Microscopy Sample Preparation Research Articles

Sample preparation for nanoanalytical electron microscopy using the FIB lift-out method and low energy ion milling

Thinning specimens to electron transparency for electron microscopy analysis can be done by conventional (2 - 4 kV) argon ion milling or focused ion beam (FIB) lift-out techniques. Both these methods tend to leave ‘‘mottling’’ visible on thin specimen areas, and this is believed to be surface damage caused by ion implantation and amorphisation. A low energy (250 - 500 V) Argon ion polish has been shown to greatly improve specimen quality for crystalline silicon samples. Here we investigate the preparation of technologically important materials for nanoanalysis using conventional and lift-out methods followed by a low energy polish in a GentleMill™ low energy ion mill. We use a low energy, low angle (6 - 8°) ion beam to remove the surface damage from previous processing steps. We assess this method for the preparation of technologically important materials, such as steel, silicon and GaAs. For these materials the ability to create specimens from specific sites, and to be able to image and analyse these specimens with the full resolution and sensitivity of the STEM, allows a significant increase of the power and flexibility of nanoanalytical electron microscopy.

Preparation of tissue samples for X-ray fluorescence microscopy

As is well-known, trace elements, especially metals, play an important role in the pathogenesis of many disorders. The topographic and quantitative elemental analysis of pathologically changed tissues may shed some new light on processes leading to the degeneration of cells in the case of selected diseases. An ideal and powerful tool for such purpose is the Synchrotron Microbeam X-ray Fluorescence technique. It enables the carrying out of investigations of the elemental composition of tissues even at the single cell level. The tissue samples for histopathological investigations are routinely fixed and embedded in paraffin. The authors try to verify the usefulness of such prepared tissue sections for elemental analysis with the use of X-ray fluorescence microscopy. Studies were performed on rat brain samples. Changes in elemental composition caused by fixation in formalin or paraformaldehyde and embedding in paraffin were examined. Measurements were carried out at the bending magnet beamline L of the Hamburger Synchrotronstrahlungslabor HASYLAB in Hamburg. The decrease in mass per unit area of K, Br and the increase in P, S, Fe, Cu and Zn in the tissue were observed as a result of the fixation. For the samples embedded in paraffin, a lower level of most elements was observed. Additionally, for these samples, changes in the composition of some elements were not uniform for different analyzed areas of rat brain.

Characterization of the magnetic properties of a GdBa2Cu3O7/La0.75Sr0.25MnO3 superlattice using off-axis electron holography

Abstract Off-axis electron holography is used to characterize the magnetic properties of a GdBa2Cu3O7/La0.75Sr0.25MnO3 superlattice below the Curie temperature of the manganite layers, in both cross-sectional and plan-view geometry. The samples were prepared for electron microscopy using focused ion beam milling. Differences between the magnetic properties of successive manganite layers are observed in the cross-sectional sample. Magnetic ripple contrast and weakly magnetic regions are observed in plan-view geometry. Although the results may be affected by sample preparation for electron microscopy, the observed differences between the magnetic properties of the manganite layers are consistent between the different samples examined.

Ultrastructural examination of dentin using focused ion-beam cross-sectioning and transmission electron microscopy

Focused ion-beam (FIB) milling is a commonly used technique for transmission electron microscopy (TEM) sample preparation of inorganic materials. In this study, we seek to evaluate the FIB as a TEM preparation tool for human dentin. Two particular problems involving dentin, a structural analog of bone that makes up the bulk of the human tooth, are examined. Firstly, the process of aging is studied through an investigation of the mineralization in ‘transparent’ dentin, which is formed naturally due to the filling up of dentinal tubules with large mineral crystals. Next, the process of fracture is examined to evaluate incipient events that occur at the collagen fiber level. For both these cases, FIB-milling was able to generate high-quality specimens that could be used for subsequent TEM examination. The changes in the mineralization suggested a simple mechanism of mineral ‘dissolution and reprecipitation’, while examination of the collagen revealed incipient damage in the form of voids within the collagen fibers. These studies help shed light on the process of aging and fracture of mineralized tissues and are useful steps in developing a framework for understanding such processes.

