3D Microscopy Research Articles

Neuroanatomical and morphometric study of S100β positive astrocytes in the entorhinal cortex during ageing in the 3xTg-Alzehimer’s disease mouse model

Astrocytes contribute to the progression of neurodegenerative diseases, including Alzheimer’s disease (AD). Here, we report the neuroanatomical and morphometric analysis of astrocytes in the entorhinal cortex (EC) of the aged wild type (WT) and triple transgenic (3xTg-AD) mouse model of AD. Using 3D confocal microscopy, we determined the surface area and volume of positive astrocytic profiles in male mice (WT and 3xTg-AD) from 1 to 18 months of age. We showed that S100β-positive astrocytes were equally distributed throughout the entire EC in both animal types and showed no changes in Nv (number of cells/mm3) nor in their distribution at the different ages studied. These positive astrocytes, demonstrated an age-dependent gradual increase in their surface area and in their volume starting at 3 months of age, in both WT and 3xTg-AD mice. This last group demonstrated a large increase in both surface area and volume at 18 months of age when the burden of pathological hallmarks of AD is present (69.74% to 76.73% in the surface area and the volume, for WT and 3xTg-AD mice respectively). We observed that these changes were due to the enlargement of the cell processes and to less extend the somata. In fact, the volume of the cell body was increased by 35.82% in 18-month-old 3xTg-AD compared to WT. On the other hand, the increase on the astrocytic processes were detected as soon as 9 months of age where we found an increase of surface area and volume (36.56% and 43.73%, respectively) sustained till 18 month of age (93.6% and 113.78%, respectively) when compared age-matched non-Tg mice. Moreover, we demonstrated that these hypertrophic S100β-positive astrocytes were mainly associated with Aβ plaques. Our results show a severe atrophy in GFAP cytoskeleton in all cognitive areas; whilst within the EC astrocytes independent to this atrophy show no changes in GS and S100β; which can play a key role in the memory impairment.

Failure Analysis of Printed Circuit Board Solder Joint under Thermal Shock

Investigating the failure mechanism of solder joints under different temperature conditions is significant to ensure the service life of a printed circuit board (PCB). In this research, the stress and strain distribution of a PCB solder joint was evaluated by high- and low-temperature thermal shock tests. The cross-section of the solder joint after thermal shock testing was measured using a 3D stereoscopic microscope and SEM equipped with EDS. The microstructure of the lead-free solder joint and the phase of the intermetallic compound (IMC) layer were studied by XRD. The working state of the PCB solder joint under thermal shock was simulated and analyzed by the finite element method. The results show that thermal shock has a great effect on the reliability of solder joints. The location of the actual crack is consistent with the maximum stress–strain concentration area of the simulated solder joint. The brittle Cu6Sn5 and Cu3Sn phases at the interface accelerate the failure of solder joints. Limiting the growth of Cu6Sn5 and Cu3Sn phases can improve the reliability of solder joints to a certain extent.

Significant enhancement of thermal conductivity and EMI shielding performance in PEI composites via constructing 3D microscopic continuous filler network

Polymer-based composites with effective heat dissipation and ideal electromagnetic interference shielding performance have gradually become an essential research focus in modern electronic industry. Herein, a method is reported by constructing 3D microscope continuous filler network into polyetherimide (PEI) matrix via incorporating function graphite nanoplatelets (GNP) with PEI microspheres, and then fillers are distributed beyond the domain area of polymer and leaded to continuous filler framework. The fabricated composites exhibit effective thermal conductivity (4.77 W m−1K−1), great EMI effectiveness (42.7 dB), ideal electrical conductivity and profound thermal property. Different scale of polymer microspheres will also change the micro-architecture from “filler-wrapped polymer” structure to “polymer-wrapped filler” structure, resulting in various thermal performance and electrical property. Finite element simulation is further employed to explore the relationship between microsphere scale and heat transfer behavior. In a word, great comprehensive performance endows PEI composites broaden prospect on the potential applications in the fields of electronic equipment.

