bluuebunny/crossref_metadata_2025 huggingface dataset:
Applications of Dispersion Staining Technique in Image Analysis of Colorless Particles
Authors: Su Shu-Chun
Published: July 2020

particles and their surrounding immersion liquid medium into a color in the When analyzing particle size distribution of colorless, translucent or transparent particles by image analysis, a major challenge is to obtain images of particles that ensure proper object detection, especially if the particles are amorphous or non-crystalline. Conventional imaging techniques, such as brightfield, darkfield, cross-polarized light, etc., might not applicable for these types of materials.

Dispersion staining (DS) is a technique that coverts the refractive index (RI) difference between visible range and renders particles optically stained with that particular color. There are two modes of dispersion staining techniques: central stop (CS) and annular Stop (AS). For image analysis, CS is preferred.

In the CS mode, the matching wavelength λm, i.e., the wavelength at which the RI of a particle equals that of liquid, is not refracted at the particle/liquid interface and therefore blocked by a 3-4 mm opaque round disk located at the center of the back focal plane of objective lens.
Placing TEM Negative Images into the Digital Domain
Authors: Mascorro JA , Frasch T , Yates RD
Published: July 2020

Several thousand images of various biological tissues have been produced by these investigators (*) over careers spanning many, many years. The majority of the images are preserved on Kodak Electron Image Film #4463, Kodak Electron Microscopy Film #4489, Kodak Electron Image Film #SO 163, or Dupont Ortho S Litho (COS-7) film. These 7 mil ESTAR thick base films were the standards utilized by the biological microscopist. The emulsion was covered with a thin layer of hardened gelatin in order to minimize abrasion. After exposure to the electron beam in the 60-80 kV range, the negatives were developed according to routine photographic procedures employing Kodak D-19 developer, hardened in Kodak fixer, washed for the appropriate time, and stored in glassine envelopes in a dust-free environment. The finished negative with the captured image proves to be long-lasting and will produce an image, today, displaying the very same excellent qualities as did original prints produced years ago.
Hybrid Imaging in Electron Microscopy
Authors: Black D L , Stoker S C , Minter J R
Published: July 2020

Imaging is an integral part of electron microscopy and is one of the mechanisms we use to share data with our colleagues. We interpret these images to understand microstructure of biological, polymeric and solid state materials. The imaging process can be viewed as a multistep process comprising image capture, processing, and storage. For example, images can be captured on film or digitally using CCD cameras. The processing step uses computers and the appropriate software and the images are stored as hard copies such as prints or a data file. Digital imaging is a dramatically evolving field that has enhanced the way we interpret and analyze these images.

Although digital imaging can be used in each step of the imaging process, one can also use a hybrid system to minimize cost and retain the advantages of digital imaging. in this presentation, we discuss using a hybrid system that allows us to retain the advantages of digital processing and storage of images.
Real-Time Observation of Phospholipid Bilayer Membrane Restructuring Induced by Protein Molecules using Atomic Force Microscopy
Authors: Xing You Hong , Qi Xiaoyang , Yu Lei
Published: July 2020

Atomic force microscopy (AFM) allows the surfaces of native biological materials to be imaged in aqueous solution with submolecular resolution. The ability to perform AFM imaging in aqueous and physiological environment has made it possible to monitor important biological processes in real time at high resolution. Currently, there is a great deal of interest in AFM studies of the structure and property of lipid bilayer membranes and protein interactions with lipid bilayer membranes. Lipid bilayer membranes in biological cells form a permeability barrier, which controls the flow of ions, water, and other molecules between biological cells and their environments, whereas membrane-bound and/or membrane-associated proteins are responsible for most of the dynamic functions carried out by the membrane. However, real-time AFM monitoring of dynamic biological processes has been challenged by the limited temporal resolution of AFM, potential physical damage to soft biological samples, and intrinsic complexity of biological processes. There are few successful examples of AFM real-time studies of dynamic biological events, particularly in the aspect of protein interactions with lipid bilayer membranes.

We have attempted to use atomic force microscopy to study interactions between a particular protein, saposin C, and phospholipid bilayer membranes in real time. Saposin C (Sap C), a small glycoprotein, is an essential co-factor for the hydrolysis of glucosylceramide by glucosylceramidase in lysosomes, and a deficiency of Sap C leads to a variant form of Gauchers’ diseases. Supported planar phospholipid bilayer membranes were used in the study.
Observation of Contact Potential Difference (CPD) on Semiconducter Surface using Ultrahigh Vacuum Scanning Kelvin Probe Force Microscope (UHV SKPM)
Authors: Kitamura S , Suzuki K , Mooney C B
Published: July 2020

The scanning Kelvin probe force microscope (SKPM) is a member of the scanning probe microscope (SPM) family, and was derived from the non-contact atomic force microscope (NCAFM) technique. The contact potential difference (CPD) originating from the work function difference between the tip and sample surfaces can be measured using SKPM with simultaneous observation of the topography image. Using SKPM the surface of semiconductor device has been observed to measure the dopant concentration as the CPD difference in two dimensions. Most of the SKPM measurements are acquired in the atmosphere. The lateral resolution is achieved less than 100 nm.