Apoptosis induction in DU-145 human prostate carcinoma cells

Due to the recent importance assumed by the apoptotic process as a tool in the therapeutic protocols, we investigated the onset and rate of drug-induced apoptosis in a cell model of prostatic tumor: the DU-145 cells. The human prostatic cancer cells, DU-145, have been treated with pro-apoptotic substances like puromycin (1 μg/ml for 4 and 6 h) and oxygen peroxide (1 mM for 1 h) or exposed to hyperthermic treatment (43 °C for 1 h followed by 2 h of recovery). The conventional techniques of preparation and observation of biological samples for electron scanning microscopy were used to estimate the sensitivity of these cells to the pro-apoptotic treatments and to establish their ability to undergo apoptosis. The ultrastructural changes occurring in the different phases of the phenomenon were characterized in parallel with the evaluation of the rate of apoptosis and in parallel with the evaluation of the expression levels of hsp-70 proteins.

Immunogold localisation of microcystins in cryosectioned cells of Microcystis

Insights into the origins, function(s), and fates of cyanobacterial toxins may be obtained by an understanding of their location within cyanobacterial cells. Here, we have localised microcystins in laboratory cultures of Microcystis PCC 7806 and PCC 7820 by immunogold labelling. Cryosectioning was used for immunoelectron microscopy since microcystins were extracted during the ethanol-based dehydration steps routinely used for sample preparation. Microcystins were specifically localised in the nucleoplasm and were associated with all major inclusions of the microcystin-producing strains Microcystis PCC 7806 (MC +) and Microcystis PCC 7820, and labelling was preferentially associated with the thylakoids and around polyphosphate bodies. A mutant strain of Microcystis PCC 7806 (MC −) which does not produce microcystins was used as a control. Distribution of total gold label within each cell region or associated with inclusions indicated that most of the cells’ microcystin pool was associated with the thylakoids (69%, PCC 7806 (MC +); 78%, PCC 7820), followed by the nucleoplasmic region (19%, PCC 7806 (MC +); 12%, PCC 7820). Cryosectioning is a useful technique since it reduces the extraction of microcystins during sample preparation for electron microscopy.

High Resolution Light Microscopy of Live Cells

Abstract All living creatures, including humans, are made of cells. The majority of life forms exist as single cells that perform all functions to continue independent life. Some cell structures, cell organelles and particularly bacteria and viruses are commonly too small to be fully observed with an optical microscope. Therefore, an electron microscope is required. Since samples examined with an electron microscope are exposed to very high vacuum, it is impossible to view living cells. The sample preparation for electron microscopy requires that living cells be killed, frozen, dehydrated, and impregnated with heavy metals.

Role of the Degree of Oligomerization in the Structure and Function of Human Surfactant Protein A

The role of the degree of oligomerization in the structure and function of human surfactant protein A (SP-A) was investigated using a human SP-A1 mutant (SP-A1(DeltaAVC,C6S)), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys(6) and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys(6) mutant lacked the NH(2)-terminal Ala(-3)-Val(-2)-Cys(-1) (DeltaAVC) extension present in some SP-A1 isoforms. SP-A1(DeltaAVC,C6S) was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1(DeltaAVC,C6S) was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated from human lung lavages. SP-A1(DeltaAVC,C6S) showed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin degradation. The T(m) was 32.7 degrees C for SP-A1(DeltaAVC,C6S) and 44.5 degrees C for SP-A1. Although SP-A1(DeltaAVC,C6S) was capable of binding to calcium, rough lipopolysaccharide, and phospholipid vesicles, this mutant was unable to induce rough lipopolysaccharide and phospholipid vesicle aggregation, to enhance the interfacial adsorption of SP-B/SP-C-surfactant membranes, and to undergo self-association in the presence of Ca(2+). On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1(DeltaAVC,C6S) to inhibit the production of tumor necrosis factor-alpha by macrophage-like U937 cells stimulated with either smooth or rough lipopolysaccharide. We conclude that supratrimeric assembly of human SP-A is essential for collagen triple helix stability at physiological temperatures, protection against proteases, protein self-association, and SP-A-induced ligand aggregation. The supratrimeric assembly is not essential for the binding of SP-A to ligands and anti-inflammatory effects of SP-A.

