From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

Highly porous particles with internal triply periodic minimal surfaces were investigated for sorption of proteins. The visualization of the complex ordered morphology requires complementary advanced methods of electron microscopy for 3D imaging, instead of a simple 2D projection: transmission electron microscopy (TEM) tomography, slice-and-view focused ion beam (FIB) and serial block face (SBF) scanning electron microscopy (SEM). The capability of each method of 3D image reconstruction was demonstrated and their potential of application to other synthetic polymeric systems was discussed. TEM has high resolution for details even smaller than 1 nm, but the imaged volume is relatively restricted (2.5 μm)3. The samples are pre-sliced in an ultramicrotome. FIB and SBF are coupled to a SEM. The sample sectioning is done in situ, respectively by an ion beam or an ultramicrotome, SBF, a method so far mostly applied only to biological systems, was particularly highly informative to reproduce the ordered morphology of block copolymer particles with 32–54 nm nanopores and sampling volume (20 μm)3.

Mitochondria play an essential metabolic role in all eukaryotic cells because they are the organelles that provide energy to drive chemical reactions. Mitochondria are highly dynamic and change their shape between discrete structures and large interconnected networks by selective fission and fusion of their membranes. The resulting fused three‐dimensional networks can permeate through an entire cell, but their advantages, relative to isolated discrete organelles, are not fully understood. It has been suggested that networks might have the following functions: dampen biochemical fluctuations; provide a mechanism for quality control and selective mitophagy; enable diffusion of proteins; and transmit calcium signals and mitochondrial membrane depolarization throughout the cell [1, 2]. Despite their highly dynamic state, useful structural information can nevertheless be obtained from electron microscopy by studying the organization of mitochondrial networks at a given time point. We have used a SIGMA VP (Zeiss Inc.) scanning electron microscope (SEM), equipped with a 3View (Gatan Inc.) serial block face (SBF) system [3], to quantify the connectivity of mitochondrial networks in entire insulin‐secreting b ‐cells of mouse pancreatic islets of Langerhans. Previous work has suggested that alterations in mitochondrial fission and fusion might play a role in nutrient‐induced b ‐cell apoptosis with possible involvement in the pathophysiology of Type 2 diabetes [4]. Previously, we found that manual segmentation of SBF‐SEM mitochondrial volumes in a single b ‐cell took about one week for a trained operator, which precluded analysis of multiple cells within a reasonable time. We have explored a faster and more practicable approach by making use of tools within the Amira visualization software package (FEI Inc.) to partially automate the segmentation process, and enabling an entire cell to be analyzed in approximately two hours (Figure 1). Using this approach we were able to analyze quantitatively mitochondrial networks in individual pancreatic b ‐cells in terms of the total mitochondrial volume, average volume per network, total network length, and average network length. We found that approximately one‐third of the mitochondrial volume was contained in networks of length less than 3 µm, one‐third in networks of length between 3 µm and 10 µm, and one‐third in highly fused networks of length between 10 µm and 60 µm (Figure 2). SBF‐SEM thus provides quantitative data on the organization of mitochondrial networks, making it feasible to test computational models for mitochondrial fusion and fission.

Extract Extended abstract of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007

