The class B, type I scavenger receptor (SR-BI) was the first molecularly well defined cell surface receptor which binds HDL and mediates the selective uptake of HDL cholesteryl ester. It is expressed primarily in liver and steroidogenic tissues. However, the recent studies also suggested a potentially important role of SR-BI in the initial steps of cholesterol efflux in the peripheral tissues. In this study, we have used immunofluorescence and immunoelectron microscopy to study the expression and subcellular localization of SR-BI in the endothelial cells and the smooth muscle cells of rat aorta in vitro and in vivo.

Reactive oxygen species (ROS) play an important role in lupus nephritis. It has been known that nuclear factor kB(NFkB) is activated by ROS, and binds to KB element in nucleus. Afterwards, inflammatory cytokines are translated and produced in lymphocytes, macrophages and glomerular cells in the kidney. In this study, we investigated the role of ROS and NFkB p65 in the pathogenesis of lupus nephritis (MRL lpr/lpr mice) by the histochemical and immuno-electron microscopic methods.

A modified Brigg’s method was used to determine the generation of H2O2 in the renal tissue of lupus mice. Tissue samples were incubate in 0.1 MTris maleate buffer (pH 7.4) with 7% sucrose, 1mM CeCl3 and 10 mM aminotriazole at 37 °C for 30 min. After completion of the cytochemical reaction, tissues were fixed in 2% w/v glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4 °C for 60 min.

The mechanism of myocardial injury in myocarditis is still unclear. Recently, nitric oxide (NO), massively produced in the inflammation, has been suggested to be harmful for tissue at excess amount as well as superoxide. However, it is still unclear the relationship between production of NO with superoxide and myocardial damage in myocarditis. In this study, we investigated whether NO and/or superoxide play an important role in the pathogenesis of myocardial injury in myocarditis.

The heart tissue specimens were taken from Lewis rats with experimentally induced myocarditis. Immunohistochemical staining was performed using polyclonal antibody for inducible NO synthase by avidin-biotin peroxidase complex method in paraffin sections of the tissue. Histochemical study for ultrastructural H2O2-producing site in the heart tissue was carried out by Brigg’ s method using CeCl3; briefly, the heart tissues were incubated in 0.1M Tris-malate buffer with ImM CeCl3 and 10mM aminotriazole. Then tissues were fixed in 2% glutaraldehyde, postfixed with 1% OsO4 and embedded in resin for electron microscopy.

Renewed interest in textile blends of wool and cotton has led to developments in union dyeing (same shade on both wool and cotton fibers) that has potential for increased markets for both fibers as trans-seasonal apparel. The difficulty of efficiently dyeing wool and cotton to the same shade has limited the availability of yarns and fabrics containing blends of both fibers. In fabrics containing blends of cellulosic cotton and proteinaceous wool, it is generally necessary to employ more than one class of dyes and the process involves several steps with adjustments of pH and temperature. The ARS process provides maximum effectiveness and efficiency for one-step union dyeing. It requires pretreatment before dyeing to impart cationic character to cellulosic cotton so that it can compete with protonated wool for a common anionic dye. Three pre-treatments have been applied.

Due to the decreasing size of components in the semiconductor industry, TEM sample preparation grows increasingly difficult. And at the same time the customer need for TEM analysis increases, along with the urgency to report the data. We have found that TEM sample preparation has been greatly improved using the combination of the Allied High Tech MultiPrep™ System (Figure 1) in conjunction with the Gatan Broad Beam Guns™ with the Graphite Holder and accessories. The technique discussed in this paper will show that this procedure for sample preparation has decreased the time it takes to prepare the sample, decreased the number of failing samples and increased the ability to do high resolution imaging.

The MultiPrep™ is used when repeatability is critical for sample preparation and eliminates the function of holding the polisher by hand. When polishing by hand the amount of force placed on the sample while polishing is inconsistent.