On the other hand, Scanning capacitance microscope (SCM) of contact mode is now most popular method to measure the dopant concentration in semiconductor device. However, the lateral resolution of SCM is limited, because of the fact that the contact mode cannot achieve a true atomic resolution, the fact that the capacitance is measured through the insulating layer on surface, and the fact that the tip and sample is damaged by the high electric field to be applied between the tip and sample. So SCM never achieves the atomic level resolution.
New Concept in Helium Cooled Transmission Microscopes Philips Tecnai F30 Helium: Design and First Results
Authors: Lücken U , Wagner R , de Jong AF , Hax W
Published: July 2020

To date it has been possible to solve the structure of several membrane proteins to atomic resolution with cryo-electron crystallography (1-4). in all studies two-dimensional crystals were required as well as special provisions on the transmission microscope. in all cases a field emission gun provided the necessary coherence and liquid-Helium cooling the specimen protection (4,5).

Although repeatedly studies have been done on the cryo-protection factor of beam sensitive specimen at liquid-Nitrogen and liquid-Helium temperatures (6-10), the data available are still too limited for a clear demonstration of the superiority of liquid-Helium. Especially investigations are needed with individual particles embedded in amorphous ice (11).

To meet the increasing demand for liquid-Helium cooling, FEI developed the Tecnai F30 Helium. The challenge was to provide an instrument with a very stable cryo-stage, which can work at liquid- Nitrogen as well as liquid-Helium temperature.
Development of Multi-Purpose Thermal Field Emission SEM
Authors: Mita M , Nokuo T , Yanagihara T , Ogura K , Iwatsuki M , Nielsen C
Published: July 2020

Past FE-SEM could obtain a high resolution image, however its probe current was not sufficiently strong enough for analytical purpose.

We have developed a multi-purpose thermal field emission scanning electron microscope (JSM- 6500F), in which a new designed “In-Lens Thermal FEG” is installed.

Fig. 1 shows a cross section images of the In-Lens Thermal FEG, comparing with the past FEG. The In-Lens Thermal FEG consists of the thermal FEG and the 1st condenser lens. The emitter is located in the magnetic field produced by the 1st condenser lens, so that electrons emitted from the emitter are condensed effectively to produced a high probe current. The maximum probe current of 200 nA is attainable at the accelerating voltage of 15 kV, ten times larger than the maximum probe current of ordinary FE-SEMs. Therefore the WDS analysis can be performed by this newly FE-SEM.

The “aperture angle control lens” has been installed in the electron optics system, for improving the resolution at a large probe current. The resolution of 3.0nm at the analytical condition (at probe current 5nA, accelerating voltage 15kV, WD 10mm: fig.2).

The ultimate resolution of the microscope is 1.5nm at 15kV and 5nm at lkV. The objective lens is not an immersion type and does not leak magnetic fields on the specimen surface, therefore this equipment was suitable for observing or analyzing magnetic materials, and also suitable for the EBSD analysis. Fig.3 shows an example of the EBSD analysis.
New Technologies for Microanalysis and Element Imaging in THJ Scanning Electron Microscope
Authors: Smith Paul , Gannon John , Eggert Frank
Published: July 2020

RÖNTEC’s UHV Dewar detectors have established new standards for high resolution, lowmaintenance, low operating cost, and reliability in Si(Li) X-ray detectors. Now, the recently introduced XFlash® series X-ray detectors are enabling new methodologies for microanalysis and element imaging in the SEM. These detectors are compact, liquid-nitrogen-free semiconductor Xray detectors that are based on Silicon Drift Diode (SDD) technology. XFlash detectors produce extraordinarily high count rates with excellent energy resolution and have introduced ultra-fast microanalysis and element mapping to the SEM world. The addition of color to SEM images enables easy visualization of element distributions and allows the microstructural features and compositional variations of different materials to be more readily identified. Persons unfamiliar with electron microscopy can more readily interpret color images than black and white or gray scale images. This new technology has great potential to revolutionize electron microscopy.

RÖNTEC’s UHV Dewar Detector offers the highest long-term stability and best energy resolution ever specified for a commercial Si(Li) detector (less than 129 eV). The UHV design leads to small size and weight (for reduced column loading) along with extremely low nitrogen consumption and low susceptibility to microphonics. The UHV detector never ices up and thus never requires defrosting or warm-ups. It is available with a variety of entrance windows for light element analysis.
XPS Evaluation of Samples Surface Cleaned by the XEI Evactron®
Authors: Walck Scott D , Strohmeier Brian R , Goralski Edward G , Vane Ronald A
Published: July 2020

With the advent of field emission gun equipped SEMs and TEMs with clean vacuum systems, it has been shown repeatedly that the surface cleanliness of samples exposed to the small, high current density electron beam available in these instruments is critical with respect to controlling hydrocarbon contamination under the electron beam. in previous studies of dedicated plasma cleaners for TEM samples by one of the authors (SDW), it was shown that surface analytical techniques are the best methods for determining the effectiveness of such cleaners in removing hydrocarbons from the samples’ surfaces. in the current study, some of the experiments that were used in these previous studies were repeated using the XEI Evactron® system in order to compare the relative effectiveness of this system for surface cleaning of EM samples.