Optical fibers have been the basis of the modern information technology since Kao and Hockham proposed glass waveguides as a practical medium for communication in 1965. A lot of different optical fiber active/passive devices including couplers, interferometers, gratings, resonators and amplifiers have been widely employed for applications on telecommunications and sensing networks (Agrawal, 2002). For a number of applications, it is important to reduce the device’s size. Small size is often attractive for particular sensing applications because of some benefits such as fast response to detecting small objection with little perturbation on the object being measured. There are two steps to obtain fiber devices as small as possible. First, it is to taper or etch the fiber and reduce its diameter. A subwavelength-scale microfiber is the basic element of miniature fiber devices and subsystems (Tong et al., 2003; Brambilla et al., 2004, 2005, 2010). The second is to engineer the microfiber to realize miniature version of conventional fiber devices. There are various fabrication methods to engineer the microfiber, such as CO2 laser, femtosecond (fs) laser, HF acid etching, arc splicing and focused ion beam (FIB). Most of these techniques have the difficulties in carving the microfiber freely because of the resolution. The latest progress in FIB technique has opened a new widow for ultra-small size fiber devices. So far, FIB is the most flexible and powerful tool for patterning, cross-sectioning or functionalizing a subwavelength circular microfiber due to its small and controllable spot size and high beam current density. FIB systems have been produced commercially for approximately thirty years, primarily for large semiconductor manufacturers. FIB systems operate in a similar fashion to a scanning electron microscope (SEM) except, rather than a beam of electrons and as the name implies, FIB systems use a finely focused beam of ions that can be operated at low beam currents for imaging or high beam currents for site specific sputtering or milling (http://en.wikipedia.org/wiki/Focused_ion_beam). The fine and controllable ion spot size and high beam current density are perfect for microand nano-fabrications with high spatial resolution (~ 10 nm). As a result, FIB has recently become a popular candidate for fabricating high-quality micro-devices or high-precision microstructures. Originally, FIB processing was used for mask repair (Liang et al., 2000), integrated circuit chip repair/modification (Liu et al., 2006), cross-sectional imaging of critical parts of

The global energy landscape is undergoing a profound transformation, shifting from traditional fossil fuel-based systems to sustainable, renewable energy sources like wind, solar, bioenergy, hydroelectric, and tidal energy. This transition signifies a significant evolution in global energy generation and consumption patterns, necessitating advanced energy storage solutions to manage the intermittent nature of renewables and ensure a stable and reliable energy supply. Advanced energy storage technologies, including batteries, supercapacitors, and other innovative systems, play a pivotal role in this transition by enabling effective load balancing, supporting the electrification of transportation through electric vehicles and charging infrastructure, managing peak demand, and facilitating the seamless integration of renewable energy into the grid. This evolution not only advances sustainability goals but also enhances energy security and independence. [1]Advanced analytical techniques are essential for developing energy storage systems, especially batteries, by providing critical insights into their complex chemical and physical processes. Battery materials' microstructure, composition, and operational dynamics can be explored using analytical techniques like scanning electron microscopy (SEM), focused ion beam (FIB), transmission electron microscopy (TEM), X-ray diffraction (XRD), and spectroscopy. The detailed analysis through these techniques is crucial for optimizing material properties, enhancing energy density, improving safety, and extending battery lifespan. By identifying degradation mechanisms and optimizing electrode and electrolyte materials, these techniques contribute to precise battery engineering. As demand grows for more efficient and higher-capacity batteries, these sophisticated tools are indispensable for advancing energy storage technology. [2]At Shell Technology Center Houston, we have developed air-controlled and cryogenic electron microscopy (EM) workflows for studying advanced energy storage systems in preparation for the energy transition. The air-controlled EM workflow, employing SEM, FIB, TEM, and XRD, plays a pivotal role in battery research by providing a controlled environment that prevents exposure to atmospheric elements that could alter material properties. These techniques are essential for examining the fine structural details and chemical compositions of battery components under near-native conditions. The cryo-EM workflow using SEM, FIB, and TEM is vital in battery research for studying materials and interfaces sensitive to environmental conditions or rapid degradation upon exposure. These cryogenic techniques involve rapid freezing of battery components to preserve their structure in a close-to-native state and prevent deleterious effects of air exposure such as oxidation or moisture interaction. [3]In our study, we present comprehensive results obtained using both air-controlled EM and cryogenic EM workflows to investigate advanced energy storage systems, including lithium-ion batteries (LIBs) and beyond-LIBs such as multivalent rechargeable batteries. The utilization of air-controlled EM techniques such as SEM, FIB, TEM, and XRD allows us to examine battery components under controlled environmental conditions, ensuring accurate analysis of structural details and chemical compositions without the risk of atmospheric contamination. Additionally, the use of cryo-EM techniques, including cryo-SEM, cryo-FIB, and cryo-TEM, enables the study of sensitive battery materials and interfaces in a preserved, near-native state. These cryogenic methods prevent degradation effects such as oxidation and moisture interaction, providing unprecedented insights into the ultrastructural properties and operational dynamics of battery materials at atomic or molecular levels. By leveraging both air-controlled and cryogenic EM workflows, we have achieved a comprehensive understanding of battery mechanics and chemistry, paving the way for improved battery design and enhanced performance in energy storage applications.Reference[1] International Renewable Energy Agent (IRENA) 2021 https://www.irena.org/publications/2021/Aug/Renewable-energy-statistics-2021[2] Tarascon, J.-M., Armand, M., Nature 414, 359–367 (2001). https://doi.org/10.1038/35104644[3] Cheng, D., Lu, B., Raghavendran, G., Zhang, M., and Meng, Y. S., Matter, Volume 5, Issue 1, p26-42, 2022 https://doi.org/10.1016/j.matt.2021.11.019