Real-Time In-Process Evaluation of Spatter Area and Depth of Aluminium Surface in a Pulsed Laser Ablation Process Using Received Radio Frequency Power from Plasma Plumes

During the pulsed laser ablation of metals, as well as other materials, the development of a plasma plume close to the ablated surface leads to the emission of radio frequency energy. In this paper, we describe a process for analysing the received radio frequency power (RFP) for an aluminium (Al) surface ablation process in atmosphere using picosecond laser pulses at a wavelength of 1064 nm. The analysis of the RFP was carried out on two sets of experiments, where two parameters of the laser (repetition rate of laser (RRL) and power of laser (PL)) were varied while other parameters remained constant. In addition to the RFP measurement during the laser processing, the spatter area (SA), which is defined in this paper, and the depth of the ablated hole were measured post-process using a 3D microscope. It was observed that there is a direct relationship between (RFP)2 and SA. Accordingly, an appropriate RF calibration was performed, which leads to the definition of a quantity called the RF regulation % (RFR%). By comparing the RFR and PL/RRL variations, to which the laser beam fluence is proportional in these experiments, a diagnostic process (i.e., flowchart) for real-time depth evaluation was proposed and experimentally confirmed. This diagnostic process can indicate if the depth of the laser ablated crater is less than or exceeds a predetermined depth, which in this study was set to 15 µm. It is also demonstrated that the SA variation can be estimated in real-time by analysing the received RF power and, secondly, the depth of ablation can be measured in real time using a combination of information from the received RF power and laser parameters.

Microstructure, mechanical and thermal properties of YSZ thermal barrier coatings deposited by axial suspension plasma spraying

Yttrium-stabilized zirconia (YSZ) thermal barrier coatings (TBCs) are indispensable elements of present-day turbine propulsion systems. The ones deposited with atmospheric plasma spraying (APS) are characterized by required low thermal conductivity, but they are unable to survive frequent thermomechanical loading and therefore their application is limited to parts remaining stationary. Expanding capability of TBCs is sought in various areas, but the one realized through modification of most proliferated apparatus used for plasma spraying (PS) (from radial to axial injection) and substituting micrometric powders with the nano-structured suspension needs least changes in the industry established procedures and offers the highest property improvement. The present experiment covered the deposition of ZrO2-8Y2O3 YSZ TBC using both atmospheric and suspension PS processes. They were performed with commercial micrometric and nano-structured YSZ (8% Y2O3) powders. The coatings morphology and microstructure were characterized with 3D profilometry, scanning and transmission electron microscopy (SEM/TEM) methods. Finally, the coating’s hardness and heat conductivity were measured. This complex approach allowed to state that PS of micrometric t’-ZrO2 powder having an admixture of m-ZrO2 phase is capable of only partial improvement in its homogenization. However, the suspension PS process of nano-structured powder eliminated any traces of the monoclinic phase from the coating. The TEM microstructure observations indicated that the suspension PS coating is built by in-flight solidified droplets as well as by the melted ones flattened on arrival. A surface layer of liquefied material on solid droplets increases their adhesion to surface asperities promoting pseudo-columnar growth of the coating. The preservation of monotonic slow increase of thermal conductivity during heating of the suspension PS coating means, that its pseudo-columnar microstructure is better suited to withstand high strains during such treatment.

Volume Exploration Using Multidimensional Bhattacharyya Flow.