Automated Cross-Sectional Sample Preparation for Scanning Capacitance Microscopy

Abstract With the growing complexity of new processes and the introduction of new materials, the need for product yield management and process control is placing unprecedented demands on failure analysis laboratories in the semiconductor industry. These demands are calling for faster and superior analytical capabilities to determine root-cause failure mechanisms in semiconductor devices fabricated using deep submicron processes. This article presents a new automated sample preparation technique that facilitates direct electrical contact to the area of interest, with a surface quality sufficient for scanning probe microscope analysis.

Scanning/transmission electron microscopy and dual-beam sample preparation for the analysis of crystalline materials

Scanning electron microscopy, scanning transmission electron microscopy and transmission electron microscopy are powerful tools in the analysis of crystalline materials. In combination with their associated analytical techniques, they roughly cover three length scales: the ‘supra-nm’ scale, the ‘nm’ scale and the ‘sub-nm’ scale, respectively. In this paper, the investigation of crystalline properties is presented, by means of 2D and 3D characterization examples. Techniques such as channeling contrast, electron backscatter diffraction, crystal orientation contrast on thin films and convergent beam electron diffraction are covered. Furthermore, time-efficient and site-specific focused ion beam and dual-beam sample preparation for any of the techniques above are introduced.

Improvements in performance of focused ion beam cross-sectioning: aspects of ion-sample interaction

A gallium (Ga) focused ion beam (FIB) has been applied increasingly to 'site-specific' preparation of cross-sectional samples for transmission electron microscopy (TEM), scanning TEM, scanning electron microscopy and scanning ion microscopy. It is absolutely required for FIB cross-sectioning to prepare higher-quality samples in a shorter time without sacrificing the site specificity. The present paper clarifies the parameters that impose limitation on the following performances of the FIB cross-sectioning: milling rate, cross-sectioning at a right angle with respect to the sample surface, curtain structures formed on the cross sections, ion implantation and ion damage. All of these are discussed from the viewpoint of ion-sample interaction. Improvements for these performances achieved by diminishing their limiting origins or by correcting the resultants are described. Especially, the FIB scanning speed is significantly utilizable to improve the milling rate. A microsampling method, which allows the FIB incidence in a sidewards or upwards direction as well as downwards with respect to the microsample surface, is very effective to minimize the curtain structures.

Ni Nanoparticles Elaborated with an Ar Ion Polishing Technique

AbstractNi nanoparticles where obtained from a Ni sample milled with Ar ions by using a Gatan precision ion polishing system, normally used for Transmission Electron Microscopy (TEM) sample preparation. Deposition of Ni nanoparticles was performed over two different surfaces: on a double sided carbon tape and, on a Cu grid covered with collodion film. A continuos film of Ni was characterized, over the carbon tape by SEM and EDAX techniques. The last surface was analysed by TEM. In both cases, a thin film composed of Ni nanoparticles, was founded and result obtained by TEM, show a nanoparticle diameter of about 4 nm.

Transmission electron microscopy sample preparation technique for sintered alloys

Powder metallurgy alloys are typically inhomogeneous with a significant amount of porosity. This complicates conventional transmission electron microscopy sample preparation. However, the use of focused ion beam milling allows site specific transmission electron microscopy samples to be prepared in a short amount of time. This paper presents a method that can be used to produce transmission electron microscopy samples from an Al-Cu-Mg PM alloy.

Improved preparation of chick embryonic samples for magnetic resonance microscopy.