High resolution investigation of the microstructure of materials and devices is very often restricted to the study of the very surface of the sample. This is because most high resolution analytical and imaging techniques like scanning electron microscopy (SEM), atomic force microscopy (AFM) or scanning tunnelling microscopy (STM) only provide information about the surface microstructure of the sample. To locally investigate the internal microstructure of the sample at high resolution, the sample has to be opened up. This can be done very precisely by the use of a focused ion beam (FIB) for cutting into the sample and the use of a field emission SEM for high resolution imaging of the internal structure. The combination of FESEM and FIB is a future key technology for semiconductor and material science related applications. A new CrossBeam tool is discussed in this presentation. Through the combination of the well known Gemini ultrahigh resolution field emission SEM column and the well known Canion31+ high performance FIB column a wide field of applications can now be accessed. This includes structural cross-sections for SEM and TEM applications, device modification, failure analysis, sublayer measurement and examination, as well as SEM and FIB related analytical techniques such as EDS, WDS, SIMS etc. Real time high resolution SEM imaging of the cutting and deposition process enables the researcher to perform very accurate three dimensional structural examinations and device modifications.

ABSTRACTKnowledge of the composition of many cultural heritage objects is limited, resulting in many unanswered questions in regards to the provenance, composition, and production methods. In this paper, our objective is to show that dual beam scanning electron microscope (SEM) and focused ion beam (FIB) can be used rapidly and non-destructively to determine the surface and bulk metal compositions in small cultural heritage objects. We show, for the first time, that this novel FIB technique can be successfully applied non-destructively to cultural heritage objects by examining three representative silver plated objects (Candelabra, “Century” spoon, and New York World’s Fair spoon) from the Dallas Museum of Art’s unparalleled collection of modern American silver. In each case, we successfully reveal and characterize the bulk metal as well as the Ag-plating, up to ∼80 µm deep and show that there is no visual damage resulting from the milling process of the FIB. This novel characterization technique can be applied, due to its ease of availability and rapid use, to many other problems in addition to silver plated objects, making dual beam SEM/FIB a possible cornerstone technique in the study of cultural heritage objects.

More than 40 years after its invention, the atomic force microscopy (AFM) can be integrated with scanning electron microscope (SEM) instruments as an increasingly capable and productive characterization tool with sub-nanometer spatial resolution. The authors have designed and developed an AFM instrument capable to be integrated into any SEM or in a combination of SEM with a focused ion-beam (FIB) tool. The combination of two or more different types of techniques like SEM, energy dispersive x-ray spectroscopy, and AFM is called correlative microscopy because analytical information from the same place of the sample can be obtained and correlated. For the first time, they introduced to the SEM/FIB tool correlative nanofabrication methods like field-emission scanning probe lithography, tip-based electron beam induced deposition, and nanomachining. The combination of all these methods provides a completely new nanotechnology instrument, which should be seen as a tool for correlative nanofabrication and microscopy. Thus, it provides for the first time the capabilities of a stand-alone instrument with the capabilities of nondestructive three-dimensional tip-based metrology and nanofabrication into the combined SEM/FIB tool. In this article, the authors describe all these methods in detail and present a brief example of the results obtained. They demonstrate that the self-sensing, self-actuating cantilevers (called active cantilevers) equipped with Diamond tip are a versatile toolkit for fast imaging and emerging nanofabrication. The AFM integrated into SEM is using active cantilevers that can characterize and generate nanostructures all in situ without the need to break-vacuum or contaminate the sample.

