Detection of epidermal growth factor receptor by scanning electron microscopy

Low-voltage scanning electron microscopes (LV-SEMs) are widely used in nanoscience. However, image resolution for SEMs is restricted by chromatic aberration due to energy spread of the electron beam at low acceleration voltage. This study introduces a new monochromator (MC) with offset cylindrical lenses (CLs) as one solution for LV-SEMs. The MC optics, with highly excited CLs in offset layouts, has advantageous high performance and simple experimental setup, making it suitable for field emission LV-SEMs. In a preliminary evaluation, our MC reduced the energy spread from 770 to 67 meV. The MC was integrated into a commercial SEM equipped with an out-lens (a conventional objective lens without immersion magnetic or retarding electric fields) and an Everhart–Thornley detector. Comparing SEM images under two conditions with the MC turned on or off, the spatial resolution was improved by 58% at 0.5 and 1 keV. The filtering effect of the MC decreased the probe current with a ratio (i.e., transmittance) of 5.7%, which was consistent with estimations based on measured energy spreads. To the best of our knowledge, this is the first report on an effective MC with higher-energy resolution than 100 meV and the results offer encouraging prospects for LV-SEM technology.

Spherical and chromatic aberration coefficients Cs and C of various immersion lenses forlow voltage SEM and LEEM are calculated. The minimun values of magnetic immersion lens are C5Cl mm. For the combined electrostatic and magnetic lenses, those values are at most C5=1 mm and CO.7 mm, when the specimen is free from the electrostatic field. Whenthe specimen is immersed in the electrostatic field, those values reduce to C50.2 mm and Cc=o.1 mm at 1 kV. Key words: SEM, LEEM, Objective lens, Spherical aberration, Chromatic aberration, Immersion lens, Cathode lens, Computer aided design 1. INTRODUCTION Importance of low accelerating voltage in scanning electron microscopy (SEM) increasesrecently for semiconductor and biological applications. A main difficulty of low voltage SEM(LVSEM) is its low spacial resolution due to the increase of the wave length and the increaseof AVIV0 ratio where AV is the energy spread and V0 the incident energy. The immersionobjective lens as well as the field emission electron gun is very useful in realizing the highresolution LVSEM. Low energy electron microscopy (LEEM) instruments are very useful forsurface studies of metals and semiconductors. The electrostatic immersion objective lensis essentially necessary for LEEM to get a low landing voltage and low aberrations.There are three kind of immersion lenses: the magnetic immersion lens, the retardingelectrostatic immersion lens and the combination of immersion electrostatic and magneticlenses. The latter two lenses are reviewed by Mullerova and Lenc (1992)1. Here we have to

This paper describes a method for applying passive voltage contrast (PVC) in the failure analysis of CMOS LSIs using a conventional scanning electron microscope (SEM), and demonstrates the effectiveness of this method. It was confirmed by measurement of the stage absorption current that the combined emission efficiency of secondary and backscattered electrons from aluminum is larger than 1 at acceleration voltages lower than about 2.5 kV. This means that local positive charges should be generated on a conductor in association with its irradiation with an electron beam at a relatively low acceleration voltage. However, a pn junction connected to the conductor would change the potential. The potential is still positive if the conductor is connected to a reverse-biased diode but becomes lower if such a diode is forward-biased. The PVC signal observed on the conductor should be defined according to the bias state of any diode connected to the conductor. Therefore, for failure analysis applications, if the bias state of a diode connected to a suspicious conductor is known, the PVC observation is useful for determining whether there is an open defect or a short-circuit defect. Some case studies are presented to demonstrate the effectiveness of the method. Cross-sectional TEM observations of defects localized in this way are also included.

