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.

The Small Angle Cleavage Technique (SACT) can produce samples from brittle and cleavable materials very quickly. It is possible for a skilled person to produce about 10 XTEM specimens in an hour. However, there are materials related limitations to the technique. These include 1) the ductile/brittle nature of the substrate, i.e. the fracture mechanics of the materials, 2) the strength of the interfaces between multi-layers, 3) the thickness of any ductile layers, and 4) the strength of the layer materials. in most of the cases where SACT fails, the sample is near electron transparency at the apex of the sample. Where there is a weak interface as in some multi-layer systems, or where the material itself is weak such as when an evaporated Ge layer is used, some of the film near the substrate is sufficiently thin, but there is a jog in the film stack and further back from the apex it is not transparent. If such a SACT sample is viewed from the surface in an SEM, this apparent delaminating of the top layers is evident and usually within 1 μm of the apex.

The purpose of this paper is to show that the geometry of a SACT-prepared sample is well suited for applying FIB milling for further thinning of the sample to electron transparency. in addition, the FIB can be easily applied to SACT specimens that fail to produce usable samples for the reasons cited above. The SACT method allows two main options for mounting the samples that are illustrated in Fig 1.

In recent years, the use of the focused-ion beam (FIB) microscope has become widespread in the areas of materials processing and materials characterizaation. Although initial commercialization of FIB instruments was driven by applications in the semiconductor industry, recently the FIB has emerged as a broad characterization tool capable of imaging, material removal and material deposition. This combination makes it a useful instrument for applications ranging from site-specific sample preparation for transmission electron microscopy1 to thin-film head manufacturing. However, since the interaction of a high-energy ion beam (e.g., 30 keV Ga) with a solid inevitably produces implantation damage and the possibility of other effects such as grain growth, dislocation motion or degradation of magnetic properties , it is important to quantify to what extent the material under examination has been modified. Simple TRIM simulations may provide an estimation of the implantation level and depth to which ions will travel into a solid, but these results may not be accurate because FIB milling is not a static situation.

In order to investigate Ga implantation depth, concentration and possible grain growth effects, three circular regions on a 500 nm thick electroplated Ni-80 at.% Fe film were milled using 30 keV Ga ions at three different beam currents, Fig. 1.

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].

The focused ion beam (FIB) miller allows preparation of site-specific transmission electron microscopy (TEM) specimens from a wide range of materials in both cross-sectional and planar configurations [1,2]. However, radiation damage during exposure to the high-energy gallium beam may result in the formation of amorphous regions on thin film specimens. The thickness of such damage layers, on both sides of a TEM specimen, is comparable with the thickness required for lattice imaging. For example, the thickness of an amorphous layer in Si after 30 kV Ga+ FIB processing has been reported in the range from 15 [3] to 28 nm [4]. This problem limits the capabilities of FIB sample fabrication.

The aim of this study was to investigate, in detail, the structure, composition and the thickness of the damage layers in Si specimens after milling with a gallium ion beam. Using a FEI xP200 FIB system, with 30 kV Ga+ ions, a row of trenches on a silicon sample was milled under different beam currents ranging from 150 to 6600 pA. The average size of such trenches was 15×10 μm wide and 1 μm deep. The trenches were then removed from the FIB and sputter coated with a thick Au film to preserve the trench surfaces from further damage during subsequent TEM specimen preparation steps. Cross-sectional TEM specimens of the trench walls were then prepared using standard FIB procedures [5]. Observations were made using a Philips CM 200 Field Emission Gun TEM operating at an accelerating voltage of 200 kV.

Critical dimensions in advanced semiconductor devices are now such that a significant fraction of some structures is contained within the thickness of a TEM foil. An important example is the vias used in multilevel metallization to interconnect transistors and other structures. Current generation vias have a diameter of 300 nm, and vias in devices under development are targeted at 180 nm. These dimensions are to be compared to the thickness of a TEM foil, which may be ∼50 nm. Vias are typically fabricated by etching holes in a dielectric layer, then depositing into these holes a barrier metal, such as Ta, followed by a Cu seed layer and finally Cu fill. Critical to via processing reliability are the thicknesses of barrier and seed layers. A TEM cross section of such a via will contain sharply curved layers which, when seen in projection, will lead to inaccurate measurement of layer thicknesses. Ordinary plan view preparation of these vias avoids this projection problem, but uncertainty is introduced in the height at which the via is sectioned.

We have developed an angled plan view (APV) FIB preparation technique that allows accurate measurement of barrier and seed thicknesses at discrete heights from bottom to top of a via.

