Sample Preparation Artifacts Research Articles

Void formation during early stages of passivation: Initial oxidation of iron nanoparticles at room temperature

The examination of nanoparticles allows study of some processes and mechanisms that are not as easily observed for films or other types of studies in which sample preparation artifacts have been the cause of some uncertainties. Microstructure of iron nanoparticles passivated with iron oxide shell was studied using high-resolution transmission electron microscopy and high-angle annular dark-field imaging in aberration-corrected scanning transmission electron microscopy. Voids were readily observed on both small single-crystal α-Fe nanoparticles formed in a sputtering process and the more complex particles created by reduction of an oxide by hydrogen. Although the formation of hollow spheres of nanoparticles has been engineered for Co at higher temperatures [Y. Yin, R. M. Riou, C. K. Erdonmez, S. Hughes, G. A. Somorjari, and A. P. Alivisatos, Science 304, 711 (2004)], they occur for iron at room temperature and provide insight into the initial oxidation processes of iron. There exists a critical size of ∼8nm for which the iron has been fully oxidized, leading to a hollow iron-oxide nanoparticle. For particles larger than the critical size, an iron/iron-oxide core-shell structure was formed and voids reside at the interface between the oxide shell and the iron core. The present observation provides new insight for tailoring of metal/metal-oxide core-shell structured nanoparticles for applications related to optics, magnetism, and nanoelectronics.

Cross-Sectioning of Plan-View Samples and Artifacts of TEM Sample Preparation

Cross-Sectioning of Plan-View Samples and Artifacts of TEM Sample Preparation

Biliary cholesterol crystallization characterized by single-crystal cryogenic electron diffraction

Cholesterol crystals are the building blocks of cholesterol gallstones. The exact structure of early-forming crystals is still controversial. We combined cryogenic-temperature transmission electron microscopy with cryogenic-temperature electron diffraction to sequentially study crystal development and structure in nucleating model and native gallbladder biles. The growth and long-term stability of classic cholesterol monohydrate (ChM) crystals in native and model biles was determined. In solutions of model bile with low phospholipid-to-cholesterol ratio, electron diffraction provided direct proof of a novel transient polymorph that had an elongated habit and unit cell parameters differing from those of classic triclinic ChM. This crystal is exactly the monoclinic ChM phase described by Solomonov and coworkers (Biophysical J., In press) in cholesterol monolayers compressed on the air-water interface. We observed no evidence of anhydrous cholesterol crystallization in any of the biles studied. In conclusion, classic ChM is the predominant and stable form in native and model biles. However, under certain (low phospholipid) conditions, transient intermediate polymorphs may form. These findings, documenting single-crystal analysis in bulk solution, provide an experimental approach to investigating factors influencing biliary cholesterol crystal nucleation and growth as well as other processes of nucleation and crystallization in liquid systems.

Characterization of a Novel Modification to Monoclonal Antibodies: Thioether Cross-link of Heavy and Light Chains

A novel, nonreducible thioether bridge between the light and heavy chains of different IgG1 monoclonal antibodies has been characterized. An additional band with an apparent molecular weight of 92 kDa was detected when monoclonal antibodies were analyzed by reducing capillary gel electrophoresis (rCGE) and reducing SDS-PAGE. To further investigate this observation, an early-eluting peak in the size exclusion chromatogram of a reduced and alkylated monoclonal antibody was collected and characterized by liquid chromatography, mass spectrometry, and gel electrophoresis. The reduced and alkylated Mab was shown to be a cross-linked adduct with a molecular weight of 75 kDa. In the adduct, the heavy and light chains of the antibody were cross-linked by a nonreducible thioether bond between Cys-223 of the heavy chain and the C-terminal Cys residue of the light chain. The thioether bond modification was confirmed in the Fab fragment of a monoclonal antibody by LC-MS and nonreduced Lys-C peptide mapping with tandem mass spectrometry. The data show that the disulfide bond modification occurred under nonreducing conditions and was not an artifact of sample preparation for the rCGE analysis. The thioether bond modification was observed in several IgG1 monoclonal antibody products. Structural characterization of this novel modification is important in understanding the mechanism of thioether bond formation.

