Specimen Preparation Artifacts Research Articles

The effect of specimen preparation on the nanostructure of SLES aqueous solutions observed by cryo-TEM.

Sodium lauryl ether sulphate (SLES) is a detergent widely used in cosmetics and personal-care industries; hence, it is of particular interest to study the self-assembled nanostructure it forms at different conditions. Cryogenic transmission electron microscopy (cryo-TEM) is the most suitable technique for the direct-imaging of such systems. However, since SLES is sensitive to flow and shear, specimen preparation artefacts may misrepresent the native state of the solution. In this paper, we present different cryo-TEM specimen preparation methods, and show how they affect the nanostructure of the system. In fact, for this system, we were able to record the native state of the solution only after sufficient time of on-the-grid relaxation (OGR) after blotting. Here, we also intend to point out the importance of considering the nature of the solution when preparing cryo-TEM specimens.

Comparing Xe+ pFIB and Ga+ FIB for TEM sample preparation of Al alloys: Minimising FIB-induced artefacts.

Recently, the dual beam Xe+ plasma focused ion beam (Xe+pFIB) instrument has attracted increasing interest for site‐specific transmission electron microscopy (TEM) sample preparation for a local region of interest as it shows several potential benefits compared to conventional Ga+FIB milling. Nevertheless, challenges and questions remain especially in terms of FIB‐induced artefacts, which hinder reliable S/TEM microstructural and compositional analysis. Here we examine the efficacy of using Xe+ pFIB as compared with conventional Ga+ FIB for TEM sample preparation of Al alloys. Three potential source of specimen preparation artefacts were examined, namely: (1) implantation‐induced defects such as amophisation, dislocations, or ‘bubble’ formation in the near‐surface region resulting from ion bombardment of the sample by the incident beam; (2) compositional artefacts due to implantation of the source ions and (3) material redeposition due to the milling process. It is shown that Xe+pFIB milling is able to produce improved STEM/TEM samples compared to those produced by Ga+ milling, and is therefore the preferred specimen preparation route. Strategies for minimising the artefacts induced by Xe+pFIB and Ga+FIB are also proposed.Lay Description FIB (focused ion beam) instruments have become one of the most important systems in the preparation of site‐specific TEM specimens, which are typically 50‐100 nm in thickness. TEM specimen preparation of Al alloys is particularly challenging, as convention Ga‐ion FIB produces artefacts in these materials that make microstructural analysis difficult or impossible. Recently, the use of noble gas ion sources, such as Xe, has markedly improved milling speeds and is being used for the preparation of various materials. Hence, it is necessary to investigate the structural defects formed during FIB milling and assess the ion‐induced chemical contamination in these TEM samples. Here we explore the feasibility and efficiency of using Xe+PFIB as a TEM sample preparation route for Al alloys in comparison with the conventional Ga+FIB.

Structure elucidation of silica-based core–shell microencapsulated drugs for topical applications by cryogenic scanning electron microscopy

We present here a technology to microencapsulate drugs by the sol–gel process, and cryo-SEM methodology that allows the nanostructural characterization of the formed capsules in their native state without any artifacts, related to their drying prior to imaging. The methodology utilizes three signals generated by the electron beam scanning the specimen: Secondary electrons, backscattered electrons, and x-rays. The first gives topographical information of the fracture-surface of the thermally-fixed specimen, the second gives contrast between elements of different atomic numbers, and the third allows the identification of those elements. Combined, the three signals provide full microstructural characterization of the studied specimen.Using this methodology, we were able to demonstrate that the sol–gel technology does indeed enable the encapsulation of two hydrophobic active molecules with a silica shell. This technology allows the active ingredient in the drug product to slowly migrate from the microcapsule onto the skin, thus obtaining the desired effect with minimal side-effects, as was exhibited in several clinical studies.The successful application of the cryo-SEM methodology in this case, demonstrates that it can be used to characterize a wide range of liquid-phase suspensions, in their native state, with minimal specimen preparation or imaging artifacts.

A discussion of the paper “Effect of design parameters on microstructure of steel-concrete interface in reinforced concrete”

The steel-concrete interface is fundamental to many aspects related to the performance of reinforced concrete, particularly for corrosion behavior. A recently published paper by Chen et al. [Effect of design parameters on microstructure of steel-concrete interface in reinforced concrete, Cem. Concr. Res. 119 (2019) 1–10] quantitatively characterized the thickness of the gap observed between the steel and concrete. Considering the low number of available publications, studies documenting the microstructure at the steel-concrete interface are important. However, in our discussion, we think that aspects related to the terminology, the specimen preparation and possible experimental artefacts in the discussed paper can be improved.

