Volume Electron Microscopy: Imaging Principles, Computational Advances and Applications in Multi-Scale Biological System

The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3'-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images. As DAB is unable to cross the yeast cell wall to react with APEX2, cell walls have been partly removed by the formation of spheroplasts. This has resulted in a clear electron-dense ER signal for the Erg11 protein using FIB-SEM. With this study, we have validated the use of the APEX2 tag for visualization of yeast proteins in electron microscopy. Furthermore, we have introduced a methodology that enables precise and three-dimensional (3D) localization studies in yeast, with nanometer resolution and without the need for antibody staining. Because of these properties, the described technique can offer valuable information on the molecular functions of studied proteins.IMPORTANCE With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometer-scale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology.

Alignment of stacks of serial images generated by Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is generally performed using translations only, either through slice-by-slice alignments with SIFT or alignment by template matching. However, limitations of these methods are two-fold: the introduction of a bias along the dataset in the z-direction which seriously alters the morphology of observed organelles and a missing compensation for pixel size variations inherent to the image acquisition itself. These pixel size variations result in local misalignments and jumps of a few nanometers in the image data that can compromise downstream image analysis. We introduce a novel approach which enables affine transformations to overcome local misalignments while avoiding the danger of introducing a scaling, rotation or shearing trend along the dataset. Our method first computes a template dataset with an alignment method restricted to translations only. This pre-aligned dataset is then smoothed selectively along the z-axis with a median filter, creating a template to which the raw data is aligned using affine transformations. Our method was applied to FIB-SEM datasets and showed clear improvement of the alignment along the z-axis resulting in a significantly more accurate automatic boundary segmentation using a convolutional neural network.

Background: Relationships between reflectivity, hardness and chemical composition of the dispersed phase, included in orthodontic composites Transbond XTTM (Trans), Light-Cure Orthodontic Paste (Leone) and Bisco Ortho Bracket Paste LC (Bisco), were investigated in vitro to evaluate whether reflectivity results can be useful in internal material composition interpretation, thus obtaining information on mechanical behaviours. Methods: Light transmission through 36 resin discs was measured with a UV/Vis spectrophotometer, evaluating the spectral range from 190–1100 nm. To have a benchmark of material hardness and internal composition, Vickers measurements and Cross-Sectional Focus Ion Beam Scanning Electron Microscopy (FIB/SEM) analysis were provided. Results: Bisco has the highest reflectivity, Leone shows an absorption pattern in the UV region similar to Bisco and Transbond has the lowest reflectivity compared to the others. This trend is confirmed by FIB/SEM imaging, showing a more similar induced roughness and internal composition for Bisco and Leone, with respect to Transbond. Higher filler presence in the composition of Bisco and Leone justifies a higher hardness of these two materials, with respect to Transbond, as confirmed by Vickers measurements. Conclusions: Bisco and Leone show similar optical responses and similarities in mechanical performance. This statement is explained by the lower and similar filler content as confirmed also by FIB/SEM analysis. The inner composition of Bisco and Leone provides a higher value of microhardness, as demonstrated by Vickers measurements. Therefore, this study confirms that the UV-Vis analysis can also offer a significant overview on the internal material composition, thus indirectly providing information on the mechanical properties of orthodontic composites.

Mixed matrix membranes (MMMs) have emerged as promising materials for various separation processes due to their tunable properties, enhanced separation performance and reproducibility. In this review paper, we provide a comprehensive overview of the methodologies, challenges, and applications associated with the characterization of MMMs using two advanced imaging techniques: Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and Transmission Electron Microscopy (TEM). We begin by outlining the principles and capabilities of FIB-SEM and TEM, emphasizing their suitability for studying the microstructure, morphology, and composition of MMMs at nanoscale resolution. Subsequently, we discuss the specific challenges and limitations encountered in the characterization of MMMs using these techniques, including sample preparation, image acquisition, and data interpretation. Furthermore, we review the diverse applications of FIB-SEM and TEM in elucidating the structure-property relationships of MMMs. Through illustrative examples, we highlight the valuable insights gained from these imaging techniques in optimizing MMMs for various separation applications. Finally, we propose future directions and emerging trends in MMM characterization, including the integration of lasers into FIB-SEM and in situ characterization techniques, to address current challenges and push the boundaries of MMM design and performance. Overall, this review provides a comprehensive overview of the state-of-the-art methodologies for characterizing MMMs using FIB-SEM and TEM, identifies key challenges, and offers insights into future research directions aimed at harnessing the full potential of MMMs for sustainable separation technologies.