PAH dissipation in a contaminated river sediment under oxic and anoxic conditions

A batch experiment was conducted to compare PAH degradation in a polluted river sediment under aerobic and anaerobic conditions, and to investigate whether input of fresh organic material (cellulose) could enhance such degradation. All measurements were checked against abiotic control treatments to exclude artifacts of sample preparation and non-biological processes like aging. Three- and four-ring PAHs could be degraded by the indigenous microbial community under aerobic conditions, but anaerobic metabolism based on iron and sulphate reduction was not coupled with PAH degradation of even the simplest 3-ring compounds like phenanthrene. Cellulose addition stimulated both aerobic and anaerobic respiration, but had no effect on PAH dissipation. We conclude that natural attenuation of PAHs in polluted river sediments under anaerobic conditions is exceedingly slow. Dredging and biodegradation on land under aerobic conditions would be required to safely remediate and restore polluted sites.

Effects of SEM Preparation Techniques on the Appearance of Bacteria and Biofilms in the Carter Sandstone

Abstract When biofilms (aggregations of bacteria and extracellular polymer secretions) in samples from the Carter Sandstone of Alabama were prepared for scanning electron microscopy (SEM) using different dehydration techniques, the organic material had visibly different textures and distributions. In order to assess whether the variation was attributable to sample preparation or to inherent biofilm heterogeneity, each of five techniques were tested 3 to 10 times on small (1 cm) pieces of the Carter Sandstone containing either a strain of bacteria cultured from and reintroduced into the rock, or an in situ biofilm grown by injection of nutrients through core samples. The techniques tested were (1) air drying alone; (2) fixation in 10% glutaraldehyde with air drying; (3) ethanol dehydration with hexamethyldisilazane (HMDS) drying [2.5% glutaraldehyde, ethanol dehydration, and HMDS]; (4) ethanol dehydration with critical-point drying; and (5) ethanol and acetone dehydration with critical-point drying. Unpreserved control samples were either imaged wet in an environmental scanning electron microscope (ESEM) or vacuum-dried for SEM. Observations were based on SEM microscopy of over 60 samples and study of over 150 photomicrographs. In both experiments, the original morphology of individual bacteria was best preserved by ethanol dehydration with HMDS drying, ethanol dehydration with critical-point drying, or ethanol-acetone dehydration with critical-point drying. Critical-point drying preserved bacteria but stripped away mucilaginous material, revealing filamentous structures within the biofilm. These filaments, along with masses of microspheres (nannobacteria?) and the smooth mucilaginous outer layer, also occur in wet samples studied by ESEM, and are, therefore, not dehydration artifacts. However, different sample preparation techniques accentuated different components of the heterogeneous biofilm, thus resulting in vastly different textures. The cultured bacteria produced a biofilm that had a different surface texture and was more susceptible to sample preparation artifacts than the in situ biofilm. Use of more than one sample preparation technique is recommended in order to avoid bias.

Uptake of arsenic by vegetables for human consumption: a study of Wellingborough allotment plots