Defect structures in solution-grown single crystals of the intermetallic compound Ag3Sn

The compound Ag3Sn adopts the ordered orthorhombic D0a Cu3Ti-type structure. It exhibits an unusual low yield stress and high ductility for an intermetallic compound, but the reasons for these effects are not clear. Here, we report an electron microscopy study on the defects present in solution-grown Ag3Sn single crystals that have deformed during the decanting and subsequent handling processes. It is found that the crystals contain two types of lenticular deformation twins: {011}-type and {211}-type. These twins interpenetrate with no evidence of cracking at the intersections. The crystals also contain high densities of dislocations including long straight dipoles with b = ± [010] and shorter curved segments and loops with b = [ $$ 10\bar{2} $$ ] and [001]. It is inferred that the dipoles are artifacts of specimen preparation that climb in from the cross-sectional sample surfaces, whereas the shorter segments are deformation debris. If a combination of twinning and dislocation glide of the types observed here were to form concurrently during general deformation of Ag3Sn, then they could provide the necessary number of independent deformation modes to accommodate an arbitrary plastic strain, which might help to explain the unusual ductility of this compound.

High-resolution imaging of biotite using focal series exit wavefunction restoration and the graphene mechanical exfoliation method

AbstractWe have applied mechanical exfoliation for the preparation of ultra-thin samples of the phyllosilicate mineral biotite. We demonstrate that the 'scotch tape' approach, which was made famous as an early method for production of single-atom-thick graphene, can be used for production of sheet-silicate specimens that are sufficiently thin to allow high-resolution transmission electron microscope (HRTEM) imaging to be achieved successfully while also being free from the specimen preparation artefacts that are often caused by ion-beam milling techniques. Exfoliation of the biotite parallel to the (001) planes has produced layers as thin as two structural TOT units thick (∼2 nm). The minimal specimen thickness enabled not only HRTEM imaging but also the application of subsequent exit wavefunction restoration to reveal the pristine biotite lattice. Exit wavefunction restoration recovers the full complex electron wave from a focal series of HRTEM images, removing the effects of coherent lens aberrations. This combination of methods therefore produces images in which the observed features are readily interpreted to obtain atomic resolution structural information.

Comparison of preparation techniques for nuclear materials for transmission electron microscopy (TEM)

Preparation of highly radioactive and irradiated nuclear fuels and materials for transmission electron microscopy (TEM) is accompanied with a set of unique challenges. The paper evaluates three specimen preparation techniques for preparation of irradiated materials and determines which technique yields to the most reliable characterization of radiation damage microstructure. Various specimen preparation artifacts associated with each technique are considered and ways of minimizing these artifacts are addressed.

Electron holography for analysis of deep submicron devices: Present status and challenges

A potential distribution analysis of source/drain (SD) regions in sub-30-nm-gate-length metal-oxide-semiconductor field-effect transistors (MOSFETs) is presented as an example of the present status of electron holography analysis. First, the authors describe experimental setups that determine the resolutions of the analysis in practice. They show that specimen preparation artifacts significantly affect the energy resolution and that the artifacts can be reduced by preparing specimens using low-energy back side ion milling. Second, they describe the SD potential analysis in sub-30-nm-gate-length MOSFETs.

Electron holography analysis of a shallow junction for planar-bulk metal-oxide-semiconductor field-effect transistors approaching the scaling limit

We investigated electrostatic potential distributions in source∕drain extensions (SDEs) in metal-oxide-semiconductor field-effect transistors (MOSFETs) fabricated using state-of-the-art junction formation technology. We first demonstrate that electron holography can directly reveal potential distribution in scaled MOSFETs when specimen preparation artifacts are reduced, which we did by using back side low-energy Ar ion milling. Second, we examine the potential distributions in SDEs in a scaled (30-nm-gate-length) MOSFET fabricated by using a combination of cluster B implantation, millisecond annealing, and multihalo implantation. The results show that these junction formation technologies enable fabrication of very abrupt and shallow (10-nm-deep) SDE junctions. In addition, our experimental analysis, in conjunction with a Monte Carlo doping-process simulation, indicates that B channeling along the ⟨110⟩ direction of the Si substrate during the implantation process significantly blurs the SD junction profiles and that multihalo implantation can increase junction abruptness. Third, we show that our experimental results describe well the roll-off characteristics of the MOSFETs.

Electron Holography Characterization of Ultra Shallow Junctions in 30-nm-Gate-Length Metal–Oxide–Semiconductor Field-Effect Transistors

We demonstrate that electron holography can be used to map the electrostatic potential in source–drain extensions (SDEs) of 30-nm-gate-length metal–oxide–semiconductor field-effect transistors (MOSFETs). To reduce specimen-preparation artifacts, which have prevented the electron holography of advanced MOSFETs, we prepared specimens using low-energy backside Ar ion milling. Our analysis revealed the potential distributions in SDEs formed by a co-implantation technique and those formed by a conventional BF2 implantation technique and showed that the potential change at the p–n junctions is more abrupt in the former. We also show that our electron holography results clearly describe the roll-off characteristics of the MOSFETs.