As the semiconductor industry paces toward 0.15-micron feature size and below, the visibility of structures becomes a challenge. As a result, the use of higher precision metrology equipment such as FESEM, STEM, TEM and SCM becomes a necessity. TEM analysis today is a less common methodology of analysis. This is due to the time and effort required for the analysis as well as the difficulty of the sample preparation. This publication will share the experience gained in sample preparation for challenging applications such as STEM, TEM and SCM. The main driver will be the focus on short turnover time and high success rates.

In order to perform high-resolution analysis, samples should be transparent to electrons. Optimal thickness would be in the range of 20 to 120 nm (1). Pre-preparation of sample for advanced imaging will determine the final step method required. We used the automated polishing technique for sample preparation and reached 1 to 2 micron thickness.

Cross sectional SEM specimens are routinely prepared for process monitoring/development, fabrication problem solving and failure analysis in semiconductor industries. A considerable variety of established methods are already in place for production of these specimens. They include: mechanical polishing, manual/automated cleaving, wet chemical etching and focused ion beam (FIB) milling. This report covers a new approach to the preparation of such specimens utilizing a broad ion beam technique. The technique consists of three steps, I) perpendicular “slope cutting” to expose the profile of the layers/features in the starting wafer, II) etching of the cut surface to delineate the microstructures and III) coating the specimen with a conductive thin film to prevent charging effects in the SEM. All three steps are carried out in a dedicated broad ion beam instrument, designed initially to etch and coat SEM specimens in one vacuum chamber [1]. The entire three-step process can be completed in one hour.

This technique is used to prepare cross sections of semiconductor devices in small samples for analysis by Transmission Electron Microscopy (TEM). These small samples, measuring 100 X 100 X 50 microns, are too small for the manual handling involved in routine mechanical cross sectioning methods. A larger sample, for easier manual handling, is made by gluing the original small sample between a larger piece of silicon and a larger piece of dimpled quartz. The dimpled depression in the quartz is just large enough to surround the original sample. The sample is then mechanically thinned down using a Tripod polisher and the wedge technique. The quartz piece, glued to the top of the original sample, allows the progress of the polish to be monitored as the first side of the cross section is being mechanically polished. The silicon piece, glued to the bottom of the original sample, is used to gauge the final thickness of the wedge produced when polishing the second side of the cross section using the wedge technique.

As device features shrink below sub-0.2 micron and the time from development to market reduces, the demand for using focused ion beam (FIB) to prepare TEM samples has increased tremendously over the last two years in semiconductor industry. There are many ways to prepare a TEM sample prior to the FIB one of them involves with a use of dicing saw. In our lab, about 80% of the samples were prepared using the dicing saw technique. Average time to cut out one sample is less than 20 minutes.

The dicing saw we used is a Disco model DAD321 equipped with a 250X optical and ccd camera viewing monitor system. To minimize chipping, a 200 um-thick diamond resin composite blade is used. A spindle rotation speed at 30,000 rpm and a stage moving speed at 100 mm/s is set for minimizing blade ware and sample chipping. The parameters for each cut are pre-programmed, so there is no setup required.

Nanometer scale, high resolution Ga+ ion probes, attainable in commercially available focused ion beam (FIB) instruments, allow imaging, sputtering and deposition operations to be performed with a high degree of spatial precision. Of particular interest is how this precision milling/deposition capability has enabled a wide range of site specific micromachining and microfabrication operations (e.g., TEM, SEM, SIMS, and AUGER specimen preparation and circuit modification). The applications of FIB instruments frequently involve the creation of high aspect ratio features (i.e., deep narrow trenches). Ideally, the sidewalls of an FIB milled feature should be vertical; however, it has been generally observed that the trenches tend to exhibit a gradual sloping. The observed deviation from vertical milling has been attributed to the redeposition of sputtered material, and is especially pervasive at high beam currents and confining trench geometries. A hole milled with an FIB tends to be widest at the top surface and taper down to a point at the bottom.

