Specimen Preparation Procedures Research Articles

Atom probe analysis of planar multilayer structures

Atom probe field ion microscopy has been used to analyze a planar-deposited layered structure in plan view. The specimens were prepared with a newly developed method that involves a combination of photolithography and focused ion-beam milling. A multilayer structure consisting of {Ta/CoFe/(Cu/CoFe)15/Ru/(CoFe/Ru)5/Ru/NiFe} was sputter deposited for use as a test stack. The corresponding thicknesses of these layers were 7/13(3/3)/50/(3/1)/50/150 nm. The nanometer-scale periodicity of the Cu/CoFe stack is readily apparent in transmission electron microscopy images of a field ion specimen fabricated from this material, suggesting that the specimen preparation procedure does not lead to destruction of the multilayer structure. Atom probe analysis of the bulk NiFe layer and the Ru/NiFe interface revealed the distribution of impurity atoms in the film, and these may affect the magnetic properties of the multilayers. Whereas a uniform distribution of C, N and Ar was observed, segregation of O was observed in the NiFe layer within ∼0.25 nm of the interphase interface, with a concentration greater than 20 times that found in the bulk of the NiFe layer.

Optimization of FIB-Milling Technique for Preparation of TEM Specimens from Copper/Low-K Materials

Focused ion beam (FIB) thinning of materials to electron transparency is now a routine procedure for preparation of specimens for transmission electron microscopy (TEM) of microelectronic materials and devices. The nano-scale structural damage, including implantation and amorphization due to this ion milling process has been well investigated and documented. In this paper, we discuss the micro-scale structural damage observed in copper/low-k materials and our efforts to minimize the extent of the damage without compromising the overall specimen preparation time.Figure 1 shows an area-specific cross-sectional specimen prepared from a copper/low-k via-chain test structure using the FIB-milling technique. The procedure involved mechanical thinning of a transverse wafer sliver followed by FIB-milling the area of interest to electron transparency according to conventional steps and conditions' using a liquid Ga+ ion source FIB system. The evidence of structural damage in terms of melting and/or sputtering of the metallization is visible at different areas.

A Versatile High-Vacuum Cryo-Transfer for Cryo-FESEM, Cryo-SPM and other Imaging Techniques

A structure preservation of biological and organic samples, close to native state, can only be reached by cryo immobilization techniques. Cryo immobilization allows not only to preserve the high structural integrity but also to arrest dynamic processes in the μs- ms range.After freezing the sample and preparing the surface of interest, it is important to prevent the sample from ice crystal damage, removal of structural water, condensation of water or other contaminants until imaging. Therefore, ideally the samples are kept below the recrystallisation temperature of water (< 147K) during the transfer from the preparation environment into the imaging chamber.For the transfer of frozen samples several concepts have been followed in the,past: a) the specimen after manipulation/preparation were submersed in liquid nitrogen and transferred to the cold stage of the microscope or b) a preparation chamber was permanently attached to the microscope column allowing the direct transfers between the preparation chamber and the cold stage in the microscope. These concepts allow either a high grade of flexibility combined with a high risk of contamination or to prevent contamination but combined with inflexibility. In addition the later also does not allow using the microscope during the specimen preparation procedure, nor transferring the specimen to an other imaging device.

Preservation of Biomembranes by High Pressure Freezing ?

Abstract Sample preparation techniques for electron-microscopy (EM) dictate our perception of the microworld: any structural information which is lost or distorted during preparation can not be regenerated later and might lead to wrong interpretation of the observed micrograph. Cryofixation based procedures for specimen preparation can avoid most of the structural alterations associated with standard techniques based on chemical fixation. The ultrastructure can be represented in a near “native state” thanks to the high time resolution for dynamic cellular events (1). High pressure freezing (2) permits to cryoimmobilize biological samples up to approx. 200μm thick, in contrast to rapid freezing procedures at ambient pressure that are useful to cryoimmobilize samples up to 10-20 μm thick. The actual samplethickness that can be adequately frozen (=without visible damage due to ice crystal formation in freeze-substituted or freeze-fractured specimens) depends on the concentration of naturally occuring substances that exhibit cryoprotective activities.

5] Mounting techniques for confocal microscopy

The chapter describes mounting methods for confocal microscopy. The quality of images taken by the confocal microscope is dependent on the specimen preparation procedure, such as fixation, embedding, cutting, staining, and mounting. Fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC) are the most commonly used fluorochromes in immunohistochemistry and in situ hybridization. By using a confocal microscope, a series of optical sections can be produced from a thick specimen labeled with fluorochromes. The number of optical sections collected is dependent on the characteristics of the specimens and is a function of objective lenses. A confocal microscope is usually equipped with oil immersion-type objective lenses for maximum resolution. Because the working distance of these lenses is very short (0.14–0.16 mm), the thickness of the coverslip impedes the observation of deeper parts of thick sections. The chapter describes methods for fixation, thin sections, thick sections cut with microslicer, whole mount specimens and thick sections cut with saw microtome, and confocal microscopy.