Figure 1 shows a picture of the sample introduction chamber of a VG Scientific ESCALAB Mkll system (XPS) with the Evactron® in place. The Evactron® system consists of an RF head unit that attaches to the vacuum chamber with an adaptable flange, an RF impedance matching unit, and an RF generator/vacuum controller. Although a 100% O2 system can be used and is recommended, for safety reasons, a 20%O2/80%Ar by volume gas mixture was selected because of the hydrocarbon oil used in the mechanical pump. Cleaning was also performed using laboratory air. The major difference between the dedicated plasma cleaners and the Evactron® configuration is that the sample is not immersed in the plasma but is exposed to the activated oxygen species in the downstream flow.
The Triebenberg Laboratory-Designed for Highest Resolution Electron Microscopy and Holography
Authors: Lichte Hannes , Schulze Dietrich , Lehmann Michael , Just Holger , Erabi Taher , Fuerst Peter , Goebel Jaqueline , Hasenpusch Andreas , Dietz Peter
Published: July 2020

Very often, the performance of modern electron microscopes is limited by the poor quality of the laboratory rather than by the specifications of the microscope. Therefore, with microscopes aiming at subangstrom resolution microscopy and highest performing holography methods, special measures have to be taken to reach the needed low level of AC-stray fields, vibrations, and acoustic noise. in an ”organically“ grown institute, this is very difficult to realize, if possible at all.

• The Triebenberg Lab is newly built well outside the city of Dresden, in an area essentially free from disturbances, i.e. free from power lines, away from roads, trains and airplanes; fortunately, the site is shielded from wind by means of a dense belt of trees. The lab houses six microscope units each consisting of a microscope room, a room for peripheral devices (power supply, cooling units, computers, etc), and an office for up to three scientists. in view of the nearly ideal environment, the main challenge is to avoid any self-made disturbances which might arise by the operation of the lab. Therefore, without any compromise using the best art of engineering, we designed a special lab virtually free from disturbances. in the following, the main guidelines for the construction are sketched.
Aberration-Corrected STEM: the Present and the Future
Authors: Krivanek OL , Dellby N , Nellist PD , Batson PE , Lupini AR
Published: July 2020

Surprising as it may seem, aberration correction for the scanning transmission electron microscope (STEM) is now a practical proposition. The first-ever commercial spherical aberration corrector for a STEM was delivered by Nion to IBM Research Center in June 2000, and other deliveries have taken place since or are imminent. At the same time, the development of corrector hardware and software is still proceeding at full speed, and our understanding of what are the most important factors for the successful operation of a corrector is deepening continuously.

Fig. 1 shows two high-angle dark field (HADF) images of [110] Si obtained with the IBM VG HB501 STEM operating at 120 kV, about 2 weeks after we fitted a quadrupole-octupole corrector into it. Fig. 1(a) shows the best HADF image that could be obtained with the corrector's quadrupoles on but its octupoles off. Sample structures were captured down to about 2.5 Å detail, easily possible in a STEM with a high resolution objective lens with a spherical aberration coefficient (Cs) of 1.3 mm. Fig. 1(b) shows a HADF image obtained after the Cs-correcting octupoles were turned on and the corrector tuned up. The resolution has now improved to 1.36 Å. This is sufficient to resolve the correct separation of the closely-spaced Si columns.
Sub-Ångstrom Transmission Electron Microscopy at 300keV
Authors: O’Keefe MA , Nelson EC , Turner JH , Thust A
Published: July 2020

Sub-Ångstrom TEM to a resolution of 0.78Å has been demonstrated by the one-Ångstrom microscope (OÅM) project at the National Center for Electron Microscopy. The OÅM combines a modified CM300FEG-UT with computer software able to generate sub-Angstrom images from experimental image series.

Sub-Ångstrom HREM is gaining in importance as researchers design and build artificially-structured nanomaterials such as semiconductor devices, ceramic coatings, and nanomachines. Commonly, such nanostructures include atoms with bond lengths shorter in projection than the point resolution of a mid-voltage HREM. in addition, image simulations have shown that structure determinations of defects such as dislocation cores require sub-Angstrom resolution, as will hold true for grain boundaries and other interfaces.

Sub-Ångstrom microscopy with a transmission electron microscope requires meticulous attention to detail. As resolution is improved, resolution-limiting parameters need to be reduced. in particular, aberrations must be minimized, power supplies must be stabilized, and the microscope environment optimized to reduce acoustic and electromagnetic noise in addition to vibration.
Correction of Aberrations of a Transmission Electron Microscope
Authors: Haider M
Published: July 2020

One of the most striking problems in electron optics, the correction of resolution limiting aberrations by means of a corrector incorporated into the electron microscope column, has been solved during the last six years by demonstrating the improvement of resolution beyond the theoretical limit of the uncorrected Electron Microscope (EM). At first, in 1995 [1] with the correction of spherical and chromatic aberration of a dedicated Low Voltage Scanning Electron Microscope (LVSEM) and later, in 1997, with the correction of only spherical aberration of a commercially available 200 kV TEM [2]. The correction of spherical aberration of a dedicated Scanning Transmission Electron Microscope (STEM) at 100 keV primary energy has been demonstrated [3] and further improvements can be anticipated within the near future.