Previous work demonstrated the power of three-dimensional (3D) magnetic resonance microscopy (MRM) to follow complicated morphologic development in the embryonic cardiovascular system. In this study we describe a new dual-contrast method for specimen preparation that combines perfusion fixation and immersion in fixative with macro- and small molecular gadolinium agents to provide enhanced definition of both the heart wall and chamber. MRM was performed at 9.4 T with image resolutions of 25, 31, and 50 microm isotropic voxels for three stages of chick embryos (day 4, day 5.5, and day 9), and compared to histological sections of the same embryos. The results show considerable improvement of image quality over previous efforts, with better signal-to-noise ratio (SNR) and contrast between the cardiac chamber and myocardial wall. Excellent correlation was shown between the MRM images and histological sections. Thus, 3D high-resolution MRM in combination with the dual-contrast technique is useful for acquiring quantitative 3D morphologic data regarding heart development.

Multidisciplinary utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects

DMSO is an amphipathic molecule with a highly polar domain and two apolar methyl groups, making it soluble in both aqueous and organic media. It is one of the most common solvents for the in vivo administration of several water-insoluble substances. Despite being frequently used as a solvent in biological studies and as a vehicle for drug therapy, the side-effects of DMSO (undesirable for these purposes) are apparent from its utilization in the laboratory (both in vivo and in vitro) and in clinical settings. DMSO is a hydrogen-bound disrupter, cell-differentiating agent, hydroxyl radical scavenger, intercellular electrical uncoupler, intracellular low-density lipoprotein-derived cholesterol mobilizing agent, cryoprotectant, solubilizing agent used in sample preparation for electron microscopy, antidote to the extravasation of vesicant anticancer agents, and topical analgesic. Additionally, it is used in the treatment of brain edema, amyloidosis, interstitial cystitis, and schizophrenia. Several systemic side-effects from the use of DMSO have been reported, namely nausea, vomiting, diarrhea, hemolysis, rashes, renal failure, hypertension, bradycardia, heart block, pulmonary edema, cardiac arrest, and bronchospasm. Looking at the multitude of effects of DMSO brought to light by these studies, it is easily understood how many researchers working with DMSO (or studying one of its specific effects) might not be fully aware of the experiences of other groups who are working with it but in a different context.

Development of a High Lateral Resolution Electron Beam Induced Current Technique for Electrical Characterization of InGaN-Based Quantum Well Light Emitting Diodes

ABSTRACTElectron Beam Induced Current (EBIC) is a Scanning Electron Microscope (SEM)-based technique that can provide information on the electrical properties of semiconductor materials and devices. This work focuses on the design and implemenation of an EBIC system in a dedicated Scanning Transmission Electron Microscope (STEM). The STEM-EBIC technique was used in the characterization of an Indium Gallium Nitride (InGaN) quantum well Light Emitting Diode (LED). The conventional “H-bar” Transmission Electron Microscopy (TEM) sample preparation method using Focused Ion Beam Micromachining (FIBM) was adapted to create an electron-transparent membrane approximately 300 nm thick on the sample while preserving the electrical activity of the device. A STEM-EBIC sample holder with two insulated electrical feedthroughs making contact to the thinned LED was designed and custom made for these experiments. The simultaneous collection of Z-contrast images, EBIC images, and In and Al elemental images allowed for the determination of the p-n junction location, AlGaN and GaN barrier layers, and the thin InGaN quantum well layer within the device. The relative position of the p-n junction with respect to the thin InGaN quantum well was found to be (19 ± 3) nm from the center of the InGaN quantum well.

Technical advance: an automated device for cryofixation of specimens of electron microscopy using liquid helium.

Metal-contact rapid freezing using liquid helium is theoretically the best method for preserving the fine structure of living cells with high temporal resolution in preparation of tissue samples for electron microscopy. However, this method is not commonly used, because of its technical difficulty and low reproducibility. We have designed and constructed an automatic device which allows simple, rapid and reproducible preparation of high-quality electron microscopic specimens by the non-specialist. We assessed the quality of cryofixation in samples prepared using this device by examining the preservation of cellular ultrastructure in relation to distance from the freezing block, and found that the region within 10 microm of the metal-contact plane was fixed with the highest quality. We applied this device, in combination with freeze-substitution methods and immunocytochemical techniques, to two phenomena involving rapid movement of subcellular components: (1) active movement of subcellular structures in the papillar cells of stigma and (2) light-induced rapid subcellular translocation of phytochrome A. Considering the importance of understanding subcellular processes of living cells for molecular and cell biology, this device will be a useful tool for diverse biological applications in the near future.

