case of femtosecond pulse lengths. This can have an impact on the spatial resolution of 3DAP analysis, due to surface diffusion, and peak temperatures must be kept sufficiently low to avoid these effects. Similarly, mass resolution can be limited in the analysis of low thermal conductivity materials, due to the slower cool-down of the specimen after the pulse. In such cases, the use of lower repetition frequencies and specimens with large shank angles is shown to improve mass resolution and reduce the noise and degradation in quantitative accuracy resulting from increases in base temperature.     

Time dependant measurements of cavitation damage

Due to the extremely long length of experiments, in most studies of cavitation erosion only damage in the incubation period is measured and the final damage (mass loss rate) is then predicted by extrapolation. The methods of extrapolation are usually very basic since there were almost no in depth time dependant measurements of cavitation erosion performed in the past. A rotating disc test rig that generates a very aggressive cavitation and pure copper specimens, as erosion sensors, were used to investigate the correlation between the damage within the incubation period and final mass loss. The damage was measured optically three times during the incubation period and by weighing the specimen during the rest of the experiment.The results confirmed that the same clear relationship between the damage in the incubation period and the final mass loss rate exists, what means, that the mass loss rate can indeed be qualitatively predicted on the basis of measurements performed within the incubation period. This is a good basis for developing laws of extrapolation from a short time scale (laboratory measurement within the incubation period) to the real time scale (machine operation).     

Electron Beam Heating Temperature Profiles in Moderately Thick Cold Stage STEM/SEM Specimens

The analytic solution for the model of electron beam heating a moderately thick STEM/SEM specimen was used to calculate the steady state temperature profiles developed in the bulk of the specimen. For thin specimens one maximum in the temperature profile was found near the surface. The location of this maximum shifted away from the surface with increasing sample thickness. For specimens of thickness approaching the electron range two maxima were found: one close to the surface (as in thin samples) due to a ‘self-insulating’ effect, and another maximum near the sample-substrate interface—a result of very rapid increase in the energy loss by the electrons near their penetration range. These results are of particular interest for X-ray microanalysis where high beam currents are used, resulting in potentially large temperature rises in the bulk of the specimen.     

Effect of surface etching on condensing heat transfer

This study conducted experiments on humid air condensation during heat transfer in an air preheating exchanger attached to a home condensing boiler to improve thermal efficiency. An etchant composed of sulfuric acid and sodium nitrate was used to create roughness on the heat exchanger surface made from STS430J1L. A counter flow heat exchanger was fabricated to test the performance of heat transfer. Results showed that the overall heat transfer coefficients of all specimens treated with etchant improved with respect to the original specimens (not treated with etchant), and the overall heat transfer coefficient of the 60 s etching specimen increased by up to 15%. However, the increasing rate of the heat transfer coefficient was disproportional to the etching time. When the etching time specifically increased above 60 s, the heat transfer coefficient decreased. This effect was assumed to be caused by surface characteristics such as contact angle. Furthermore, a smaller contact angle or higher hydrophilicity leads to higher heat transfer coefficient.

     

Effect of Gradient Nanostructures on Tribological Properties of 316L Stainless Steel with High Energy Ion Implantation Tungsten Carbide

Surface gradient nanostructure of 316L stainless steel was prepared by ultrasonic surface rolling processing (USRP). WC were implanted into the gradient nanostructure surface by using high energy ion implantation technology (HEII). First, surface characteristics of two sets of specimens were investigated through hardness distribution, sectional morphology and microstructure. Then friction and wear mechanism was discussed. Results are as follows: the ion impregnated channels were obviously increased under USRP; hardness value of the top surface of USRP?+?HEII specimen was increased by 57.8% and thickness of hardened layer was increased by 100% comparing to HEII specimen; wear mass loss of USRP?+?HEII specimen was greatly reduced compared with HEII specimen; friction and wear mechanism of HEII specimen was changed from oxidation wear to fatigue wear, while the oxidation wear and adhesion wear were the main wear mechanisms for USRP?+?HEII specimen as the increase of rotational speed.     

Application of the ultrasonic propagation imaging system to an immersed metallic structure with a crack under a randomly oscillating water surface

Ultrasound is widely used and studied to satisfy the increased demands of the Non-destructive evaluation (NDE) and testing of underwater structures. However, because of the large size and mass of underwater structures, such as submarines, ship hulls, or nuclear reactor pipe lines, it is difficult to inspect the structures during operation. This underwater NDE technology is challenging but could be highly beneficial because the time and cost of maintenance will be effectively reduced. We propose an NDE method for immersed structures using an ultrasonic propagation imaging system with a piezoelectric sensor. The underwater sensing capability of a piezoelectric sensor is experimentally demonstrated using an aluminum plate specimen. A piezoelectric sensor can compensate for the decreased signal amplitudes due to leaky waves that are generated on interfaces between structures and water, since water transmits signals better than air. Additionally, a piezoelectric sensor can be applied even if the water surface is oscillating. Using these properties, the laser induced guided Ultrasonic propagation imager (UPI) inspected a T-shaped steel structure with a 2-mm crack on the weld zone. The inspection was implemented in three cases: a specimen without water, a specimen immersed in water and a specimen immersed in water with a randomly oscillating surface. The crack was visualized and measured using the ultrasonic wave propagation imaging algorithm, the adjacent wave subtraction algorithm, and the variable time window amplitude mapping algorithm. In the case with a randomly oscillating water surface, the laser pulse was refracted randomly based on Snell’s law. This phenomenon may cause degradation of the inspecting results. However, a repeated scanning process and outlier elimination led to an improved signal-to-noise ratio such that it was able to detect the crack. These results demonstrate the possibility to apply the laser UPI to submerged structures even if the water surface is randomly oscillating.     