STUDY OF METALLIC ELEMENT DISTRIBUTION IN CANCELLOUS BONE OF FEMORAL HEAD BOTH YOUTH AND OLD PEOPLE BY PIXE

Proton Induced X-ray Emission (PIXE) method is used for the determination of elemental concentration in a cancellous bone of human femoral head from five autopsies and seven patients with femoral neck broken. Specimen preparation and experimental procedure are described in detail. Using t test, the results show that the concentrations of P , Ca , Fe , Cu , Sr in a control group are higher than those in a patient group, but the concentrations of S , K , Zn , Mn are not significantly different. The physiological functions of metal elements in human bone are also discussed.

Ultrasensitive reverse transcription-PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma.

With the recent introduction of combination therapy, human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma have been dramatically reduced, frequently to below the limit of quantitation (400 copies/ml of plasma) of the AMPLICOR HIV-1 MONITOR Test (Roche Diagnostic Systems). To achieve enhanced sensitivity of the AMPLICOR HIV-1 MONITOR Test, a modified specimen preparation procedure that allows input of RNA from 10-fold more plasma per amplification reaction was developed. This "ultrasensitive" method allows the accurate quantitation of plasma HIV-1 RNA levels as low as 50 copies/ml. A precision study yielded average within-run and between-run coefficients of variation (CV) of 24.8 and 9.6%, respectively. A multicenter reproducibility study demonstrated that the laboratory-to-laboratory reproducibility of this assay is good, with an average CV of 32%. The linear range of this test is between 50 and 50,000 copies/ml of plasma. RNA concentrations measured by the ultrasensitive and standard HIV-1 MONITOR tests exhibited good agreement within the shared linear range of the two methods. The two measurements were within a factor of 2 for 91% of the specimens tested, with the concentration measured by the ultrasensitive method being only slightly lower (median, 22% lower). Preliminary studies suggest that this assay will prove to be useful for predicting the stability of viral suppression in patients whose RNA levels drop below 400 copies/ml in response to highly active antiretroviral therapy.

Elemental contents in vacuolar granules of ectomycorrhizal fungi measured by EELS and EDXS. A comparison of different methods and preparation techniques

The elemental composition of vacuolar granules in different ectomycorrhizal fungi, Pisolithus tinctorius, Suillus bovinus and Xerocomus badius, were determined by electron energy loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDXS). The investigations dealt with advantages and limits of the ECXS and EELS technique with respect to the analysis of the elemental composition of vacuolar granules and the effect of different specimen preparation techniques. Axenic cultures of these fungi as well as field mycorrhizae were used for the analysis. Results after conventional chemical fixation and dehydration of material were compared to results obtained after cryofixation followed by freeze-drying of the samples. Light microscopical studies were also carried out to control the occurrence of vacuolar granules in living hyphae. The results showed that vacuolar granules exist in living hyphae of different ectomycorrhizal fungi and are not an artifact of the fixation or other specimen preparation procedures of cells. EDXS and EELS differed in their ability to detect the elemental composition of these granules. Both analytical techniques found P in the vacuolar bodies, which indicates a deposition of polyphosphates. Polyphosphate granules are strongly negative polyanions, which contain different cations to balance the negative charge. These cations were often difficult to determine by EELS and could only be shown by EDXS, but the cations varied considerably depending on the technique used for specimen preparation. In chemically fixed and dehydrated material, especially Mg, K and Ca were detected in the granules. However, measurements of cryofixed and freeze-dried specimens showed that the most abundant cations in polyphosphate granules were K and Mg and the incorporation of Ca has to be interpreted as a result of the chemical specimen preparation. EELS revealed that N was also incorporated in the vacuolar granules independently of the specimen preparation. Based on these results, it can be assumed (1) that the N-containing granules, detected by EELS, and the P-rich granules, analysed by EDXS, were identical; (2) that these vacuolar inclusions were polyphosphate granules or metachromatic granules, which were often described in different mycorrhizal associations; and (3) that polyphosphate granules can also act as a binding site for large amounts of N.

On the spurious endoproteolytic processing of the presenilin proteins in cultured cells and tissues.

It has been widely reported that the presenilin proteins PS-1 and PS-2 in extracts derived from a variety of cultured cells and from tissues are fragmented extensively by endoproteolytic processing events. It generally has been presumed that this endoproteolysis is a physiologically normal intracellular event following presenilin expression, which might play an important role in the still unknown functions of these molecules in connection with Alzheimer disease. We demonstrate herein, however, that, if a variety of cultured cells and several mouse tissues are examined under conditions minimizing cell trauma, the presenilin molecules in the extracts are found to be intact but that, if the cells and tissues are prepared under somewhat more stressful conditions, the endoproteolytic fragments are then observed. We conclude that these particular endoproteolytic events are not the result of physiologically normal processing of the presenilins but are rather artifacts occurring during the common procedures of specimen preparation.