Microstructural features of a mortar as seen by computed microtomography

While low resolution limits the use of computed microtomography for microstructural investigation, the method has a unique advantage in that no specimen preparation is required; the images produced are thus entirely free of specimen preparation artifacts. In the present communication we report examination of the microstructure of a w:c 0.6 mortar, with the tomographic information resolved on a set of more than 500 successive planar images each 1.2 μm apart. Selected images are compared with backscatter SEM images taken from similar materials at comparable magnifications. The same features are observed, including sand grains, air voids, residual cement grains, hydrated cement paste components, and hollow-shell pores. The “patchy” microstructure of different areas of hardened cement paste, previously reported for mortars and concretes in backscatter SEM, is clearly also present in images derived from computed microtomography, and are not artifacts of SEM specimen preparation.

High-Pressure Freezing, Cellular Tomography, and Structural Cell Biology

Structural cell biology, which we define as electron microscopic analysis of intact cells, suffered a loss of interest and activity following the advances in light microscopy beginning in the 1990s. Interestingly, it is the wealth of detailed observation in the light microscope that is one of the driving forces for the current renewed interest in electron microscopy (EM). A great many cellular details are simply beyond the resolving power of the light microscope. In this article, we describe how electron microscopists are responding to the demands for better preservation of cells and for ways to view cell ultrastructure in three dimensions at high resolution. We discuss how low temperature methods, especially high-pressure freezing and freeze substitution, reduce the artifacts of conventional EM specimen preparation. We also give a brief introduction to cellular electron tomography, a powerful analytical method that can give near-atomic resolution of cell ultrastructure in three-dimensional (3-D) models.

Freezing Biological Samples

Abstract Freezing damages cell membranes and reduces the histological readability of biological specimens. This is a discussion of why, the consequences, and how to minimize or avoid the damage introduced when using freezing as the means to harden biological tissue in order to cut thin sections for histology. Pure water can exist in the solid state in three forms. Two forms are crystalline: one with a hexagonal lattice and the other with a cubic lattice. The third form capitalizes on the fact that water can be frozen so rapidly that it does not have time to form a crystal lattice and remains amorphous (vitreous form). Crystal formation is responsible for the expansion of water as it freezes (a property unique of water), which creates specimen preparation artifacts. Vitreous ice does not expand upon solidification. This makes it the only desirable form of ice appropriate for biological specimens.

Electron Tomography and Three-Dimensional Aspects of Transmission Electron Microscopy

Abstract Transmission electron microscopy (TEM) plays an important role semiconductor process development, defect identification, yield improvement, and root-cause failure analysis. At the same time, however, certain artifacts of specimen preparation and imaging present barriers for linear scaling of TEM techniques. This article assesses these challenges and explains how electron tomography is being used to overcome them.

Cryoelectron microscopy reveals new features in the three‐dimensional structure of phosphorylase kinase

Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3-MDa hexadecameric complex, (alphabetagammadelta)(4). PhK comprises two arched octameric (alphabetagammadelta)(2) lobes that are oriented back-to-back with overall D(2) symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three-dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three-dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 A. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90 degrees instead of 68 degrees, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure.

Callose in Frankia‐Infected Tissue of Datisca glomerata is an Artifact of Specimen Preparation

Abstract: Callose, or β‐1,3‐glucan, is a plant cell wall polysaccharide that occurs endogenously at distinct sites in a variety of tissues. Callose is also formed in response to stress involving cell membrane perturbation. In sections of chemically‐fixed nodule tissue of the actinorhizal host, Datisca glomerata, callose was cytochemically detected within the Frankia‐infected cortical cells, as an extensive network of wall material surrounding the microsymbiont, but not in uninfected cortical cells. Callose formation was completely inhibited within the infected cells when 2‐deoxy‐D‐glucose, an inhibitor of callose formation, was included in the tissue fixative. The study concludes that callose deposition in the Datisca nodule infected zone is apparently a stress response to tissue preparation and fixation. However, the rapidity and extent of callose deposition primarily at the symbiotic interface in Frankia‐infected cells suggests an unusual predisposition to biosynthesis of β‐1,3‐glucan in the nodule cortical cells that is related to their interaction with the microsymbiont.