Data on the bioaccessibility and soil-to-plant concentration factors for arsenic at seven allotments within the Wellingborough area were collected. The data were used to evaluate the suitability of such site-specific data for use in detailed quantitative risk assessments, and their effect on the site-specific assessment criteria was derived. The evaluation indicated that: • The uses of site-specific bioaccessibility and soil-to-plant concentration factors have a considerable impact on the assessment of risk from arsenic at the various allotments.  The results of physiologically-based extraction tests indicate that the bioaccessibility of arsenic in soil at the allotments tested is considerably less than the 100% assumed by the CLEA model, even accounting for the limitations and applicability of this test.  Once the reduced bioaccessibility of arsenic is taken into account, the dominant exposure pathway becomes the consumption of contaminated vegetables.  Physical and metabolic characteristics of some vegetable types lead to exceptionally high soil-to-plant concentration factors. Whether or not these represent real risks to human health, or are merely artefacts of sample preparation and handling, has not been determined. However, this issue should be resolved in order to demonstrate the reliability of the data produced and to ensure that a risk assessment based on these data is protective of human health but is not overly conservative.  Analytical limitations also have a considerable influence on the soil-to-plant concentration factors (CF) derived and can, in some cases, lead to highly conservative values for CFleafy and CFroot being used to assess the risk to human health.  Further investigation and guidance are needed on deriving site-specific soil-to-plant concentration factors in order to ensure that a standard methodology is employed and that human health is adequately protected.  This work provides the Borough Council of Wellingborough (BCW) with local evidence of the applicability of the generic advice contained in the CLR reports, and helps BCW determine when to pursue detailed quantitative risk assessment and when to engage in remediation in order to ensure that arsenic does not pose an unacceptable risk to human health.

Imaging of fullerene-like structures in CN x thin films by electron microscopy; sample preparation artefacts due to ion-beam milling

The microstructure of CN x thin films, deposited by reactive magnetron sputtering, was investigated by transmission electron microscopy (TEM) at 200 kV in plan-view and cross-sectional samples. Imaging artefacts arise in high-resolution TEM due to overlap of nm-sized fullerene-like features for specimen thickness above 5 nm. The thinnest and apparently artefact-free areas were obtained at the fracture edges of plan-view specimens floated-off from NaCl substrates. Cross-sectional samples were prepared by ion-beam milling at low energy to minimize sample preparation artefacts. The depth of the ion-bombardment-induced surface amorphization was determined by TEM cross sections of ion-milled fullerene-like CN x surfaces. The thickness of the damaged surface layer at 5° grazing incidence was 13 and 10 nm at 3 and 0.8 keV, respectively, which is approximately three times larger than that observed on Si prepared under the same conditions. The shallowest damage depth, observed for 0.25 keV, was less than 1 nm. Chemical changes due to N loss and graphitization were also observed by X-ray photoelectron spectroscopy. As a consequence of chemical effects, sputtering rates of CN x films were similar to that of Si, which enables relatively fast ion-milling procedure compared to carbon compounds. No electron beam damage of fullerene-like CN x was observed at 200 kV.

Identification of Sample Preparation Artifacts in Powder Analysis

Abstract The synthesis of inorganic materials of a specific size and shape is a key aspect in modern powder technology. During the past two decades, solution-based precipitation reactions have been explored for controlled particle formation. A wide variety of particle shapes, sizes, and compositions has been demonstrated. However, several questions, especially concerning particle nucleation, remain. Some of these questions are currently addressed by investigating precipitate (ex situ) at different reaction stages by means of powder x-ray diffraction (PXRD), scanning (SEM) and transmission electron microscopy (TEM).

Electron microscopic investigation of the morphology and calcium-induced fusion of lipid vesicles with an oligomerised inner leaflet

The lipid head groups in the inner leaflet of unilamellar bilayer vesicles of the synthetic lipids DHPBNS and DDPBNS can be selectively oligomerised. Earlier studies have established that these vesicles fuse much slower and less extensively upon oligomerisation of the lipid head groups. The morphology and calcium-induced fusion of vesicles of DHPBNS and DDPBNS were investigated using cryo-electron microscopy. DHPBNS vesicles are not spherical but flattened, ellipsoidal structures. Upon addition of CaCl 2, DHPBNS vesicles with an oligomerised inner leaflet were occasionally observed in an arrested hemifused state. However, the evidence for hemifusion is not equivocal due to potential artefacts of sample preparation. DDPBNS vesicles show the expected spherical morphology. Upon addition of excess CaCl 2, DDPBNS vesicles fuse into dense aggregates that show a regular spacing corresponding to the bilayer width. Upon addition of EDTA, the aggregates readily disperse into large unilamellar vesicles. At low concentration of calcium ion, DDPBNS vesicles with an oligomerised inner leaflet form small multilamellar aggregates, in which a spacing corresponding to the bilayer width appears. Addition of excess EDTA results in slow dispersal of the Ca 2+-lipid aggregates into a heterogeneous mixture of bilamellar, spherical vesicles and networks of thread-like vesicles. These lipid bilayer rearrangements are discussed within the context of shape transformations and fusion of lipid membranes.