These achievements could only be obtained due to the emergence of new computer technology and especially CCD-cameras in the case of TEM correctors. These two developments made it possible first to calculate the electron optical components more precisely and hence, to achieve a better understanding of the requirements on the hardware and second, to have a better computer control of the electron microscope and the corrector itself. The combination of these two new technologies made it possible to go towards an automatisation of the alignment. This simplification of the alignment of an even more complex system is achieved by means of a proper combination of image acquisition and dedicated software in order to analyze and measure the aberrations of an electron optical system on one side and on the other to have appropriate tools to compensate these aberrations by computer controlled power supplies [4,5].
A New High Performance Electron Energy Loss Spectrometer for use with Monochromated Microscopes
Authors: Brink HA , Barfels M , Edwards B , Burgner P
Published: July 2020

A new type of electron energy loss spectrometer for use with monochromated microscopes is presented. The energy resolution of the spectrometer is better than 0.100 eV. A completely new electron optical design with a number of extra optical elements and advanced tuning software makes it possible to correct spectrum aberrations to 4th order, which increases sensitivity and collection angles. New high-stability electronics make it possible to maintain energy resolution over a period of several minutes in a practical laboratory environment.

The energy resolution of Transmission Electron Microscopes (TEMs) equipped with electron energy loss spectrometers is determined by a combination of the energy spread of the electron source, the stability of the microscope’s high voltage power supply, and the energy resolution of the spectrometer. Commercial microscopes usually employ electron sources with an energy distributions of around 0.5 eV or more (FWHM), limiting the energy ultimate energy resolution that can be achieved. Recently FEI constructed a special 200 kV TEM with a built-in monochromator which makes it possible to monochromize the electron source to better than 0.100 eV. A prototype of the presented spectrometer has been installed on this microscope.
Reconstruction of the Projected Potential from a through Voltage Series of Dynamical Electron Diffraction Patterns Including Absorption
Authors: Koch C , Spence JCH
Published: July 2020

Recently several different methods have been proposed to reconstruct the projected crystal potential from electron diffraction patterns. These methods envolve either diffraction patterns at many different orientations (as many orientations as beams in the pattern) and/or images, which makes their experimental realization difficult. We propose an entirely new method for reconstructing the projected crystal potential from fully dynamical [including absorption and multiple scattering effects to all orders] diffraction patterns from only a single crystal orientation and no image at all. Knowledge of the specimen thickness is not necessary. However, it requires diffraction patterns at many different accelarating voltages, which is a parameter that can easily be varied (within a certain range) in most modern electron microscopes. Since the intensities in the electron diffraction pattern are not affected by lens abberations this method is capable of reconstructing the projected potential with a resolution far better than that of any method using HRTEM images.
Ultrahigh-Vacuum Electron Microscopy for Gold Nanostructures
Authors: Kondo Yukihito , Okamoto Kimiharu , Naruse Mikio , Honda Toshikazu , Kersker Mike
Published: July 2020

Ultrahigh-vacuum transmission electron microscopy (UHVTEM) has become increasingly popular for the direct observation of nanostructures having clean surfaces, since industrial requirements to make and research nano-scale materials have been rapidly growing for quantum or nanoscale electronic devices. Since we have first developed high resolution UHVTEM in 1986, the UHVTEMs have been evolved with steady advances such as UHV compatible goniometer, field emission gun or etc. Furthermore, the UHVTEM started to combine analytical capabilities such as energy dispersive X-ray spectrometer, in-column type energy filter and etc., and to combine STM (scanning tunneling microscope). The UHV technology is essential for the analysis, because the portion of contaminant in a nano-scale specimen increases as the size of the specimen goes down. This paper reports the results of gold nanostructures by recently the developed UHVTEM.

Figure 1 shows recently developed UHVTEM with Schottky type field emission gun.
Design and Performance of the Sesame I
Authors: Krahl D , Kujawa S , Rilk A , Benner G , Hahn P , Lang G , Essers E , Probst W
Published: July 2020

The strong technological trend towards higher and higher integrated systems is resulting in considerably increasing demands on nanotechnique and -electronics. One of the most important sets of information is the characterization of the inner interfaces within such materials not only concerning the atomic structure but also concerning chemical composition and bonding and concerning local strain. New generation energy filtering TEMs using corrected in-column spectrometers and providing considerably increased power of resolution concerning imaging and analysis are thus essential in order to be able to understand, define, built and characterize such devices.