We present a novel approach for volume exploration that is versatile yet effective in isolating semantic structures in both noisy and clean data. Specifically, we describe a hierarchical active contours approach based on Bhattacharyya gradient flow which is easier to control, robust to noise, and can incorporate various types of statistical information to drive an edge-agnostic exploration process. To facilitate a time-bound user-driven volume exploration process that is applicable to a wide variety of data sources, we present an efficient multi-GPU implementation that (1) is approximately 400 times faster than a single thread CPU implementation, (2) allows hierarchical exploration of 2D and 3D images, (3) supports customization through multidimensional attribute spaces, and (4) is applicable to a variety of data sources and semantic structures. The exploration system follows a 2-step process. It first applies active contours to isolate semantically meaningful subsets of the volume. It then applies transfer functions to the isolated regions locally to produce clear and clutter-free visualizations. We show the effectiveness of our approach in isolating and visualizing structures-of-interest without needing any specialized segmentation methods on a variety of data sources, including 3D optical microscopy, multi-channel optical volumes, abdominal and chest CT, micro-CT, MRI, simulation, and synthetic data. We also gathered feedback from a medical trainee regarding the usefulness of our approach and discussion on potential applications in clinical workflows.

Workflows for assessing electronic devices with 3D X-ray microscopy and nanoscale computed tomography

Modern 2D and 3D X-ray technologies are among the most useful non-destructive testing (NDT) methods that allow inspection of an object’s internal features without cutting or disassembling a sample. This paper presents workflows that combine 3D X-ray microscopy and nanoscale tomography to generate a detailed three-dimensional visualization of the interior of electronic devices and assemblies, without destroying them, to enable the study of internal components for failure analysis (FA). These imaging workflows can be run independently or complementary to other multiscale correlative microscopy evaluations and provide valuable insights into the inner workings of electronic packages and integrated circuits at multiple length scales, from macroscopic features on electronic devices (i.e., hundreds of mm) to microscopic details in electronic components (in the tens of nm). Understanding advanced electronic systems through X-ray imaging, possibly complemented with additional correlative microscopy investigations, can speed development time, increase cost efficiency, and simplify FA and quality inspection of semiconductor packaging, printed circuit boards (PCBs), and electronic devices assembled with new emerging technologies.

Real-time process monitoring and closed-loop control on laser power via a customized laser powder bed fusion platform

Additive manufacturing (AM) is one of the most effective ways to fabricate parts with complex geometries using various materials. However, AM also suffers from printing quality issues resulting from the defects such as over-melt, lack of fusion, swelling, etc. One of the root causes of those issues is that the process parameters remain constant during the entire printing process, regardless of the dynamic heat accumulation and various printing feature sizes. For instance, raster is the most common scanning strategy in the laser powder bed fusion (L-PBF) process. The length of the raster line varies depending on the printing feature size. When scanning small features, the raster line is short, resulting in heat accumulations and over-melt. These variabilities may cause severe quality issues and thus suggest adaptive process parameters be applied. Aiming to address this challenge, this study develops a closed-loop control system to regulate the laser power based on melt pool thermal emission to avoid over-melt, balling, and high surface roughness. The control target is determined by correlating the printing quality (dimensional printing error in this study) with the thermal emission through thin-line printing trials using variable power. A high-speed thermal sensor and controller are designed, tuned, and implemented on a newly developed L-PBF testbed. The system successfully maintains a low dimensional error by regulating the laser power at 2 kHz. A significant improvement in printing quality was achieved, as validated by both microscopic imaging and 3D scanning.

Correlative microscopy and block-face imaging (CoMBI): a 3D imaging method with wide applicability in the field of biological science.

Correlative microscopy and block-face imaging (CoMBI) is an imaging method, which is characterized by the ability to obtain both serial block-face images as a 3-dimentional (3D) dataset and sections for 2-dimentional (2D) light microscopic analysis. These 3D and 2D morphological data can be correlated with each other to facilitate data interpretation. CoMBI is an easy-to-install and low-cost 3D imaging method since its system can be assembled by the researcher using a regular microtome, consumer digital camera, and some self-made devices, and its installation and instruction manuals are open-source. After the first release of CoMBI method from our laboratory, CoMBI systems have been installed in more than a dozen laboratories and are used for 3D analysis of various biological specimens. Typical application of CoMBI is 3D anatomical analysis using the natural color and contrast of the specimen. We have been using CoMBI for analyzing human brain to obtain the fine 3D anatomy as a reference to determine the causes of neurological diseases and to improve the effectiveness of surgery. Recently, we have been using CoMBI for detecting the colors of chromogens, which are used for labeling specific molecules. Mouse embryos colored with X-gal, a conventional chromogen for detecting LacZ products, were imaged using CoMBI, and the 3D distribution of X-gal was successfully visualized. Thus, CoMBI can now be used for many purposes, including 3D anatomical analysis, 2D microscopy using sections, and 3D distribution of specific molecules. These suggest that CoMBI should be more widely used in the field of biological research.