Preparation, Transmission Electron Microscopy, and Microanalytical Investigations of Metal-III-V Semiconductor Interfaces

Ohmic metallizations on semiconductors such as In0.53Ga0.47As and GaAs have been analyzed by cross-sectional transmission electron microscopy. To obtain a lattice image of multilayer structures in the high-resolution mode, a cross-sectional specimen preparation procedure was developed. The phase formation after thermal treatment of the ohmic contacts has been studied by applying different microanalytical techniques. The results have been utilized for improving the metallurgical stability and the electrical properties of the contacts.

Electron Backscattered Diffraction (EBSD) with a Cold Held Emission Gun (cFEG) SEM: Resolution, Sensitivity and Applications

A thorough and complete assessment of microstructure of materials requires a wide variety of analytical techniques, which should be sensitive to at all length scales - from mms to atomic scale. While there have been rapid advances in imaging and spectroscopy techniques for microstructural analysis - at all length scales, techniques for crystallographic analysis of microstructure have primarily relied on bulk x-ray/neutron diffraction and TEM. X-ray and neutron diffraction techniques, though very powerful, are primarily bulk techniques and extraction of local crystallography is formidable if not impossible. On the other hand, TEM diffraction techniques provide precise crystallographic information, but at a much smaller length-scale and suffer from poor statistics and tedious specimen preparation procedures. With the advent of commercially available electron backscattered diffraction (EBSD) and orientational imaging (OIM) systems for SEM, and sophisticated pattern recognition procedures, it is now possible to bridge the length-scale gap between bulk and TEM diffraction techniques.

Methodology and apparatus to determine material properties of the knee joint meniscus

This paper describes a soft tissue test method and apparatus which determines the constants necessary to characterise the elastic properties of a transversely isotropic material such as the knee joint meniscus. The tensile machine was designed to test small delicate samples in a humidified environment and measure specimen deformation with a CCD video camera. The method described here employs a common specimen preparation procedure and test protocol using specimens with four different fibre orientations. Discrete markers (0.075 mm graphite particles) permit repeatable measurements of two-dimensional strain on the specimen surface enabling the determination of Poisson's ratio characteristics. Shear strain was also measured in tensile test specimens prepared with an oblique collagen fibre orientation, offering an alternative method to investigate shear properties. Regional strain measurements provide an assessment of the uniformity of the tissue deformation. Preliminary results on bovine menisci are presented as an application of the test system. Material properties characterising the meniscus tissue were obtained with one test procedure and identical coupon size. This has the advantage of avoiding systematic errors caused by different preparation techniques, test procedures and equipment.

Comparison of Laboratory-Measured GCL Hydraulic Conductivity Based on Three Permeameter Types

Abstract Specimen preparation, installation, and testing procedures are presented for testing a needle-punched geosynthetic clay liner (GCL) in a consolidation-type, fixed-ring constant-flow-rate hydraulic conductivity apparatus. The proposed approach is illustrated by reporting the results obtained for a GCL statically confined to effective stresses of ∼3 and ∼35 kPa, hydrated and permeated with distilled water and concentrated aqueous salt solutions (0.6 and 2.0 N NaCl), and sequentially permeated with concentrated aqueous salt solutions (0.6 and 2.0 N NaCl) after initial permeation with distilled water. The issue of potential preferential sidewall flow in the fixed-ring permeameter tests was then addressed by comparing results with those conducted under otherwise similar testing conditions in a double-ring and flexible-wall permeameter. Reasonable reproducibility of the test results was obtained between the different types of permeameters, suggesting that, if present, preferential sidewall flow was negligibly small and was not significant on the calculated hydraulic conductivity values. This conclusion was found to be valid for the range of permeant types (distilled water and 0.6 and 2.0 N NaCl), static-confining stresses, and hydrating mediums considered. Premature arrival of the salt solution front in the effluent (i.e., solute breakthrough) during fixed-ring hydraulic conductivity testing was explained as a probable diffusion-dominated transport mechanism through the relatively thin GCL specimens.

Preparation of multilayered materials in cross-section for in situ TEM tensile deformation studies.

The success of in situ transmission electron microscopy experimentation is often dictated by proper specimen preparation and sample design procedures. We have developed a novel technique permitting the production of tensile specimens of multilayered films in cross-section for in situ deformation studies. Of primary, importance in the development of this technique is the production of an electron transparent micro-gauge section. This micro-gauge section predetermines the position at which plastic deformation, crack nucleation and growth, and failure are observed. In short, we report in detail, a unique combination of specimen preparation procedural steps and the design of a multilayer foil sample. The ability of these procedures to facilitate the success of in situ TEM tensile studies of layered materials in cross-section is demonstrated using a Cu-Zr multilayer foil.