Effect of shot peening on rolling contact fatigue and lubricant film thickness within mixed lubricated non-conformal rolling/sliding contacts

The effect of shot peening on rolling contact fatigue (RCF) and lubricant film thickness within non-conformal rolling/sliding contacts operated under mixed lubrication conditions was observed in this study. Rolling contact fatigue tests and film thickness measurements were carried out using specimens with modified surface topography by shot peening process using glass beads having diameter between 0.07 and 0.11 mm. It has been shown that the effect of shot peening on RCF has no positive effect even if shot peened surface of the roller exhibited somewhat higher hardness in contrast to the grounded surface. The reduction of RCF may be caused due to asperities interactions because after shot peening the surface roughness of the roller was increased. Film thickness measurements confirmed that the contact is realized actually only between asperity peaks of shot peened ball and smooth disc.Conversely, no negative effect on RCF was observed when the shot peened surface of the roller was polished. The polish of asperity peaks causes the creation of lands and micro-cavities, which may be employed as lubricant micro-reservoirs. From film thickness measurements it has been observed that lubricant emitted by shallow micro-cavities can provide the local increase in lubrication film thickness, which thereby reduces asperities interactions. Similar results were obtained for start-up conditions where the squeeze lubricant enlarges film thickness and reduces surface interactions.From the obtained results, it can be suggested that properly designed surface topography modification could help to increase the efficiency of lubrication films leading to the enhancement of contact fatigue life of non-conformal mixed lubricated rolling/sliding contacts.     

Quantitative electron holographic tomography for the 3D characterisation of semiconductor device structures

Electron tomography and electron holography experiments have been combined to investigate the 3D electrostatic potential distribution in semiconductor devices. The experimental procedure for the acquisition and data reconstruction of holographic tilt series of silicon p-n junction specimens is described. A quantitative analysis of the experimental results from specimens of two different thicknesses is presented, revealing the 3D electrostatic potential variations arising from the presence of surfaces and damage generated by focused ion beam (FIB) sample preparation. Close to bulk-like properties are measured in the centre of the tomographic reconstruction of the specimen, revealing higher electrically active dopant concentrations compared to the measurements obtained at the specimen surfaces. A comparison of the experimental results from the different thickness specimens has revealed a 'critical' thickness for this specimen preparation method of 350nm that is required for this device structure to retain 'bulk'-like properties in the centre of the membrane.     

X-ray microanalysis of freeze-dried and frozen-hydrated cryosections

The elemental composition and the ultrastructure of biological cells were studied by scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray microanalysis. The preparation technique involves cryofixation, cryoultramicrotomy, cryotransfer, and freeze-drying of samples. Freeze-dried cryosections 100-nm thick appeared to be appropriate for measuring the distribution of diffusible elements and water in different compartments of the cells. The lateral analytical resolution was less than 50 nm, depending on ice crystal damage and section thickness. The detection limit was in the range of 10 mmol/kg dry weight for all elements with an atomic number higher than 12; for sodium and magnesium the detection limits were about 30 and 20 mmol/kg dry weight, respectively. The darkfield intensity in STEM is linearly related to the mass thickness. Thus, it becomes possible to measure the water content in intracellular compartments by using the darkfield signal of the dry mass remaining after freeze-drying. By combining the X-ray microanalytical data expressed as dry weight concentrations with the measurements of the water content, physiologically more meaningful wet weight concentrations of elements were determined. In comparison to freeze-dried cryosections frozen-hydrated sections showed poor contrast and were very sensitive against radiation damage, resulting in mass loss. The high electron exposure required for recording X-ray spectra made reproducible microanalysis of ultrathin (about 100-nm thick) frozen-hydrated sections impossible. The mass loss could be reduced by carbon coating; however, the improvement achieved thus far is still insufficient for applications in X-ray microanalysis. Therefore, at present only bulk specimens or at least 1-μm thick sections can be used for X-ray microanalysis of frozen-hydrated biological samples.     

Removal of epoxy-attached thin sections from glass slides by plasma etching

Thin-sectioned samples mounted on glass slides with common petrographic epoxies cannot be easily removed (for subsequent ion-milling) by standard methods such as heating or dissolution in solvents. A method for the removal of such samples using a radio frequency (RF) generated oxygen plasma has been investigated for a number of typical petrographic and ceramic thin sections. Sample integrity and thickness were critical factors that determined the etching rate of adhesive and the survivability of the sample. Several tests were performed on a variety of materials in order to estimate possible heating or oxidation damage from the plasma. Temperatures in the plasma chamber remained below 138°C and weight changes in mineral powders etched for 76 hr were less than ±4%. A crystal of optical grade calcite showed no apparent surface damage after 48 hr of etching. Any damage from the oxygen plasma is apparently confined to the surface of the sample, and is removed during the ion-milling stage of transmission electron microscopy (TEM) sample preparation.     

Methods for specimen thickness determination in electron microscopy. II. Changes in thickness with dose

The electron diffraction patterns of tilted thin crystals were used to determine the unit cell size in the direction normal to the supporting film. The method revealed a considerable dose-dependent thinning or shrinkage. Using a variety of specimens and stains, we found that this amounted to a 50% reduction in volume and could be attributable to two causes. Firstly, the specimen is held to the supporting film so that volume changes can only occur through changes in thickness. Secondly, the decrease in volume is associated with a dose-induced mass loss which is greatly suppressed at liquid nitrogen temperatures.