Nondestructive 3D Pathology with Light-Sheet Fluorescence Microscopy for Translational Research and Clinical Assays.

In recent years, there has been a revived appreciation for the importance of spatial context and morphological phenotypes for both understanding disease progression and guiding treatment decisions. Compared with conventional 2D histopathology, which is the current gold standard of medical diagnostics, nondestructive 3D pathology offers researchers and clinicians the ability to visualize orders of magnitude more tissue within their natural volumetric context. This has been enabled by rapid advances in tissue-preparation methods, high-throughput 3D microscopy instrumentation, and computational tools for processing these massive feature-rich data sets. Here, we provide a brief overview of many of these technical advances along with remaining challenges to be overcome. We also speculate on the future of 3D pathology as applied in translational investigations, preclinical drug development, and clinical decision-support assays. Expected final online publication date for the Annual Review of Analytical Chemistry, Volume 16 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Understanding the Degradation Mechanisms of Pt Electrocatalysts in PEMFCs by Combining 2D and 3D Identical Location TEM.

The evolution of Pt nanoparticles in proton-exchanged membrane fuel cells is monitored before and after electrochemical potential cycling, using 2D and 3D identical location aberration-corrected transmission electron microscopy. This work demonstrates that 2D images might be a challenge to interpret due to the 3D nature of the carbon support. Thus, it is critical to combine both 2D and 3D observations to be able to fully understand the mechanisms associated with the durability of Pt catalyst nanoparticles. In particular, this investigation reveals that the mechanism of particle migration followed by coalescence is operative mainly across short distances (<0.5 nm). This work also shows that new Pt particles appear on the carbon support, as the result of Pt dissolution, followed by the formation of clusters, which grow by Ostwald ripening. This mechanism of Ostwald ripening is also responsible for changes in shape and particle growth, which later may result in coalescence.

An investigation of the PMEDM processing and surface characterizations of AZ61 matrix composites via experimental and optimization methods

In this study, AZ61/15 wt%TiB2 and AZ61/15 wt%TiB2-0.5 wt%GNPs composites were manufactured by hot pressing, and the machinability of the produced samples was carried out by powder mixed electrical discharge machining (PMEDM). The influence of PMEDM parameters, namely pulse on time, current and materials, were studied by surface roughness (SR) and material removal rate (MRR) using the Taguchi design. The microstructure and surface quality of the machined surfaces and cross-sections were investigated using 3D microscopy, scanning electron microscopy (SEM), energy dispersive spectrometry (EDS) and X-ray diffraction (XRD). Results showed that PMEDM produced a melting, white layer and transition zone direct proportionally to the processing parameters. The white layer did not differ from the metal integrity of the transition zone. Additionally, volcanic craters, holes, cracks, debris covered with molten metal, and reinforcement particles were observed at the machined surface and cross-section. A high amount of oxygen was detected on the machined surface as a result of the interaction between kerosene and generated heat changing proportionally to the amount of the EDM parameters. The analysis of variance (ANOVA) showed that the pulse on time and materials, with 65.46% and 40.86%, were the most significant parameters on the SR and MRR, respectively. For regression models, the determination coefficient (R2) for the prediction of SR and MRR was noted to be 0.98 and 0.85, respectively.

First person – Joanna Pylvänäinen

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Joanna Pylvänäinen is first author on ‘ Fast4DReg – fast registration of 4D microscopy datasets’, published in JCS. Joanna is a PhD student in the lab of Guillaume Jacquemet at Åbo Akademi University, Turku, Finland, where she is interested in microscopy images and how to analyze them in the best possible way.