Characterization of emulsions and suspensions in the petroleum industry using cryo-SEM and CLSM

Confocal laser scanning and cryogenic scanning electron microscopy techniques for imaging colloidal systems are discussed. The advantages of each technique are shown along with examples of emulsions and suspensions from the petroleum industry. Given the sample preparation and manipulation that is sometimes required for cryo-SEM observation, the degree to which the images actually represent the sample morphology can be in doubt. An example from the study of oil sands tailings suspensions shows that sample preparation artifacts can be subtle and deceptive. Ultimately, direct observation should be used in conjunction with, or as support for conventional characterization techniques of colloidal systems, not as a substitute.

Use of the Vital Stain FM4-64 for Visualizing Membrane Dynamics in Living Fungal Cells

Abstract Allomyces macrogynus is a zoosporic fungus (Chytridiomycota) that exhibits a true alternation of generations between the diploid and haploid thalli. During the diploid stage in the life cycle, sporothalli are produced which are capable of forming asexual zoosporangia containing a multinucleate protoplast bound by a single plasma membrane and cell wall. Though able to remain in a quiescent, coenocytic state for an extended period of time, under certain environmental conditions the cytoplasm of mature zoosporangia is cleaved and gives rise to numerous uninucleate, uniflagellate zoospores within 40 to 50 min. Previous studies of cleavage membrane development m Allomyces zoosporangia have been conducted using light microscopy and transmission electron microscopy (TEM). Despite these investigations, our understanding of cytokinesis in zoosporangia of Allomyces and other zoosporic organisms remains incomplete. This is due, in part, to the difficulty of detecting cleavage elements using standard light microscope optics (i.e., phase contrast, differential interference contrast) and because ultrastructural analysis often provides limited temporal and spatial detail and may suffer from artifacts of sample preparation.

NMR studies of calcium induced alginate gelation. Part II. The internal bead structure

Proton NMR relaxation, diffusion and microimaging techniques were used to investigate the internal structure of calcium alginate gel beads formed by dripping a sodium alginate solution into a calcium chloride solution. It is shown that many of the pore structures previously reported are probably artefacts of sample preparation. Evidence is presented for a non-uniform calcium alginate concentration profile even in beads that have been ‘equilibrated’ in calcium chloride solution for over 24 h. It is found that the internal structure depends on the ratio of calcium and alginate concentrations and a microstructural phase diagram can be constructed. Copyright © 2000 John Wiley & Sons, Ltd.

An NMR analysis of the reaction of ubiquitin with [ acetyl-1- 13C]aspirin

The acetylation of ubiquitin by [ acetyl-1- 13C]aspirin has been studied using 2D NMR methods. Studies performed in a 50:50 H 2O:D 2O medium show doubling of the acetyl carbonyl resonances, indicating that all of the stable adducts formed involved amide linkages. Assignment of the heteronuclear multiple quantum coherence (HMQC) resonances was accomplished based on comparison of resonance intensities with the results of an Edman degradation analysis, pH titration studies of acetylated ubiquitin, and analysis of two ubiquitin mutants, K33R and K63R. The presence of a single tyrosine residue in close proximity to lysine-48 suggested another assignment strategy. Nitration of tyrosine-59 resulted in a small, pH-dependent shift of the resonance assigned to lysine-48, with a pK of 7.0, close to that expected for the nitrotyrosyl hydroxyl group. An additional adduct resonance with very low intensity also was observed and tentatively assigned to the acetylated N-terminal methionine residue. The relative rates of acetylation of the various lysine residues were obtained from time-dependent HMQC studies. Since no sample preparation artifacts were introduced, the levels of modification of the various residues could be determined with relatively high accuracy. Based on the time-dependent intensity data, the relative rate constants for modification of K6, K48, K63, K11, K33, and M1 were 1.0, 0.59, 0.43, 0.26, 0.23, and 0.03, respectively. These results were in much better agreement with amino accessibility predictions based on the crystal structure of the ubiquitin monomer than with predictions based on the ubiquitin structure in the crystallized dimeric and tetrameric forms. This approach provides a useful basis for understanding how local environmental factors can influence protein adduct formation, as well as for comparing the extent and specificity of various acetylation reagents.