Within the SESAMe (Sub-eV-sub-Angstrom Microscope) [1] project, LEO and CEOS realize such a microscope for the MPI of Metal Research, Stuttgart in 3 steps. For step 1 and 2 an in second order completely corrected and in third order minimized 90° OMEGA energy filter has been developed. This filter is operating since one year in a 200 kV LEO EFTEM with LaB6 emitter (Step 1) [4]. Results will be presented by W. Sigle during this meeting. This filter will also be incorporated in the Sub-Angstrom-TEM (SATEM) with Cs corrector for the Research Center Jülich.
Chemically Assisted Focused ION Beam Micromachining: Overview, Recent Developments and Current Needs
Authors: Russell PE , Stark TJ , Griffis DP , Gonzales JC
Published: July 2020

Recent developments in FIB have included optics and automation improvements as well as technique development. in the past decade, ion beam optics have improved rapidly to the point where the beam size and profile does not dramatically limit its use in many applications ranging from failure analysis to manufacturing. Developments in FIB can therefore focus on techniques to increase removal rates, enhance selectivity and improve surface finish. in this work we present various efforts to improve FIB micromachining using chemical enhancement, where the incident ions initiate chemical reactions with surface adsorbates; and using geometrical optimization of milling patterns. Chemical enhanced FIB micromachining (CE-FIBM) techniques include the use of water vapor to improve FIB micromachining of various materials including polymers, integrated circuits and diamond as well as the use of various halogen based (F, Cl and I) precursors for machining of materials ranging from Si to Fe-Ni alloys. The machining of a free edge provides a geometrical yield enhancement of more than 5 and, when combined with simultaneous chemical enhancement, can increase total yield by as much as 30 times. The major applications of FIB for preparing cross sectional analysis samples for FIB or SEM imaging, or for TEM analysis benefit in may ways from the specialized gas chemistry assistance methods developed over the past 10 years. Integrated circuit and MEMS device editing techniques have also been enhanced through the use of unique materials specific gas assisted FIB solutions.
3D Determination of Grain Shape in FeAl by Focused Ion Beam (FIB) Tomography
Authors: Inkson BJ , Mobus G
Published: July 2020

Techniques for the 3D analysis of materials at the sub-micron level are of major importance for understanding the structure and performance of new nanoengineered materials. Established techniques for determining 3D shapes of individual grains include X-ray tomography and light microscopy for grains ≫ 1μm, and 3D-atom probe for conducting grains < 50nm size. A new technique of TEM EELS tomography is being developed for particles 2-300nm in a chemically distinct matrix.

For the grain size range 200nm-20μm, we have applied a new technique of 3D analysis using focused ion beam (FIB) sectioning to the site-specific analysis of the shape of individual grains in a single phase matrix. in the 3D FIB technique, a sample surface may be serially sectioned using a highly focused beam of Ga+ ions (Fig. 1). A sequential set of 2D secondary electron images through the sample can be aligned in 3D by cross-correlation of the position of reference markers in the 2D images.
A Method for Thinning FIB Prepared TEM Specimens after Lift-Out
Authors: Rossie B B , Shofner T L , Brown S R , Anderson S D , Jamison M M , Stevie F A
Published: July 2020

Advantages of the FIB lift-out technique over traditional H-bar TEM specimen preparation have been recognized. The ability to rapidly (< 1 hour) prepare a site specific TEM specimen without destroying the entire bulk specimen has led to a wide spread reliance on this method. The main disadvantage of this technique is an inability to accomplish additional membrane thinning if required. Traditional H-bar preparation allows additional thinning. However, mechanical polishing is time consuming and the bulk sample is destroyed. A method has been developed which combines the efficient, site specific advantages of the lift-out method with the H-bar's ability to accomplish additional thinning. in this procedure a lift-out specimen is removed from the bulk sample and mounted onto a half-grid in a configuration similar to that employed by the H-bar technique.

A 1.0-micron thick lift-out specimen was prepared using a FEI Strata DB-235 FIB dual-beam workstation by sputtering away bulk material leaving a thin membrane containing a desired feature (FIG 1).
Application of the FIB Lift-Out Technique for the TEM of Cold Worked Fracture Surfaces
Authors: Giannuzzi Lucille A , White Henry J , Chen Wayne C
Published: July 2020

The FIB lift-out (LO) technique has been applied to a sample that been pulled to failure by tensile testing. An aluminum alloy (5083) was cold deformed using DSI's Maxistrain technology [1]. The alloy was cut with a low speed diamond coated wafering blade such that the fracture surface would fit inside the focused ion beam (FIB) specimen chamber. The sample was then mounted and placed in an FEI 200TEM FIB instrument. An FIB image of the tip of the fracture surface is shown in figure 1. A region for TEM specimen preparation was chosen such that the specimen would be in the plane of the tensile axis. The area of interest was chosen to ensure that FIB undercutting of the specimen could be accomplished upon tilting inside the FIB, and to ensure that the micromanipulator needle would have access to the FIB prepared specimen. A region of interest was coated with Pt using the FIB. A TEM specimen was then prepared in the usual LO manner [2,3]. The location of the finished LO specimen is circled in figure 1. After FIB milling was completed, the sample was removed from the FIB and placed on a light optical microscope stage. The electron transparent membrane was removed from the deformed sample using a glass rod attached to a micromanipulator. While the successful LO was performed, it should be noted that the micromanipulation was non-trivial due to the rough fracture surface combined with the poor depth of field of the light optical microscope.
Combined Focused Ion Beam, Energy Filtered TEM and STEM Techniques for Semiconductor Device Defects Observation
Authors: Pantel R , Sondergard E , Delille D , Kwakman LFTz
Published: July 2020

In this communication we present a method using Focused Ion Beam (FIB), Energy Filtered TEM (EFTEM) and STEM for semiconductor defects interception and high resolution observation. This method takes benefit of the specific imaging properties of respectively EFTEM and STEM techniques. A demonstration is presented for the observation of dislocations in a silicon integrated circuit.