Fast4DReg - fast registration of 4D microscopy datasets.

Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal visualization and quantification of biological processes. Although multiple methods and tools exist to correct images post acquisition, performing drift correction of three-dimensional (3D) videos using open-source solutions remains challenging and time consuming. Here, we present a new tool developed for ImageJ or Fiji called Fast4DReg that can quickly correct axial and lateral drift in 3D video-microscopy datasets. Fast4DReg works by creating intensity projections along multiple axes and estimating the drift between frames using two-dimensional cross-correlations. Using synthetic and acquired datasets, we demonstrate that Fast4DReg can perform better than other state-of-the-art open-source drift-correction tools and significantly outperforms them in speed. We also demonstrate that Fast4DReg can be used to register misaligned channels in 3D using either calibration slides or misaligned images directly. Altogether, Fast4DReg provides a quick and easy-to-use method to correct 3D imaging data before further visualization and analysis.

Wear Mechanism of Fe/Cu Self-Lubricating Composite Coatings Fabricated by Electro-Explosive Spraying under Dry Friction

In the present work, Fe/Cu composite coatings were fabricated by electro-explosive spraying technology (EEST), with good lubrication performance and high wear resistance. The microstructure and morphology were characterized by an energy-dispersive spectrometer (EDS), 3D digital microscope, and scanning electron microscope (SEM) coupled with electron backscattered diffraction (EBSD). Mechanical properties and tribological performance were measured using a micro Vickers hardness tester, universal testing machine, and universal friction and wear testing machine. The composite coating had low porosity with a minimum value of 0.7%, high microhardness with a maximum value of 729.9 HV0.2, high bonding strength with a maximum average value of 55.25 MPa, and good wear resistance and self-lubrication, and the ratio of soft metal and hard metal in the coatings was controllable. Under dry friction conditions, the friction reduction mechanism was that there were both metallic oxide particles and soft metal attached in the friction pairs. When the ratio of soft metal in the composite coating was higher, the self-lubricating performance of the coating was better, with adhesive wear as the main wear mechanism; when the ratio of hard metal was higher, the wear resistance was better and the wear mechanism was mainly abrasive wear.

Self-aligned multi-layer X-ray absorption grating using large-area fabrication methods for X-ray phase-contrast imaging

X-ray phase-contrast (XPCi) imaging methods are an emerging medical imaging approach that provide significantly better soft tissue contrast and could function as a viable extension to conventional X-ray, CT, and even some MRI. Absorption gratings play a central role in grating-based XPCi systems, especially because they enable the acquisition of three images in a single exposure: transmission, refraction, and dark-field. An impediment to commercial development and adoption of XPCi imaging systems is the lack of large area, high aspect ratio absorption gratings. Grating technology development, primarily due to technological limitations, has lagged system development and today prevents the scaling up of XPCi system into a footprint and price point acceptable to the medical market. In this work, we report on a self-aligned multi-layer grating fabrication process that can enable large-area X-ray absorption gratings with micron-scale feature sizes. We leverage large-area fabrication techniques commonly employed by the thin-film transistor (TFT) display industry. Conventional ITO-on-glass substrates are used with a patterned film of Cr/Au/Cr that serves as a self-aligned lithography mask for backside exposure. Commonly available SU-8 photoresist is patterned using the backside exposure mask followed by an electroplating step to fill the gaps in the SU-8 with X-ray attenuating material. Consequently, the electroplated patterned material acts as a self-aligned photomask for subsequent SU-8 layer patterning and so forth. The repeatability of the reported process makes it suitable for achieving higher aspect ratio structures and is advantageous over previously reported X-ray LIGA approaches. A prototype three-layer grating, with a thickness of around 40,upmu text{m}, having a visibility of 0.28 at 60,text{kV}_p with a 70,text{mm}times 70,text{mm} active area was fabricated on a 4-inch glass substrate and demonstrated by modifying a commercially available 3D propagation-based XPCi Microscope. The scalable and cost-effective approach to build larger area X-ray gratings reported in this work can help expedite the commercial development and adoption of previously reported Talbot-Lau, speckle-tracking, as well as coded-aperture XPCi systems for large-area clinical and industrial applications.