The considerable improvements in computational power and the increased sophistication of programming techniques now make it possible to automate many aspects of electron microscopy [1]. Automation offers several important advantages over manual operation. Automation of microscope operation permits novice microscopists to obtain data relatively easily thus increasing instrument utilization. The experienced microscopist should be able to acquire data more rapidly and with greater ease compared to manual operation, resulting in increased efficiency. Alignment of energyfilters and proposed Cs correctors are difficult and time consuming even for experienced microscopist and stable alignment algorithms are critical for practical use. Automation is essential for extensive statistical sampling of microstructures and can permit experiments to be performed that were previously impractical. For example, spectrum imaging involves acquisition of thousands of spectra from a predefined area. The acquisition and processing of such large volumes of data cannot be accomplished without the aid of automation. The future prospects for automation are exciting, although it is important to carefully consider the relationship between the computer controlled microscope and the operator. Here I give a personal perspective on approaches to automation which increase the power and efficiency of modern transmission electron microscopes.

The degree of automation is an important consideration that is related to the complexity of the analysis strategy. Some analysis situations call for repetitive sampling of a well-defined quantity such as layer thickness or particle size. These simple repetitive operations are ideal candidates for total automation. For many applications of automation it is necessary not only to acquire the data but also to process the data and extract the quantity of interest.

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.

The second generation of computer-controlled scanning electron microscopes (SEMs), i.e. instruments directed by software executing on conventional personal computers (PC-SEMs), has become the industry standard. Although limited automation has been incorporated by most manufacturers, these features do not significantly alleviate the requirement for operator expertise to attain optimal performance from the microscope. Our approach to the expertise deficit has been to pursue a knowledge-based approach to microscope control, encapsulating human knowledge in software to achieve expert performance. The task of SEM operation was decomposed into five phases - Specimen Preparation, SEM Preparation, SEM Initialisation, SEM Optimisation and External Analysis - and the central three phases were modelled in detail. A stand-alone prototype system (XpertEze) was developed to utilise this model for the LEO 400 series. A web-based version was implemented to support intelligent remote microscopy.

A stand-alone knowledge-based system has several disadvantages: its user interface competes for screen space with the conventional control software, its speed of execution is reduced as its instructions must be transferred via application programming interfaces to the microscope, and the operator must learn and manage two sets of software. Our ongoing work has been to remove these problems by incorporating the XpertEze system directly within the control system.

The integration of computers and transmission electron microscopes (TEM) in combination with the availability of computer networks evolves in various fields of computer-controlled electron microscopy. Three layers can be discriminated: control of electron-optical elements in the column, automation of specific microscope operation procedures and display of user interfaces. The first layer of development concerns the computer-control of the optical elements of the transmission electron microscope (TEM). Most of the TEM manufacturers have transformed their optical instruments into computer-controlled image capturing devices. Nowadays, the required controls for the currents through lenses and coils of the optical column can be accessed by computer software. The second layer of development is aimed toward further automation of instrument operation. For specific microscope applications, dedicated automated microscope-control procedures are carried out. in this paper, we will discuss our ongoing efforts on this second level towards fully automated electron tomography. The third layer of development concerns virtual- or telemicroscopy. Most telemicroscopy applications duplicate the computer-screen (with accessory controls) at the microscope-site to a computer-screen at another site. This approach allows sharing of equipment, monitoring of instruments by supervisors, as well as collaboration between experts at remote locations.

Electron tomography is a three-dimensional (3D) imaging method with transmission electron microscopy (TEM) that provides high-resolution 3D images of structural arrangements. with electron tomography a series of images is acquired of a sample that is tilted over a large angular range (±70°) with small angular tilt increments.

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.

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.

Recently, SEM (Scanning Electron Microscope) and the other observation devices are coming to use a LAN (Local Area Network) to save the image in the database. We developed a remote control system in which SEM image and Control interface is indicated on the WEB Browser. in this system, SEM can be controlled by an external (client) PC installed in a general WEB Browser(Internet Explorer). Accordingly, operation interface can be indicated on the WEB browser.

A JSM-6700F is connected to a LAN, and so the microscope can be controlled by a client PC. Figure. 1 shows the block diagram of SEM remote control system. The JSM-6700F has two lines to the LAN for image transfer and communication with the SEM control. in order to transfer the image, the image size squeezes from 1280 × 1024-pixels (SEM image size) to 640 × 480-pixels for quick transfer. The image signal (640 × 480-pixels) is connected to the video server only, and then the image transfers to the client PC via LAN . The SEM control communicates with client PC for external command. On the other hand, the SEM control interface and the image are indicated on WEB Browser (Internet explorer) as shown Figure 2. The SEM control interface is composed of the SEM image area and the SEM control part. The SEM image indicates the 640×480-pixels live image. This pixel number was an image quality sufficient to adjust the focus. When a high quality image is necessary, the image of 1280 × 1024-pixels is saved on the the SEM(server) side by choosing the image save button.

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.

The annual concrete consumption of two tons for every U.S. citizen makes it the most widely used construction material. Preserving this large concrete infrastructure requires an understanding of the deterioration and failure processes. Concrete exhibits composite behavior at several length scales (millimeter, micron, and submicron). in conjunction with this complex micro structure, damage processes such as crack formation and void collapse occurs at these various length scales. The diverse length scales combined with the large dimensions (meters) of concrete systems necessitates examination of macroscopic areas/volumes at microscopic magnifications to characterize damaged structures. This translates into examining large specimens, capturing a large number of images, and performing analysis on each image. Automating the image capture and image analysis greatly reduces the labor in evaluating damaged concrete structures.