Silicon defects such as dislocations are one of the major issues in Integrated Circuit fabrication. Due to their low distribution density these silicon defects are not easily observed using TEM cross section. in this paper we present a more appropriate technique using: FIB, EFTEM and STEM for such defects observation. First, in order to improve the probability of defects being present in the specimen, a very thick lamella is prepared using FIB. Then, using an energy filter for eliminating the inelastic electrons needed to allow thick specimen TEM imaging, direct defect localization and observation at medium resolution is carried out. After that, taking advantage of the beam broadening in the STEM, the defect position inside the specimen is more accurately evaluated. For that purpose two STEM observations are carried out from the two opposite specimen surfaces. The comparison of image contrast is used for further FIB specimen thinning. The process can be continued down to a thickness compatible with an acceptable spatial resolution TEM imaging.
A Method for Preparation of Site-Specific Multiple Samples of Semiconductor Material for Transmission Electron Microscopy
Authors: Rai R S , Bagchi S , Duncan L , Prabhu L , Beck J , Conner J
Published: July 2020

In recent years, the availability of focused ion beam (FIB) milling systems has given a much-needed boost for transmission electron microscopy (TEM) as a technique for site-specific analysis. Much progress has been made in the area of site-specific cross-sectional and planar TEM sample preparation techniques. However, a continuing need exists to reduce the sample preparation time, in order to improve TEM cycle time for better support of process development, yield improvement and production in a high-volume industrial environment. Thus, a faster TEM sample preparation technique is always desirable to meet this demand. A new approach to TEM sample preparation is described in this paper.

Following the new approach developed in the present work, one can prepare on a single TEM grid at least two different cross-sectional samples of site-specific device structures or up to four different cross-sectional samples of blanket films. Two different samples, each containing an area of interest near the center, are cleaved or cut to a width of about 1.25 mm; these samples may be from two separate locations of a wafer, or from two different wafers where TEM analyses are required.
TEM of Sub-Micrometer Particles using the FIB Lift-Out Technique
Authors: Lomness Janice K , Kempshall Brian W , Giannuzzi Lucille A , Watson Mark B
Published: July 2020

The ability to acquire TEM specimens from micrometer sized particles using an innovative FIB LO technique has been previously demonstrated [1-3]. This technique allowed for the preparation of a TEM LO specimen from regions which were smaller than the conventional dimensions of the LO specimen (∼5 μm × 20 μm). This paper shows how the LO technique previously developed for the preparation of micrometer sized particles [3] may be extended and utilized for the preparation of submicrometer sized particles for TEM analysis.

In this study, TiO2 particles on the order of 200 nm in width were investigated. A small portion of the TiO2 particles was dusted onto double stick carbon tape adhered to a sample stud and then sputtered coated with Au-Pd to protect the outer surface from FIB imaging. The sample stud containing the TiO2 particles was placed in an FEI 200TEM FIB workstation. Figure 1 is a FIB image of the Ti02 particles of interest. Gaps around these particles were deposited with Pt using the FIB assisted CVD process as shown in Figure 2. in this procedure, the Pt acts as a “weld” to hold the particles together, and to allow for a larger region to be milled (e.g., >10 μm) for subsequent specimen preparation.
Redeposition Effects During the FIB Milling of Silicon
Authors: Rubanov S , Munroe PR
Published: July 2020

The technique for the preparation of specimens for transmission electron microscopy (TEM) using the focused ion beam (FIB) miller typically consists of a series of milling steps performed over both sides of an area of interest until an electron transparent membrane is achieved [1]. This process is often accompanied by the formation of damage layers on the surfaces of the specimen. The origins of any damage layer are still not clear. On one hand the process of amorphisation of the target material by the highly energetic ion beam is well known. Alternatively, other workers have reported that this damage layer can be connected with redeposition of milled material. [2,3]. in this paper we have studied redeposition effects during FIB milling of silicon TEM specimens.

A FEI xP200 FIB system with a Ga+ ion source operating at 30 kV was used in this work. to study redeposition effects a row of trenches on a silicon specimen was milled under different beam currents ranging from 1000 to 6600 pA. The size of such trenches was 15x10 μm wide and 1 μm deep. The specimen was then removed from the FIB and sputter coated with a ∼50-100 nm thick Au film to preserve the trench surfaces from further damage during subsequent milling. The specimen was then placed back in the FIB system and a second set of trenches 5×8 μm wide and 0.6 μm deep was milled on the bottom of first set of trenches (Fig. 1a). The specimens were sputter coated with Au again and were placed back in the FIB system and the trenches were then covered with 1 μ thick Pt strips using the metal deposition facility of the FIB. The presence of these protection layers (Au and Pt) ensures that the final TEM specimen have unmodified original damage layers resulting from the initial milling steps. Cross-sectional TEM specimens of the trench walls were then prepared using normal FIB procedures (Fig. 1b) [2].
A System for Computer-based Reconstruction of 3-Dimensional Structures from Serial Tissue Sections: an Application to the Study of Normal and Neoplastic Mammary Gland Biology
Authors: Fernandez-Gonzalez Rodrigo , Jones Arthur , Garcia-Rodriguez Enrique , Knowles Davis , Sudar Damir , Ortiz de Solorzano Carlos
Published: July 2020