Synaptic transmission poses a major energy consuming process in the brain, but how neurons maintain a constant energy supply during extended synaptic activity and how presynaptic mitochondria contribute to the energy supply remains elusive. The mitochondria are key organelles to account for the majority of ATP newly synthesised in neuronal cells. Hence, the present study aimed at unravelling the structural adaptation of mitochondria to an increase in presynaptic energy demand. To this end, I first characterized the mitochondrial membrane architecture and how it changes in response to different energy sources using transmission electron microscopy. As a model system, several cancer cell lines were used in addition to primary hippocampal neuronal cultures isolated from rat brain. In all tested cancer cell lines, a long-term metabolic switch to ketosis induced significant changes in the mitochondrial structure reflected by an increase of the mitochondrial diameter as well as a significant increase in the abundance of crista membranes. In contrast, a metabolic switch to glycolysis resulted in a decrease of the mitochondrial diameter and a reorientation of the crista membranes in parallel to the mitochondrial outer membrane in these cells. Likewise, in cultivated hippocampal neurons, the metabolic switch to ketosis induced a 20 % increase of crista membranes. Interestingly, only about 35 % of the neuronal presynapses showed a mitochondrial occupation which was not influenced by chemical depolarization with high concentrations of potassium chloride (KCl), suggesting that induced presynaptic activity does not affect the travelling of axonal mitochondria to the presynapses of hippocampal neurons. Furthermore, I used the cochlear nucleus of mice before and after the onset of hearing as a model system for presynaptic states of low and high-energy demand. Here, the synaptic morphology together with the presynaptic mitochondrial structure using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) was assessed. The volume of the synaptic boutons, the synaptic vesicle pool as well as the post-synaptic density were increased in synapses after the onset of hearing. Beyond that, the mitochondrial volume in these presynapses increased significantly. This suggests that synapses and mitochondria undergo major structural changes to serve the higher energy demands after the onset of hearing. Intriguingly, synapses containing mitochondria displayed an overall larger volume, synaptic vesicle pool and postsynaptic density when compared to synapses lacking mitochondria in mice before and after the onset of hearing. In conclusion, mitochondria play a key role in shaping presynaptic structure and are thus pivotal for synaptic transmission. In order to gain functional insights into the role of mitochondria in synaptic transmission in addition to the structural information obtained by TEM, I developed novel correlative imaging V tools. These tools include live cell 3D correlative light and electron microscopy (3D CLEM), which allows to monitor relevant mitochondrial targets by fluorescent labelling and subsequently correlate the functional information with structural aspects, and high-accuracy CLEM allowing for high spatial resolution of the detected features in both, light and electron microscopy. These protocols built the foundation for an extended correlative approach. There, I combined high-accuracy CLEM with nanoscale secondary ion mass spectrometry (NanoSIMS). This, for the first time, allowed extracting the functional and structural information together with the chemical composition of subcellular areas of the same resin-embedded specimen. These tools could now be applied to study individual players in mitochondrial function by simultaneously getting information of their impact on synaptic structure and chemical composition.

Imaging technologies have played a pivotal role in advancing biological research by enabling visualization of biological structures and processes. While traditional electron microscopy (EM) produces two-dimensional images, emerging techniques now allow high-resolution three-dimensional (3D) characterization of specimens in situ, meeting growing needs in molecular and cellular biology. Combining transmission electron microscopy (TEM) with serial sectioning inaugurated 3D imaging, attracting biologists seeking to explore cell ultrastructure and driving advancement of 3D EM reconstruction. By comprehensively and precisely rendering internal structure and distribution, 3D TEM reconstruction provides unparalleled ultrastructural insights into cells and molecules, holding tremendous value for elucidating structure-function relationships and broadly propelling structural biology. Here, we first introduce the principle of 3D reconstruction of cells and tissues by classical approaches in TEM and then discuss modern technologies utilizing TEM and on new SEM-based as well as cryo-electron microscope (cryo-EM) techniques. 3D reconstruction techniques from serial sections, electron tomography (ET), and the recent single-particle analysis (SPA) are examined; the focused ion beam scanning electron microscopy (FIB-SEM), the serial block-face scanning electron microscopy (SBF-SEM), and automatic tape-collecting lathe ultramicrotome (ATUM-SEM) for 3D reconstruction of large volumes are discussed. Finally, we review the challenges and development prospects of these technologies in life science. It aims to provide an informative reference for biological researchers.