Influence of Field Variables on Laboratory Performance of Silane Treated Concrete

Differences in performance of silane treated concrete are sometimes noted between laboratory tests and field observations. Laboratory tests are usually conducted using a standard reference concrete with specific specimen preparation procedures that may not be representative of field conditions. This research examined the effects of concrete mix design, concrete surface finish, and curing and cleaning procedures on the performance of silane treated concrete using laboratory tests. Results indicate that to obtain better prediction of performance, laboratory tests should be conducted using the concrete mix design and surface finish anticipated for the field application.

Using an Automated Rowe Cell for Constant Rate of Strain Consolidation Testing

Abstract The paper describes the automation and calibration of a hydraulic Rowe consolidation cell that tests 151-mm-diameter by 60-mm-high specimens. The associated measurement instrumentation for the cell and specimen preparation procedures are also described. The cell was used to perform a set of one-dimensional constant rate of strain (CRS) tests at two vertical strain rates, 0.1 and 1%/h. The nonlinear CRS theory of Wissa et al. (1971) is presented, including methods for interpreting test results at higher strain rates. The results from the Rowe cell tests conducted on resedimented Boston Blue Clay are compared to those from more conventional incremental loading (IL) oedometer tests (63.5-mm-diameter by 23-mm-high specimens). For both the 0.1%/h tests (in which negligible excess pore pressure was generated at the undrained specimen base) and the 1%/h tests, the compression curves compare well with the baseline IL oedometer test curves. Values of preconsolidation pressure, σ′p, averaged 94.0 kPa in the Rowe tests and 94.5 kPa in the IL oedometer tests. The compression ratios, CR = Δεv/Δ log σ′v, in the virgin compression range were slightly lower in the Rowe cell tests (average 0.156 versus an average CR = 0.173 in the IL oedometer tests), which may be due to different testing conditions (e.g., back pressure in the Rowe cell) or the larger Rowe cell specimen. The coefficient of consolidation and hydraulic conductivity values computed from the Rowe cell tests are essentially identical with regard to stress level to the IL oedometer tests. The automated Rowe cell data indicate that the device offers a reliable and potentially rapid method for determining consolidation parameters, including hydraulic conductivity.

Influence of the confinement, soil density and displacement rate on soil-geogrid interaction

The testing equipment, specimen preparation and testing procedures are described for a series of pull-out tests on geogrids in granular soil. The interaction between a well graded very gravelly sand and a uniaxial geogrid made of high density polyethylene is studied in a pull-out box with dimensions of 1.53 m length, 1·00m width and 0·80m height. The influence of the confinement pressure, soil density and displacement rate on the pull-out resistance of the geogrid is discussed by analysing the results of the pull-out tests.

Preparation, transmission electron microscopy, and microanalytical investigations of metal-III-V semiconductor interfaces

Ohmic metallizations on semiconductors such as In0.53Ga0.47As and GaAs have been analyzed by cross-sectional transmission electron microscopy. To obtain a lattice image of multilayer structures in the high-resolution mode, a cross-sectional specimen preparation procedure was developed. The phase formation after thermal treatment of the ohmic contacts has been studied by applying different microanalytical techniques. The results have been utilized for improving the metallurgical stability and the electrical properties of the contacts.

In vivo calcineurin crystals formed using the baculovirus expression system.

Calcineurin is a heterodimeric phosphatase involved in the signal transduction of antigen-activated T cells. Coexpression of its two subunits, the regulatory subunit from human and the catalytic subunit from Neurospora crassa in cultured insect cells using the baculovirus expression system results in the formation of very large crystals in the cytoplasm. The crystals are formed initially in vesicles, but their subsequent growth appears to be uninhibited and continues without the need of an enclosing membrane until the host cell lyses. Although these in vivo crystals are low in population, ranging only 0-3 per cell, they are extremely large, over 10 mu m in some cases. Biochemical assays confirm their calcineurin origin, with the regulatory subunit incorporated being myristoylated, although both the myristoylated and unmyristoylated forms are expressed. The lattice structure of the in vivo crystals, with a spacing of 5.5 nm, is preserved with the regular electron microscopic (EM) specimen preparation procedure.

Multi-element determination in cancellous bone of human femoral head by PIXE

Proton Induced X-ray Emission (PIXE) method is used for the determination of elemental concentrations in cancellous bone of human femoral head from five autopsies and seven patients with femoral neck broken. The specimen preparation and experimental procedure are described in detail. Using the t test, the results show that the concentrations of P, Ca, Fe, Cu, Sr in control group are higher than those in patient group, but the concentrations of S, K, Zn, Mn are not significantly different. The physiological functions of metallic elements in human bone are also discussed.