Tissue heterogeneity and three-dimensionality are generally neglected by most traditional analytical microscopy methods in Biology. These often disregard contextual information important for understanding most biological systems. in breast cancer, which is a tissue level disease, heterogeneity and three dimensionality are at the very base of cancer initiation and clonal progression. Thus, a three dimensional quantitative system that allows low resolution virtual reconstruction of the mammary gland from serial sections, followed by high resolution cell-level reconstruction and quantitative analysis of the ductal epithelium emerges as an essential tool in studying the disease. We present here a distributed microscopic imaging system which allows acquiring and registering low magnification (1 pixel = 5 μm) conventional (bright field or fluorescence) images of entire tissue sections; then it allows tracing (in 3D) the ducts of the mammary gland from adjacent sections, to create a 3D virtual reconstruction of the gland; finally it allows revisiting areas of interest for high resolution (1 pixel = 0.5 μm) imaging and automatic analysis. We illustrate the use of the system for the reconstruction of a small volume of breast tissue.
Automation for Cryo-TEM: from Specimen Grid to 3D Map
Authors: Carragher B , Fellmann D , Kisseberth N , Milligan RA , Potter CS , Pulokas J , Zhu Y
Published: July 2020

Cryo-electron microscopy is becoming an increasingly powerful tool for solving the structure of protein complexes and has the potential to address problems that cannot be solved using other methods. The field however suffers from several major disadvantages related to the time required to acquire, process and analyze the data and the tedium of using the current prevailing methods. We have for some time been working towards the goal of developing a system that will result in a 3D map of a macromolecular structure automatically and within hours of inserting a specimen into a transmission electron microscope. We propose that these automated methods for data collection and analysis will have a significant impact in transferring the cryo-electron microscopy technology to the general biological community as well as in increasing the volume of data that can be collected during a single session at the microscope.

The Leginon system that we have developed is designed to emulate all of the decisions and actions of a highly trained microscopist in collecting data from a vitreous ice specimen. These include identifying suitable areas of vitreous ice at low magnification, determining the presence and location of specimen on the grid, automatically adjusting imaging parameters (focus, astigmatism) under low dose conditions and acquiring images at high magnification to either film or a digital camera.
Automated Data Collection with a Tecnai 12 Electron Microscope for Applications in Structural Biology
Authors: Zhang Peijun , Beatty Alexis , Milne Jacqueline LS , Subramaniam Sriram
Published: July 2020

In high-resolution biological electron microscopy, the speed of collection of large numbers of micrographs of the required quality is a rate-limiting step in the overall process of structure determination. Approaches to speed up data collection can be very useful especially in “single molecule” microscopy of large multi-protein and protein-nucleic acid complexes, where many thousands of individual molecular images need to be averaged to determine the threedimensional structure.

Here, we report the development of an automated low-dose image acquisition procedure on a Tecnai 12 electron microscope using the scripting functionality available on the microscope computer. The program that we have developed and implemented, AutoEM, is presently aimed at providing automation capabilities for the user familiar with normal operation of the microscope. However, the user’s participation is relatively minimal, and involves the following steps. After preparing a specimen grid and inserting it into the microscope as is normal for a cryo transfer session, the user simply downloads a Low-Dose alignment file that contains information on the relative beam shifts between the Search, Focus and Exposure modes that would normally be employed in a manual low-dose data collection experiment.
Automatic Page-Layout Scripts for Gatan Digital Micrograph®
Authors: Hou Vincent D-H
Published: July 2020

The software DigitalMicrograph (DM) by Gatan, Inc., is a popular software platform for digital imaging in microscopy. in a service-oriented microscopy laboratory, a large number of images from many different samples are generated each day. It is critical that each printed image is properly labeled with sample identification and a description before printing. with DM, a script language is provided: from this, various analyses can be designed or customized and repetitive tasks can be automated. This paper presents the procedures and DM scripts needed to perform these tasks. Due to the major software architecture change between version 2.5x and version 3.5x, each will be discussed separately.