The potential utility of FIB for routine (and novel) applications has come to forefront recently due to advances in ion optics which now allow formation of focused ion probe of better than ∼10-20 nm containing current density exceeding several A/cm2, with a liquid metal source (typically Gallium). The small ion probe size, coupled with shallow sputtering depth - yet high sputtering yield of ions, has opened several opportunities in machining, lithography and ion-assisted deposition[ 1-3] These developments, including automation, multi-specimen stages, cross-compatible specimen holders for FIB/TEM/SEM, use of in-situ electron beam (so-called dual beam), coupled with innovations such as the “lift-off process[4], have provided an invaluable set of tools for microelectronic defect characterization. However, re-deposition (contamination), ion implantation/damage especially for desirable thinner sections (<∼50 nm) remain major concerns for further applications.

While much of the excitement in TEM community for FIB is due to thin foil specimen preparation (especially in microelectronics),

Failure analysis in the microelectronic industry via TEM has long dealt with the specimen preparation spatial resolution problem—the ability to prepare useful TEM specimens at very small pre-selected locations. This problem is made more difficult with time because, while device structures become smaller and more dense, the failure rate of individual devices is extremely low and dropping. TEM analysts, therefore, must prepare specimens that optimally present a single faulty component within an ocean of a billion or more similar components. Sophisticated electrical testers can find the faulty component and transfer its coordinates to a FIB tool where the failure can be marked. Cross section TEM specimens are appropriate when it is suspected that the failure is caused by a defect in the stack of metal and insulators constructed on the top of the device substrate (usually silicon). Plan view TEM specimens are appropriate when the electrical signature of the failure indicates that the fault lies in the Si itself-dislocations or some other crystal defect.

In recent years, the focused ion beam (FIB) instrument has developed into a mainstay tool for the production of specimens for both scanning and transmission electron microscopy ((S)TEM). The inception and subsequent development of the FIB TEM lift-out (LO) technique has enabled electron transparent membranes of generally uniform thickness to be produced for TEM analysis. In general, the primary advantage of the FIB is that site specific sections may be fabricated quickly (e.g., < 1 hour) and reproducibly. Specifically, the FIB LO technique has been used extensively in our laboratories to produce on the order of a thousand Si-based specimens per year and hundreds of other specimens per year that have included metals, ceramics, composites, biological materials, geological materials, polymers, particles, and fibers, prepared in cross-section, plan view, and from fracture surfaces.

The focused ion beam(FIB) technique, developed for the microelectronics industry has become a major method for site specific transmission electron microscopy(TEM) specimen preparation in a wide range of materials[l]. The FIB lift-out technique has improved the specimen preparation procedures by removing complicated initial fabrication required prior to the FIB milling[2]. However, conventional FIB techniques are still having increased difficulty in meeting failure analysis needs from high technology industries such as microelectronics.

We have developed a site specific TEM specimen preparation method using a combination of an FIB instrument and an intermediate voltage TEM equipped with a scanning attachment [3]. In this method, the specimen is mounted on an FIB-TEM compatible specimen holder, so that localization of the specific site can be carried out in the FIB and TEM using the same holder. The scanning electron imaging mode may be used to observe surface structures of the milled area, and the scanning transmission electron microscopy(STEM) mode may be used to observe structures inside of the milled surface.

The use of focused ion beam (FIB) systems is well established as a sample preparation and imaging tool in a wide range of applications, most notably, in the semiconductor and data storage industries, but also within material and biological sciences (Figs. 1-3). The real benefit of the FIB is that the same ion beam that is used for material removal and deposition is also used for imaging the sample, which results in highly precise and localized sample preparation. In addition, the FIB can be used to prepare samples through multiple layers with different material properties, and allows the rest of specimen to be kept intact for further analysis or processing. FIB is most commonly used to prepare samples for the transmission electron microscope (TEM), the scanning electron microscope (SEM), and for the FIB itself. The FIB, which is an imaging tool in its own right, can produce secondary-electron and -ion images and collect secondary ion mass spectrometry (SIMS) data.