Methods for characterizing block copolymers and polymer blends have been developed. Results from unstained and ruthenium tetroxide‐stained samples obtained by scanning electron microscopy at various acceleration voltage and by transmission electron microscopy are presented. The contrast in secondary images between components in stained polymer blends, where one component is preferentially stained, is maximized at higher acceleration voltage (10–25 keV). For measurement of particle size and shape, this is the preferred operating condition. To obtain high‐resolution images showing surface topography and fine structure, such as 20 nm domains in block copolymers, low‐voltage (<5 keV) imaging is preferred. Observation of the 20 nm domain structure in hydrogenated styrenebutadiene‐styrene shows that the spatial resolution now possible by scanning electron microscopy is comparable to that obtained by the traditional method of transmission electron microscopy.

Evidence for Aggregation in Oxalate Stone Formation: Atomic Force and Low Voltage Scanning Electron Microscopy

By providing higher image contrast and reduced charging artifacts, the low voltage scanning electron microscope (LVSEM) is a valuable tool for surface characterization, of particular importance on nonconductive material such as biological specimens. Several SEM designs optimized for use at low voltage have been proposed.Recently, we have designed a new high resolution LVSEM using a field emission gun. The key problem is to decrease both the spherical and chromatic aberration coefficients by using a magnetic lens of small bore diameters(5mm and 10mm) and a narrow gap (7.5mm) (FIG. 1). In our first design, the magnetic lens is built around the side-entry stage of a Philips 300kV TEM and performs as well as that in the present Hitachi SEM H-900 or H-900S. Its simple design has been chosen for reliability and flexibility in farbrication.

Recent developments in scanning electron microscope design

Donor corneas are processed in eye banks and used for transplantation as a standard routine. The maximum time limit post-mortem for harvesting donor tissue varies greatly between eye banks. This study aimed to examine the corneal epithelium for structural changes post-mortem. A total of 51 corneas harvested between 14 and 163 hours post-mortem were examined using scanning electron and light microscopy. Cell loss occurred through desquamation of flat superficial cells during the first days. In corneas with a post-mortem time of more than 2-3 days, large superficial cell sheets and deeper cells detached, starting centrally. Deep peripheral cells remained. The loss of the superficial cells revealed the 3-dimensional structure of the epithelium and the membrane characteristics of deeper cells. The longer the time post-mortem, the greater the epithelial cell loss. However, a rim of peripheral cells remained, even after 7 days. The superficial cell layer showed signs of strong lateral attachment and broke up in a sheet-like fashion. The intercellular adhesion between deeper cells and adhesion between the basal cells and the basement membrane appeared to be weak post-mortem. The cell membrane structures of the remaining cells were surprisingly well retained. The clinical implication of the study is discussed.

An electrostatic aberration corrector for improved Low-Voltage SEM imaging.

Scanning electron microscopy (SEM), especially low-voltage (1 KeV) high-resolution SEM, can be used in conjunction with stereo pair high-voltage (1 MeV) transmission electron microscopy (HVEM) of whole spread cells or thick sections effectively to correlate surface structure with internal structure. Surface features such as microvilli, pits, pseudopodia, ruffles, attached virus, and other surface-related morphologic characteristics can be identified using SEM, while underlying cytoskeletal structure and organelle organization can be viewed by HVEM of the same preparation. However, the need to "prepare" cells for electron microscopy precludes observation in the living state. The use of several types of video-enhanced light microscopy (VLM) permits observation of living cells such that certain surface and internal features can be observed at a relatively high level of resolution or detection. Thus, changes in living cells can be followed, and at appropriate times the cells may be chemically fixed or rapidly frozen and prepared for ultrastructural examination by electron microscopy. We have utilized VLM in conjunction with SEM and HVEM to correlate changes in shape and surface structure with changes in the internal structure of platelets. In addition, we have found it advantageous to use colloidal gold-labeling procedures, because these markers are detectable by all three forms of microscopy. Using this approach we have labeled platelet membrane GPIIb/IIIa, a receptor for RGD-containing adhesive proteins, with gold-fibrinogen or gold-anti-IIb/IIIa. The initial binding and subsequent movement of gold-fibrinogen-IIb/IIIa complexes in living platelets was followed by VLM. The movement of individual labels could be mapped. Subsequent observation by low-voltage (1 KeV) high-resolution SEM and HVEM permits visualization of the same individual receptors tracked by LM. The final position on the membrane or the position-in-transit when fixative was added was determined relative to surface ultrastructure (SEM) and internal, particularly cytoskeletal, ultrastructure (HVEM).