Argon behaviour in gem-quality orthoclase from Madagascar: Experiments and some consequences for [formula omitted] geochronology

In order to investigate argon diffusion in as simple a K-feldspar structure as possible, a single crystal of the gem-quality Itrongay K-feldspar from Madagascar has been studied using cycled step heating, ultra-violet (UV) laser depth profiling, in vacuo crushing, Electron Microprobe Analysis (EMPA) and X-ray diffraction (XRD) techniques. The results have been modelled using both the multidomain model and a multipath model invoking pipe or short circuit (SC) diffusion. Cycle heating (both forward and reversed) indicates a very retentive K-feldspar, in which the great majority of argon release conforms to a simple model of volume diffusion with a single domain size, a single activation energy, and is associated with a plateau age of 435 ± 8 Ma. However, the first 1% of argon release exhibits younger ages and the same type of complex behaviour seen in other K-feldspars. Modelling the argon release in terms of multidomains yields good fits to data with four domains of varying activation energies. An alternative model for the argon release, involving not only volume diffusion through the lattice but also SC or pipe diffusion and mass transfer between lattice and rapid diffusion paths, provides a good though less sophisticated model to explain the argon release. Argon concentration/depth profiles of a previously outgassed sample, measured using the UV laser ablation technique, exhibited argon loss in only the upper 5–10 μm. Argon loss calculated from the measured profiles suggests that the low temperature domains outgassed during step heating were within 5–10 μm of the grain surfaces and thus may be artefacts of sample preparation and surface texture effects. In vacuo crushing of an untreated sample released argon with an older age than the plateau value, which was not detected by any of the other analytical techniques. However, previously cycled step heated samples contained very little excess argon, suggesting the existence of traps within the feldspar structure into which 40Ar diffused in nature to yield apparent excess argon, but which were then filled with corresponding 39Ar during cycle heating. The K-feldspar structure illuminated by these studies is one in which argon diffuses through the lattice over distances of at least 100 pm probably via a volume diffusion mechanism to the grain boundaries. However, close to the surface argon may also diffuse along fast diffusion paths resulting from natural traps opened during sample preparation. Though restricted to very small volumes of gas in the case of the Itrongay orthoclase gem, the existence of the traps has potentially important consequences for 40 Ar 39 Ar geochronology applied to K-feldspars.

Illite Polytype Quantification Using Wildfire© Calculated X-Ray Diffraction Patterns

AbstractIllite polytype quantification allows the differentiation of diagenetic and detrital illite components. In Paleozoic shales from the Illinois Basin, we observe 3 polytypes: 1Md, 1M and 2M1. 1Md and 1M are of diagenetic origin and 2M1 is of detrital origin. In this paper, we compare experimental X-ray diffraction (XRD) traces with traces calculated using WILDFIRE© and quantify mixtures of all 3 polytypes, adjusting the effects of preferred orientation and overlapping peaks. The broad intensity (“illite hump”) around the illite 003, which is very common in illite from shales, is caused by the presence of 1Md illite and mixing of illite polytypes and is not an artifact of sample preparation or other impurities in the sample. Illite polytype quantification provides a tool to extrapolate the K/Ar age and chemistry of the detrital and diagenetic end-members by analysis of different size fractions containing different proportions of diagenetic and detrital illite polytypes.