Ten Nanometer Surface Intrusions in Room-Temperature Silicon

As gigascale integrated circuit devices approach nanometer dimensions, the bulk and surface structure of single crystal silicon is attracting finer scrutiny. In this context, recent searches for bulk defects 10 nm in size required that we consider more carefully some ubiquitous but weakly strained specimen preparation artifacts that for studies of larger bulk defects we had ignored. Transmission electron microscopy ~TEM! observations show that these imperfections, which form in variously oriented silicon surfaces after exposure to air, are strikingly similar after thinning with physically different methods. Moreover, the process of their formation can be slowed to laboratory time scales at room temperature if air exposure is sufficiently short. This allows in situ study of nucleation and growth, offering a view of dynamic processes in silicon which take place on laboratory time scales at room temperature, including the movement of self-interstitials and surface diffusion barrier formation. Both of these are subjects of widespread fundamental and applied interest. 1-4

Elemental composition and function of polyphosphates in ectomycorrhizal fungi — an X-ray microanalytical study

The elemental composition of vacuolar granules in different ectomycorrhizal fungi, Suillus bovinus, Paxillus involutus, Pisolithus tinctorius and Laccaria laccata was analysed by energy dispersive X-ray spectroscopy (EDXS) either after chemical preparation or cryofixation, freeze-drying and pressure infiltration. Vacuolar inclusions were present in living hyphae and were not an artifact of specimen preparation, and they can be referred to as polyphosphate granules. These granules were localized in fungal vacuoles and the main counter-ions were K and Mg. The postulated association of these granules with the divalent cation Ca has to be interpreted as an artifact of the specimen preparation and was caused by the chemical preparation of former EDXS studies. The incorporation of cations, such as K, Na and Zn, depended on the external supply and emphasized the importance of these granules for the intracellular homeostasis of fungal cells. EDXS studies of polyphosphate granules in ectomycorrhizal associations of Pinus sylvestris showed that the elemental composition of granules differed between the sheath and the Hartig net. In polyphosphate granules of the Hartig net a higher content of K was detectable, whereas the incorporation of Mg was reduced. These results indicated a possible role of K in the transfer of short chained, mobile polyphosphates through the hyphae to the Hartig net. The concentration of potassium and phosphate in the fungal vacuole and the cytoplasm was closely correlated, which can be explained by a possible linkage between the phosphate and K uptake, which would maintain the charge balance and the pH of the fungal cell. The estimation of different phosphate pools in axenic cultures and mycorrhizal roots revealed that the fungal metabolism was changed by the association with an ectomycorrhizal host plant. In a mycorrhizal association higher proportions of absorbed phosphate were translocated into the metabolically inactive polyphosphate pool, which demonstrates the importance of this pool for the nutrition of the ectomycorrhizal host plant.

A brief history of the lysigenous gland hypothesis

This paper summarizes the early history of the lysigenous gland concept, traces its evolution to recent times, and explores some possible causes of conflicting reports of lysigeny and schizogeny. Secretory cavities and ducts are generally thought to form either through the schizogenous separation of cells or through lysigeny (cell lysis). Gland lysigeny was first proposed in 1857 by Karsten, who believed that plant tissues represent solidifications of humoral fluids and that plant secretions are formed by the resorption of previously solidified cells. The lysigenous gland concept has modernized as our understanding of cytology has improved, but it was established early, from Karsten’s hypothesis, long before the influence of artifacts of specimen preparation was appreciated. Different methods of specimen preparation, including variations in the mounting media used to mount freehand sections, may have caused some of the discrepancies between the findings of lysigenists and schizogenists. Tschirch and Haberlandt promoted the schizolysigeny concept, and believed that the conflicting reports resulted from incomplete observations of a developmental process that included both schizogenous separation of cells and cell lysis to form secretory cavities and ducts. Both lysigeny and schizogeny have been reported in the recent literature, although most reports of lysigeny have been opposed by conflicting observations of schizogeny, and lysigeny may represent a false category of gland development caused by the misinterpretation of artifacts.

High Resolution X-Ray Microanalysis of Nb/Al Multilayer Thin Films

A study of reactive phase formation in thin films requires the understanding of the thermodynamics and kinetics of the reactions. Previous investigations of phase formation in Nb/Al multilayers using calorimetry and x-ray diffraction have observed that despite the presence of rather large driving forces for phase formation, a pronounced nucleation event occurs. This behavior has been reported in other systems including Ti/Al and Ni/Al. The initial nature of the interface can affect the nucleation, therefore careful studies of the structure and composition of the initial interfacial region may explain the reason for a kinetically separated nucleation and lateral growth step preceding normal phase growth. In addition, examination of the interface may also lend insight into the effects of different deposition processes and can provide evidence for the formation of solid solutions or compounds that can lower driving forces available for phase formation. Finally, the cross-sectional multilayer sample provides a means of characterizing spatial resolution and isolating specimen preparation artifacts.