New method for the characterization of domain morphology of polymer blends using ruthenium tetroxide staining and low voltage scanning electron microscopy (LVSEM)

The scanning electron microscope (SEM) techniques play a key role in the characterization of various inorganic and/or organic semiconducting materials, micro-/nanostructures and devices. The power of the SEM methods is mainly in imaging, characterization and diagnostics of local near surface properties. Among a variety of the SEM methods, Cathodoluminescence (CL) and Electron Beam Induced Current (EBIC) methods have been extensively used for characterization of generation/recombination phenomena and electrical properties of bulk semiconductors and semiconductor structures [1]. Exploitation of these methods allow to visualize the electrically active defects such as dislocations, grain boundaries and inhomogeneities and to estimate the diffusion length, lifetime and surface recombination velocity of minority carriers. In addition, EBIC can be used for investigation and diagnostics of SCR of Schottky and p-n junctions, which extends its applications, in combination with other techniques such as Voltage Contrast (VC), to functional diagnostics of electronic devices biased at operation point. Currently, various modifications and improvements of “standard” SEM methods are developed to improve their resolution and sensitivity for investigations of advanced structures and devices structural, optical and electrical properties. Various sample preparation and micromanipulation / microprobe techniques are examined to localize appropriate signals with the aim to improve the spatial resolution while for extreme depth resolution low acceleration voltages of SEM techniques are utilized [2]. In addition, complex signal acquisition and processing techniques are implemented to achieve time and frequency resolution and to gain required information about the sample properties.

Particulate growth processes in Ar+SiH4 RF plasmas are studied using ultraviolet light-absorption and scanning electron microscopic (SEM) methods. Measurements of particulate size and density using the SEM method show that they grow through three phases: nucleation, rapid growth and growth saturation. Such features of temporal evolutions of their size and density are very similar to those for He+SiH4 RF plasmas previously obtained by SEM and in situ laser light scattering methods. It is also found that particulates in two different size ranges coexist after the initiation of the rapid growth phase, and their coagulation plays an important role in this phase. Close similarities among spatial profiles of Si emission intensity, Si density, and amount of particulates are revealed in wide discharge-parameter ranges of 1–100% SiH4, 11–20 Pa, and 40–100 W. These results suggest that many short-lifetime neutral radicals such as SiHn (n=0–2) contribute to nucleation and subsequent initial growth of a particulate, since other short-lifetime radicals are also expected to have similar spatial profiles.

Low-voltage, high-resolution (LVHR) scanning electron microscopy (SEM) of polymers is becoming more widespread as the new LVHRSEMs become more available to the general microscopy public. Although low-voltage SEM has been known for many years, the availability of new high-resolution microscopes with high-brightness field emission electron guns (FEG) and lens systems optimized for lower operating voltages (especially the immersion lens designs) has revolutionized the field of polymer surface morphology. The adequate beam current and excellent resolution at low voltages (4.0 nm at 1.0 keV) results in a dramatic improvement in image contrast at low voltages due to reduced beam spread and smaller interaction volume. In addition, imaging can be performed near the E2 crossover voltage, which means there is little or no sample charging, necessitating little or no sample coating with conducting metals or carbon, thus simplifying sample preparation.

Extracellular vesicles secreted by cells represent an almost spherical membrane structures enriched with biological molecules of different types. The number and molecular composition of these structures depend on both the physiological state of an organism and underlying diseases. Despite extracellular vesicles playing an important role in intercellular communication and being potential biomarkers of pathological processes, the mechanisms of their formation, their functions, and their morphological characteristics are poorly studied. Low-voltage scanning electron microscopy is a promising method for studying extracellular vesicles, since it does not need a layer of conductive covering and, consequently, permits morphological details of studied objects to be vizualized at a high resolution in a nanometer range. The results of investigation of the morphology and sizes of objects in blood-plasma fractions by low-voltage scanning electron microscopy are presented in this study.