As-deposited mixed zone in thermally grown oxide beneath a thermal barrier coating

Gas turbine designers are increasingly using electron-beam physical vapor deposited (EB-PVD) thermal barrier coatings (TBC) to meet the challenge of higher efficiency gas turbine engine requirements. A key feature for expanding the use of TBCs is increased spallation life and reduced spallation life variability. Such a coating system comprises a substrate (Ni-based single crystal alloy), a bond coat (diffusion aluminide or MCrAlY), a ceramic (7 wt.% yttria stabilized zirconia), and a thin thermally grown oxide (TGO) between the bond coat and the ceramic. The TGO is intended to be α-alumina, but evidence reported by other researchers suggests that in some cases the as-deposited TGO may not be entirely α-alumina. The thin nature of the TGO in as-deposited TBCs (<0.5 μm) makes analysis of the phases present and morphology difficult. Advancements in transmission electron microscopy (TEM) sample preparation and photo-stimulated luminescence spectroscopy (PSLS) have allowed higher quality and easier characterization of the TGO. In this study, EB-PVD TBCs were applied to platinum–aluminide bond coats on a Ni-based superalloy. Three types of coatings were produced by changing one PVD process variable. The as-processed TGO layer was characterized utilizing scanning transmission electron microscopy (STEM) and PSLS for each of the three coating process variables used. Results of this work found that the TGO comprised two sublayers; (1) a continuous layer of γ-Al 2O 3 between the mixed oxide zone and the bond coat; and (2) a mixed oxide zone between the continuous γ-Al 2O 3 and the TBC layer. An explanation for the creation of the mixed oxide zone found in these TGO morphologies is presented.

Applications of atomic force microscopy/scanning capacitance microscopy in imaging implant structures of semiconductor devices

Atomic force microscopy [G. Binnig et al., Phys. Rev. Lett. 56, 930 (1986)] and scanning capacitance microscopy [C. C. Williams et al., Appl. Phys. Lett. 55, 203 (1989)] (AFM/SCM) have been widely used to investigate the two-dimensional carrier profile of semiconductor devices. In this work, AFM/SCM was applied to both process control and failure analysis. Several different cases were studied. First, a commercially available integrated circuit device was cross sectioned and polished for the AFM/SCM investigation using standard scanning electron microscopy sample preparation techniques. Implant structures near the gate were clearly resolved. Second, a GaAs device with Zn thermally diffused through the opening of a Si3N4 mask was studied. The lateral diffusion length of Zn was determined. Third, two semiconductor devices, one good and one that had failed, were prepared by cross sectioning, followed by polishing. Implant profiles of similar structures on both devices were imaged. The thickness of the N-well structure was found to be thinner in the failed device by about 0.4 μm. These cases demonstrate applications of AFM/SCM in process control and device failure analysis.

Surface damage formation during ion-beam thinning of samples for transmission electron microscopy

All techniques employed in the preparation of samples for transmission electron microscopy (TEM) introduce or include artifacts that can degrade the images of the materials being studied. One significant cause of this image degradation is surface amorphization. The damaged top and bottom surface layers of TEM samples can obscure subtle detail, particularly at high magnification. Of the techniques typically used for TEM sample preparation of semiconducting materials, cleaving produces samples with the least surface amorphization, followed by low-angle ion milling, conventional ion milling, and focused ion beam (FIB) preparation. In this work, we present direct measurements of surface damage on silicon produced during TEM sample preparation utilizing these techniques. The thinnest damaged layer formed on a silicon surface was measured as 1.5 nm thick, while an optimized FIB sample preparation process results in the formation of a 22 nm thick damaged layer. Lattice images are obtainable from all samples.