Characterisation of inner surface and adsorption phenomena in fused-silica capillary electrophoresis capillaries

Method validation in capillary electrophoresis (CE) is substantial for the routine use in quality control. Thus all phenomena that can influence results in CE must be carefully characterised. Especially the inner surface of the fused-silica capillaries plays an important role. Sometimes there is a big difference in the properties of capillaries from different batches. Inner capillary surface can be pictured by scanning electron microscopy (SEM). Nature, pattern and number of cracks and other surface defects altered from batch to batch. These surface defects were no artefacts of sample preparation, which could be excluded by comparing different preparation techniques and results from literature. Treatments with hydrofluoric acid cannot completely remove the surface cracks and defects in subsurface areas. Hydrofluoric acid treatment not only changes the surface roughness but also the inner capillary diameter. A correlation between etching time and increase of diameter is investigated. SEM also pictures adsorption phenomena, e.g. proteins are not adsorbed as a homogenous layer. Clusters at different regions are observed. Thus the ζ potential differs considerably with space, leading to losses in separation efficiency. SEM can readily be used to analyse the effectiveness of rinsing procedures. There are residuals of adsorbed compounds even after using aggressive rinsing reagents.

Improving transmission electron microscopy characterization of semiconductors by minimizing sample preparation artifacts

Techniques employed for the preparation of transmission electron microscopy (TEM) samples can introduce artifacts that obscure subtle detail in the materials being studied. Traditional semiconductor sample preparation techniques rely heavily on ion milling, which leaves amorphous layers on ion milled surfaces and some intermixing across interfaces, thus degrading the TEM images of these samples. Experimental results of the extent of this amorphization and intermixing are presented for silicon-based semiconductor samples, and methods to minimize these effects are suggested. These methods include variations in ion milling parameters that reduce the extent of the artifacts, and improvements in the small-angle cleavage technique that eliminate these artifacts completely.

Scanning tunneling microscopy and atomic force microscopy on organic and biomolecules

The state of the art in imaging organic molecules with STM and AFM is discussed on the basis of selected examples. For large biological samples the problems of sample preparation and possible artifacts have yet to be solved in order to produce valuable new scientific results. Preliminary results on larger objects like human chromosomes are presented. Clearcut experimental results are obtained when small organic molecules from ordered surface layers on a crystalline substrate. Examples of two dimensional arrays of small organic molecules like liquid crystals and DNA bases on these substrates are shown.

Uterine Estrogen Receptorin Vivo: Phosphorylation of Nuclear Specific Forms on Serine Residues

We have characterized further the heterogeneous nuclear-specific doublet forms of the mouse uterine estrogen receptor (ER). Estrogen treatment produced the multiple nuclear ER forms of 65 and 66.5 kDa, which were isolated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soluble ER preparations exhibited only a single 65-kDa form. Isolation of the individual nuclear ER forms and reanalysis demonstrated that formation of the multiple bands was not due to artifacts of nuclear sample preparation or the presence of contaminating proteins. Analysis of individual uterine cell types (epithelial and stromal/myometrium) indicated that both ER forms were present in both cell fractions. Fractionation of nuclear components with low salt showed that both ER forms were found in the salt-resistant fraction. Extraction of nuclei with high salt (0.6 M KCl) solubilized both ER forms. Phosphorylation was studied as a protein modification to account for the multiple forms. Incorporation of 32P into uterine protein both in vivo and in intact tissue incubation indicated 32P labeling of uterine nuclear ER after hormone treatment. Both nuclear ER forms are labeled, although the 66.5-kDa form appears to be more heavily labeled. Phosphoamino acid analysis of the immunopurified 32P-labeled ER from intact uterine tissue indicated phosphoserine as the only phospholabeled residue. These data suggest that phosphorylation is associated with the physiological functioning of the ER in response to hormone and produces the heterogeneous ER forms in the nucleus.