Focused ion beam-scanning electron microscopy provides novel insights of drug delivery phenomena

Numerical analysis on transport properties of self-humidifying dual catalyst layer via 3-D reconstruction technique

Insects’ sound and vibration detection including proprioception rely on the scolopidium—a mechanosensory unit enclosing the sensory cilium of chordotonal organ neurons. The cilium, enclosed by a scolopale cell, contains mechanosensitive ion channels with the ciliary tip embedded in a cap. Despite knowledge of the scolopidial structure in multiple insects, the mechanism by which mechanical force elicits transduction remains speculative. We examined scolopidia in the auditory Müller’s organ of the desert locust and present a comprehensive three-dimensional (3D) ultrastructure of a scolopidium using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). Next, we characterised sound-evoked motions of Müller’s organ and the scolopidium using Optical Coherence Tomography (OCT) and high-speed light microscopy. Finally, we measured transduction currents via patch clamp electrophysiology during mechanical stimulation of individual scolopidia. By combining ultrastructure, sound-evoked motions, and transduction current recordings, our finding suggests that the scolopidium is activated best by stretch along the ciliary axis.

Heteroresistance in Cryptococcus neoformans is an intrinsic adaptive resistance to azoles and the heteroresistant phenotype is associated with disomic chromosomes. Two chromosome 1 (Chr1) genes, ERG11, the fluconazole target, and AFR1, a drug transporter, were reported as major factors in the emergence of Chr1 disomy. In the present study, we show Chr4 to be the second most frequently formed disomy at high concentrations of fluconazole (FLC) and characterize the importance of resident genes contributing to disomy formation. We deleted nine Chr4 genes presumed to have functions in ergosterol biosynthesis, membrane composition/integrity or drug transportation that could influence Chr4 disomy under FLC stress. Of these nine, disruption of three genes homologous to Sey1 (a GTPase), Glo3 and Gcs2 (the ADP-ribosylation factor GTPase activating proteins) significantly reduced the frequency of Chr4 disomy in heteroresistant clones. Furthermore, FLC resistant clones derived from sey1Δglo3Δ did not show disomy of either Chr4 or Chr1 but instead had increased the copy number of the genes proximal to ERG11 locus on Chr1. Since the three genes are critical for the integrity of endoplasmic reticulum (ER) in Saccharomyces cerevisiae, we used Sec61ß-GFP fusion as a marker to study the ER in the mutants. The cytoplasmic ER was found to be elongated in sey1Δ but without any discernable alteration in gcs2Δ and glo3Δ under fluorescence microscopy. The aberrant ER morphology of all three mutant strains, however, was discernable by transmission electron microscopy. A 3D reconstruction using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) revealed considerably reduced reticulation in the ER of glo3Δ and gcs2Δ strains. In sey1Δ, ER reticulation was barely detectable and cisternae were expanded extensively compared to the wild type strains. These data suggest that the genes required for maintenance of ER integrity are important for the formation of disomic chromosomes in C. neoformans under azole stress.

Zero-shot and prompt-based models have excelled at visual reasoning tasks by leveraging large-scale natural image corpora, but they often fail on sparse and domain-specific scientific image data. We introduce Zenesis, a no-code interactive computer vision platform designed to reduce data readiness bottlenecks in scientific imaging workflows. Zenesis integrates lightweight multimodal adaptation for zero-shot inference on raw scientific data, human-in-the-loop refinement, and heuristic-based temporal enhancement. We validate our approach on Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) datasets of catalyst-loaded membranes. Zenesis outperforms baselines, achieving an average accuracy of 0.947, Intersection over Union (IoU) of 0.858, and Dice score of 0.923 on amorphous catalyst samples; and 0.987 accuracy, 0.857 IoU, and 0.923 Dice on crystalline samples. These results represent a significant performance gain over conventional methods such as Otsu thresholding and standalone models like the Segment Anything Model (SAM). Zenesis enables effective image segmentation in domains where annotated datasets are limited, offering a scalable solution for scientific discovery.