Investigation on the effective factor calculation of electropolishing using full factorial design and mechanism model by microscopic analysis for super austenitic stainless steel

This investigation is to calculate the effective factors of the electropolishing by using the full factorial design for UNS N08367. In addition, based on the calculated effective factors, the electropolishing mechanism model was researched on 3D microscopy and scanning electron microscopy analysis. The parameters were selected as electrolyte composition ratio, electropolishing process time and applied current density. Calculation of effective factors was performed through analysis of variance (ANOVA). As a result of ANOVA, it was observed that the electrolyte composition ratio had the greatest effect on surface planarization. As a result of surface analysis after electropolishing, the maximum height difference is about 54 times. Also, the minimum value of surface roughness after electropolishing was 0.129 μm, which was improved by more than 70% compared to mechanical polishing. As a result of all analysis, it was most flat to perform electropolishing with a sulfuric acid and phosphoric acid ratio of 3:7, 300 mA/cm2 and 5 min.

Viral infection impacts the 3D subcellular structure of the abundant marine diatom Guinardia delicatula

Viruses are key players in marine ecosystems where they infect abundant marine microbes. RNA viruses are emerging as key members of the marine virosphere. They have recently been identified as a potential source of mortality in diatoms, a group of microalgae that accounts for roughly 40% of the primary production in the ocean. Despite their likely importance, their impacts on host populations and ecosystems remain difficult to assess. In this study, we introduce an innovative approach that combines automated 3D confocal microscopy with quantitative image analysis and physiological measurements to expand our understanding of viral infection. We followed different stages of infection of the bloom-forming diatom Guinardia delicatula by the RNA virus GdelRNAV-04 until the complete lysis of the host. From 20h after infection, we observed quantifiable changes in subcellular host morphology and biomass. Our microscopy monitoring also showed that viral infection of G. delicatula induced the formation of auxospores as a probable defense strategy against viruses. Our method enables the detection of discriminative morphological features on the subcellular scale and at high throughput for comparing populations, making it a promising approach for the quantification of viral infections in the field in the future.

Clathrin coats partially preassemble and subsequently bend during endocytosis.

Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargo. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis. We find that clathrin coats assemble first on flat membranes to 50% of the coat area before they become rapidly and continuously bent, and this mechanism is confirmed in three cell lines. We introduce the cooperative curvature model, which is based on positive feedback for curvature generation. It accurately describes the measured shapes and dynamics of the clathrin coat and could represent a general mechanism for clathrin coat remodeling on the plasma membrane.

ESTABLISHMENT AND ANALYSIS OF FRACTAL CONTACT MODEL FOR POINT GRINDING PARTS

In this work, we establish a fractal contact model and analyze the effect of the parameters on stress. The study provided a method to make a link between contact characteristics and surface condition. The surface texture model was built based on the process mechanism of point grinding. The experiment for the effect of the surface texture on contour height was carried out on TR300 roughness shape-measuring instrument and the structural function method of fractal theory was used to obtain the fractal dimensions and scale coefficients of surfaces with different surface textures. The fractal contact model was established based on the fractal characteristics of the surface point ground and the simulation was carried out using Matlab. Furthermore, the validation of compression experiment was carried out by WAW-600C universal testing machine, the high-definition pictures before and after the experiment were obtained by KEYENCE VH-Z500R ultra-depth-of-field 3D microscope and the surface topographies before and after the experiment were obtained by NANOVEA PS50 three-dimensional noncontact surface profile meter. The obtained findings can help to choose appropriate surface with the best contact characteristics under certain condition.