The focused ion beam miller (FIB) has been widely used in the semiconductor industry for many years, but only recently has its potential as a tool for materials science been recognised. The FIB uses a highly energetic beam of gallium ions to sputter material such that it can precisely section, as well as image, areas of interest. The FIB can be used to create crosssections, which can be examined in the FIB or in a scanning electron microscope (SEM). Cross sections can be made from delicate samples or samples in which a specific area needs to be viewed, for example to check the thickness of coatings or for failure analysis.

The FIB may also be used to prepare transmission electron microscope (TEM) specimens [1]. Extremely site-specific thin areas may be prepared with high positional accuracy from heterogeneous samples such as composites or layered structures.

Focused ion beam lift-out technique has become increasingly attractive to the TEM community due to its unique advantage of no mechanical grinding/polishing involved in the process [1-3]. The technique essentially consists of two parts: preparation of membrane using focused ion beam (FIB) and transfer of the membrane (lift-out) to a grid. Up to date, this technique has only been demonstrated on single beam FIB systems. From a practical standpoint, overall sample quality (thickness) and lack of end-point precision are two major issues associated with the conventional single beam FIB technique. These issues are primarily related to ion beam damage and endpoint control encountered during the final stages of specimen thinning. As a result, the widespread use of FIB lift-out technique for high precision TEM specimen preparation has been limited. Recent technological advances have made it possible to combine both an electron beam column and an ion beam column into an integrated dual beam-focused ion beam (DB-FIB) system.

The focused ion beam (FIB) instrument offers an efficient and reproducible approach for obtaining electron transparent membranes of uniform thickness for TEM analysis. Thus, the use of the FIB to investigate numerous types of materials has grown tremendously recently. Prenitzer et al. demonstrated the ability to acquire TEM specimens from individual Zn powder particles using the FIB lift-out (LO) specimen preparation technique. The 10 μm × 100 μm Zn powders particles were larger than the 5 μrn x 20 μm FIB LO specimen and therefore the LO technique could be directly applied to an individual powder particle. This paper will discuss a novel approach for the preparation of site specific micrometer-sized particles for TEM analysis using the FIB LO technique. The uniqueness of the technique described herein is that site specific TEM LO specimens may be obtained from regions which are smaller than the conventional dimensions of the LO specimen.

Defects present in microelectronic devices are often present in test structures as well. This makes test structures useful in identifying defect mechanisms. Chain patterns consists of thousands of contacts and plugs in series. The presence of an open contact in a chain can be detected by a loss of electrical continuity. The specific site of an open contact is difficult to locate for further analysis.

The application of the focused ion beam (FIB) for passive voltage contrast (PVC) provides an effective method for contact defect location. Once the defect is located the FIB facilitates efficient site-specific specimen preparation for scanning electron microscopy (SEM), transmission electron microscopy (TEM) or Auger analysis.

The FIB is capable of PVC analysis by distinguishing electrically isolated conductors from grounded conductors. Isolated conductors charge as a result of ion beam interaction. Once charged, the quantity of secondary electrons available for detection is greatly reduced.

Focused ion beam (FIB) instruments have become essential for the preparation of atom probe samples from heterogeneous materials. Previous sample preparation methods such as electropolishing are limited in their application due to either geometrical or electrochemical constraints. Recent developments in sample preparation using a FIB have enabled the production of AP samples from various materials and, more importantly, from non-traditional sample geometries that contain multilayered thin film structures (MLF).

Most sample preparation using a FIB first involves a sample that has been reduced in size through some manual sample preparation technique like tripod polishing or cutting. Smaller, thinner samples require less milling time in the FIB. A silicon wafer etched with the “Bosch” process was used to produce a surface that contains millions of 20, 16, 12, 8, and 4 μm square by -180 μrn long “posts”, Fig. 1. A multilayer film structure is deposited on the flat surface of the silicon posts in a standard deposition process.