The availability of the focused ion beam (FIB) microscope with its excellent imaging resolution, depth of focus and ion milling capability has made it an appealing platform for materials characterization at the sub-micron, or "nano" level. This article focuses on nanomechanical characterization in the FIB, which is an extension of the FIB capabilities into the realm of nano-technology. It presents examples that demonstrate the power and flexibility of nanomechanical testing in the FIB or scanning electron microscope with a probe shaft that includes a built-in strain gauge. Loads that range from grams to micrograms are achievable. Calibration is limited only by the availability of calibrated load cells in the smallest load ranges. Deflections in the range of a few nanometers range can be accurately applied. Simultaneous electrical, mechanical, and visual data can be combined to provide a revealing study of physical behavior of complex and dynamic nanostructures.

Focused Ion Beams (FIBs) provide a cross-sectioning tool for submicron dissection of organs, tissues, cells and subcellular structures. In combination with a Scanning Electron Microscope (SEM), FIB makes electron and ion microscopy available at the same time, for complementary morphological information, that moreover can be completed by EDS (Energy Dispersive X-ray Spectroscopy). In a previous paper [1] we have shown that Secondary Electron Imaging (SEI) and Back Scattered Electron Imaging (BEI) allow to recognize sample morphology; these techniques can rely on preliminary FIB operations (such as FIB sectioning, smoothing and lamellae formation) that provide specific manipulation of the sample in order to enhance the out-coming results. Here we discuss X ray analysis (EDS) to check the chemical composition of FIB exposed digestive gland cells of a terrestrial isopod Porcellio scaber.

Combined Focused Ion Beam – Scanning Electron Microscope and Synchrotron X-ray Fluorescence analysis of multi-solid and melt inclusions from the super-giant Grasberg Cu[sbnd]Au deposit, Indonesia

Focused ion beam (FIB) milling through carbon nanotube (CNT) yarns and bucky-papers followed by scanning electron microscopy has recently emerged as a powerful tool for eliciting details of their internal structure. The internal arrangement of CNTs in bucky-papers and yarns directly affects their performance and characteristics. Consequently this information is critical for further optimisation of these structures and to tailor their properties for specific applications. This chapter describes in detail FIB milling of CNT yarns and bucky-papers and gives a range of examples where FIB milling has enabled a better understanding of how processing conditions and treatments affect the internal structure. Emphasis is placed on how FIB milling elucidates the influence of fabrication conditions on the internal arrangement of CNTs and how this influences the material’s macroscopic properties.

Focused ion beams (FIBs) have been previously used as tools for such diverse tasks as high-resolution lithography, where their sub-10 nm spot sizes enable the patterning of diverse nanostructures, and surface compositional analysis, where their ability to sputter material in a localized area allows discrete components of a device or circuit to be characterized. Recently, the authors have capitalized on the FIB’s versatility by using it for microelectromechanical sensor fabrication as well as post-processing device characterization. The HRL FIB nanoprobe system has been used in the fabrication of high-performance surface-micromachined accelerometers operating on the principle of tunneling between a cantilevered beam and a sub-0.1-μm-diam tip lying beneath it on a Si substrate. The 8-nm-diam FIB has been used to pattern small dots in a bilevel negative-positive resist layer which are then transferred into a Au layer to form pyramid-shaped tunneling tip structures whose narrow dimensions are essential to high device performance and stability. High-purity, contamination-free Au on both the tunneling tip and cantilever underside is also critical to high-sensitivity tunneling devices. Because the undersides of the beams cannot be viewed with a scanning electron microscopy, even at high mechanical tilt angles, the cantilevers must be physically peeled back in order to expose their bottom surfaces and analyze them for cleanliness. Depending on the material used for fabricating the cantilevers, the rigidity of the structures can render them difficult to bend. We have used a commercial FIB milling system to cut through a portion of the cantilever width, thus creating a hinge, which facilitates the subsequent peeling back of the structure. Comparison of Auger spectroscopy data on peeled-back beams with and without a FIB-milled hinge shows similar surface contamination levels, indicating that redeposited material due to ion milling is localized enough to not affect the compositional analysis of the tunneling region.