DM Version 2.5.8 (on Macintosh®)A “Data Bar” mechanism is provided in this version of DM. Using the “Edit→Data Bar→Define and Add Data Bar...“ menu command specifies data bar items (e.g., scale bar, microscope operator) to be included in the image. in addition, other annotations (text, line, rectangle, and oval) can be included as part of “Data Bar.” This is done by first selecting the desired annotation on the image and then using the “Edit→Data Bar→use AS Default Data Bar...” menu command. After defining data bar items, executing the menu command adds these data bar items to the image.
Automated Determination of Tc for a High Tc Superconductor During Electron Irradiation Experiments in the Argonne HVEM
Authors: Ockers Stanley T , Ryan Edward A
Published: July 2020

Over the more than twenty years of operation of the 1.2 MeV AEI/Kratos HVEM at Argonne (Fig 1.), the primary emphasis by the user community has been on a wide variety of in situ irradiation effects studies involving both electrons and ions. in many cases it has been essential for the success of experiments for the professional technical staff of the Facility to develop specialty devices and techniques. This contribution deals with one such system which we have developed, namely, the design, assembly and operation of a system for in situ automated measurement of specimen electrical resistivity as a function of temperature in the range 15-135 K. A major problem is associated with this situation, which involves a small gauge-section conductor, requiring stable amplification and measurement of very minute electrical signals. The specific set of experiments for which this application was developed involved determining the effect of electron irradiation on the superconducting transition temperature Tc of YBaCuprate (YBCO) in order to distinguish between two possible theories of the conduction mechanism. in this type of experiment the critical temperature Tc is determined from a plot of electrical resistivity as a function of temperature following irradiation at 100 K at zero applied magnetic field to preclude the Meissner Effect. in these experiments Tc is 89 K prior to electron irradiation and drops to <15 K with increasing electron fluence. The analysis also revealed an oxygen-disorder-induced superconductor to normal-metal transition after sufficient electron fluence. Fig. 2 shows typical measurement results for two temperature ramps. The detailed results of these experiments are described elsewhere by Giapintzakis et al.
Eliminating effects of biprism drift in electron holography
Authors: Kahl F , Voelkl E
Published: July 2020

Reconstruction of phase images from off-axis electron holograms recorded in a field-emission TEM was for many years a tedious and time-consuming process. The advent of digital imaging systems and ever-faster desktop computers has recently resulted in processing times of a few seconds for a useful phase reconstruction. However, a goal of the “holographer” has been to view the phase images essentially in “live-time”, that is, at least at a few frames per second. This enables effective observation of phase change, through “a window to the phase world,” while the sample is being searched. Chen et al. used a mixture of digital and analog techniques to obtain phase images at nearly TV scan rates. Because optical lens systems provide the fast Fourier processing required for phase reconstructions, their geometry allows phase images to be displayed while the sample is being moved and/or timedependent specimen changes are occurring. Voelkl et al. have described a purely digital system for phase reconstructions from electron holograms that provides several phase images per second, and has several advantages over the mixed system. The refresh rate is presently limited more by the readout time of the slow-scan digital camera than by the computational time of the computer.
Structure-Property Correlation in Genetically-Engineered Mouse Enamel
Authors: Fong Hanson , Heidel Daniel , Sarikaya Mehmet , Paine Michael , Lou Wen , White Shane N , Snead Malcolm L
Published: July 2020

Dental enamel is the most durable bioceramics produced by a vertebrate as it is designed to perform masticatory functions throughout its lifetime. The understanding of the mechanism of enamel formation and effects of proteins during the biomineralization process are fundamental issues, essential for both potential enamel regeneration and as a base for synthesis, via self-assembly, of biomimetic composites.

The biomineralization process of enamel is carried out by ameloblast cells that line the inner enamel epithelium and secrete an extracellular protein matrix onto a mineralized dentin surface at the dentin-enamel junction (DEJ). A major matrix protein, amelogenin, is believed to regulate the mineralization of hydroxyapatite (HAP) in the enamel tissue. It has been shown to undergo self-assembly in vitro and in vivo to form nanospheres of ∼20nm in diameter. Previous TEM studies have shown that the nanospheres align along the length (c-axis) of hydroxyapatite (HA) crystals. There are two domains, namely A (residues 1-42) and B (residues 157-173), that control the self-assembly behavior of the nanospheres.
Trace Metal Detection by X-ray Microanalysis in Vascular Cells of Roots and Leaves from Field-Grown Sugar Maple Seedlings
Authors: McQuattie CJ , Long RP
Published: July 2020

The low survival of sugar maple (Acer saccharum) seedlings growing in unglaciated acidic forest soils (pH<4.0) in north-central Pennsylvania may be due in part to aluminum (Al) or manganese (Mn) phytotoxicity. Although foliar analysis has shown high concentrations (>2,000 ppm) of Mn, the location of potentially toxic elements in leaves or absorptive fine roots has not been studied in field-grown seedlings. This study of vascular (xylem and phloem) cells was conducted to determine by energy-dispersive X-ray microanalysis (EDX) whether Mn and/or Al could be detected in fine root or leaf midvein vascular cells. A secondary objective was to determine if vascular cells prepared by chemical fixation (CF) for conventional TEM or by freeze-substitution (FS) for EDX showed similar ultrastructural characteristics.

Four sugar maple seedlings from an unglaciated field site (Potter County PA) were excavated, and roots were washed carefully. Two mm-long segments of fine roots and leaf midveins from each seedling were immersed in 3% glutaraldehyde for 6 hours, transferred to 0.1 M phosphate buffer, and transported to the Delaware Research Lab where tissues were post-fixed in OsO4 (in buffer), dehydrated in ethanol, and embedded in PolyBed-Araldite epoxy resin (CF method).