Automated specimen positioning and image capture from an environmental scanning electron microscope (ESEM, ElectroScan 2020) and automated image analysis (Noesis Visilog 5.3 image analysis software) were synthesized with statistical and stereo logical principles into a comprehensive system for quantitative micro structural investigation of concrete.

We have developed a new Electron Probe Microanalyzer (EPMA), JXA-8100/8200, with improved basic capabilities such as X-ray intensities of wavelength dispersive spectrometers (WDS), imaging functions, automated functions and analysis software. Fig. 1 shows the appearance of JXA-8200, WD/ED combined microanalyzer. in this session, we report mainly on the improved imaging functions, automated functions and analysis software.

The JXA-8100/8200 is the first EPMA in the world to feature 1280 x 1024 pixels high resolution live scanning image display. Regarding scanning image, two or four different signal live images, of course including X-ray images, can be displayed simultaneously. Further, image mixing is also possible to display. On the high resolution image, an operator can choose the probe position or the stage position by mouse clicking. The stage position can also be chosen on the optical microscope (OM) live image. Another new “Swing Mouse” function, which is the seamless movement of mouse pointer between the scanning image display and the computer display, has been developed.

Advanced automated functions, such as autofocus, auto astigmatism and auto contrast brightness, are effective to optimize the scanning image.

A typical TEM specimen grid provides approximately a 2×2 mm area that is available for imaging. in order to identify and locate suitable targets on the grid the microscopist must usually inspect the grids at magnifications that allow for only a small area of the grid in the field of view at a time. Systematically searching the grid and mentally keeping track of relative locations presents a challenge for a microscopist especially as the image normally rotates as the magnification is changed. We present an automated technique that creates a very low magnification (VLM) image of the entire available imaging area on the grid. The VLM image can then be used as a reference map for searching the grid at high magnification.

VLM images were obtained at a nominal magnification of 57x using a Philips CM200 TEM equipped with a Gatan CCD camera. The VLM image of the entire 2 mm specimen grid can be created using a mosaic of 49 images, each 512×512 pixels in size. The sampling distance between the images in the 7×7 array is 300μm and the pixel size is 680nm. The images are then automatically tiled and re-sampled to form a final VLM matrix of 4K×4K with a pixel size of 730nm. The resampling factors for the tiling operation include the scale and the relative angle between the camera and goniometer axes. These factors are determined automatically from a calibration process that characterizes the goniometer.

We have been developing a software system, called Leginon, for the control and acquisition of images from a transmission electron microscope. This software allows for the automatic acquisition of images under low dose conditions from a specimen embedded in vitreous ice. Thousands of images are collected during each session at the microscope. These images are collected at various magnifications and under a variety of conditions. For example, images are collected of the entire grid, individual grid squares, individual holes within the square, and several high magnification images are acquired for one to several targets within each hole (figure 1). in addition, for each identified hole an image is acquired at the low dose focus position. Power spectra are also calculated for each of the high magnification images as well as for the focus images. For managing these images we are using a relational database management system. We have also developed a bench of tools to store, access, and display the data as well as tools for extracting information from the database.

A database is a structured collection of data in which the information is classified by categories. Each category is then stored in a table and these tables are linked together by defined relations. Data may be combined from several tables on request. We have been using MySQL as our relational database management system. This allows us both to manage the images and to refer each image to its real context. in other words, through the database, we can keep track of information related to the microscope settings (e.g. defocus, electron dose, magnification, goniometer coordinates etc.), the type of image, and overall information about the specimen and the experiment.

For several years, we have been involved in developing a completely automated system for cryoelectron microscopy, which will allow a 3D electron density map of a macromolecular structure to be generated from a specimen inserted into the microscope with no manual intervention required by an operator. Towards this goal, we have developed a system, called Leginon, to automatically acquire images from the electron microscope. In order to integrate the 3D reconstruction process with the data acquisition step we need then to automatically identify and segment objects from the electron micrographs. This presents a challenge, particularly in the case when micrographs are acquired very close to focus, which results in an image with extremely low contrast. We present here the results of a two-stage automated process for detecting and selecting filamentous structures from images of this kind. Images are acquired in defocus pairs; a near-to-focus (NTF) image is acquired first, followed by a far-from-focus (FFF) image. In the first stage of the process, a threelevel perceptual organization algorithm is used to identify and segment filaments in the FFF image. In the second stage the NTF image is aligned to the FFF image using phase correlation techniques and the filaments are selected at the same coordinates as were identified in the FFF image.

In humans, perceptual organization is the ability to immediately detect structural relationships such as co-linearity, parallelism, and connectivity among image elements. Researchers in human vision perception have long recognized the importance of the common rules by which our visual system attempts to group information. Some of these rules include proximity, similarity, common fate, continuation and closure.

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.