Dry powder inhaler formulation of Cu,Zn-superoxide dismutase by spray drying: A proof-of-concept

Three-dimensional microstructure reconstruction is indispensable for understanding the performance and reliability of SOFC electrodes. Not only the microstructure parameters, but also SOFC electrochemical characteristics such as overpotentials can be calculated when combined with numerical simulations. The most common method to analyze 3-D structures with a feature size of 0.01 – 10 μm is a focused ion beam - scanning electron microscope (FIB-SEM). However, there’s a tradeoff between reconstruction volume size and resolution. Besides, in order to obtain high quality reconstruction, accumulation of know-hows for pre and post-processing is required. Therefore, fast screening of the microstructures is not easy for FIB-SEM due to the difficulty of measurement. On the other hand, a single SEM image can be obtained very easily. An arising question is if it is possible to extract properties of microstructures and numerically synthesize 3-D model directly from a single 2-D SEM image.In recent years, machine learning techniques have become a powerful tool for image processing. Nevertheless, applications of machine learning in the field of porous materials, in particular fuel cells and batteries are still limited. The previous works concentrated on relatively simple artificial neural networks (ANN), but the class of neural networks that can effectively analyze visual images is the convolutional neural network (CNN). Gayon-Lombardo et al. (1) demonstrated a successful application of generative adversarial network (GAN) based on CNN layers to fabricate synthetic 3-D SOFC microstructures. It is indicated that the GAN-based approach can be very effective, but the algorithm requires 3-D teaching data (GAN3Dto3D).In the scope of this research, a novel method for fabrication of synthetic 3-D microstructure directly from a 2-D SEM image is proposed (Fig. 1A). The GAN2Dto3D network consists of a 3-D microstructure generator and a discriminator which operate on 2-D data. Each microstructure fabricated by the generator is treated as a set of individual cross-sectional 2-D images. The discriminator checks the validity of each 2-D slice in the generated microstructure in all spatial directions (height, width and thickness). Patches extracted from a real 2-D cross-section image are used as training data. In each training iteration, new random patches are extracted to increase the number of available training data. Additionally, the training images are augmented by random horizontal and vertical flipping. As all of the cross-section of the newly generated microstructure has to be statistically equivalent to the real 2-D cross-section, the generated 3-D microstructure is smooth in all spatial directions.The GAN2Dto3D approach is tested on the dataset of real SOFC microstructures. For the real microstructure, a set of nickel (Ni) – gadolinium doped ceria (GDC) microstructures was fabricated. All composite anode samples were prepared with NiO (AGC Seimi, Japan) and GDC (Gd0.1Ce0.9O ShinEtsu, Japan) powders mixed in a designated proportion and sintered at 1350 oC. Differences in the structure are due to the initial anode slurry composition and packing density. The solid phase fractions were controlled by GDC share in the composite in the range of 30 – 70 vol.%, and their porosity was controlled by isostatic pressing (2). 3-D microstructures were reconstructed by FIB-SEM and used as the ground truth data for the validation of GAN2Dto3D model. The results of the GAN2Dto3D algorithm applied to generate two synthetic microstructures with different GDC share and the porosity are shown in Fig. 1B and 1C. The size of the generated microstructures is 64 × 64 × 64 voxels, which is equivalent to 6.4 × 6.4 × 6.4 µm. The real microstructures reconstructed with FIB-SEM are shown for comparison. The proposed GAN algorithm produces realistic microstructures, which indicates that GAN has a great potential to enable fast evaluation of various samples without carrying out complex FIB-SEM measurements.1. Gayon-Lombardo, L. Mosser, N. P. Brandon, and S. J. Cooper, npj Comput. Mater., 6, 1–11 (2020).2. Komatsu, A. Sciazko, and N. Shikazono, J. Power Sources, 485, 229317 (2021). Figure 1