The aim of this work was to develop a plasma nitriding process for a bio-grade CoCrMo alloy using a High Power Impulse Magnetron Sputtering (HIPIMS) discharge. An industrial-sized PVD machine (Hauzer techno Coating 1000-4, The Netherlands) machine equipped with two HIPIMS and two DC power supplies was utilised for this objective. Low-pressure plasma nitriding was carried out in a H2:N2 (15:85) environment with different nitriding voltages (-500 V to -1100 V). Nitrided specimens thoroughly were characterised using various techniques. To understand the effect of nitriding voltage on the phase composition and texture (T*) x-ray diffraction (XRD) was used. Microstructural changes due to the variation in nitriding voltage w investigated using a focused ion beam Scanning electron microscope (FIB-SEM) equipped with various detectors such as secondary electron (SE) and back scattered (BSE). Extent of nitrogen diffusion was analysed using GDOES and SIMS. Elemental analysis of the virgin nitrided layer and analysed surfaces were performed using point, line, and colour mapping electron diffraction spectroscopy (EDS). Nano/Micro hardness testers were used to understand the effect of nitriding voltage on hardness (Hp and HV) and fracture toughness (KIc). A high frequency macro impact load tester was used to understand the effect of nitriding voltage on impact fatigue behaviour of the compound layers. Fractured surfaces were analysed using SEM and FIBSEM after static and cyclic loading to understand the deformation mechanism such as fatigue cracking underneath nitrided layer. Potentiodyanmic polarisation study was performed in 3.5 wt.% NaCl and Hank’s solution to analyse the corrosion performance of these nitrided layers. At the end, HIPIMS plasma nitrided specimens were compared with benchmarked DC plasma nitrided specimens. Sample nitrided using HIPIMS discharge at -900 V exhibited novel phase composition, superior hardness and fracture toughness, better wear resistance, improved corrosion resistance and reduced metal ion release compared to the benchmarked DCPN.

Qualitative and quantitative characterization of a transitional shale reservoir: A case study from the Upper Carboniferous Taiyuan shale in the eastern uplift of Liaohe Depression, China

Focused ion beam scanning electron microscopy is well suited for volumetric extractions and 3D reconstructions of plant cells and its organelles. The three-dimensional (3D) reconstruction of individual plant cells is an important tool to extract volumetric data of organelles and is necessary to fully understand ultrastructural changes and adaptations of plants to their environment. Methods such as the 3D reconstruction of cells based on light microscopical images often lack the resolution necessary to clearly reconstruct all cell compartments within a cell. The 3D reconstruction of cells through serial sectioning transmission electron microscopy (ssTEM) and focused ion beam scanning electron microscopy (FIB-SEM) are powerful alternatives but not widely used in plant sciences. Here, we present a method for the 3D reconstruction and volumetric extraction of plant cells based on FIB milling and compare the results with 3D reconstructions obtained with ssTEM. When compared to 3D reconstruction based on ssTEM, FIB-SEM delivered similar results. The data extracted in this study demonstrated that tobacco cells were larger (31410 µm3) than pumpkin cells (20697 µm3) and contained more chloroplasts (175 vs. 124), mitochondria (1317 vs. 291) and peroxisomes (745 vs. 79). While individual chloroplasts, mitochondria, peroxisomes were larger in pumpkin plants (25, 53, and 50%, respectively) they covered more total volume in tobacco plants (5390, 395, 374 µm3, respectively) due to their higher number per cell when compared to pumpkin plants (4762, 134, 59 µm3, respectively). While image acquisition with FIB-SEM was automated, software controlled, and less difficult than ssTEM, FIB milling was slower and sections couldnot be revised or re-imaged as they were destroyed by the ion beam. Nevertheless, the results in this study demonstrated that both, FIB-SEM and ssTEM, are powerful tools for the 3D reconstruction of and volumetric extraction from plant cells and that there were large differences in size, number, and organelle composition between pumpkin and tobacco cells.

Correlative light and electron microscopy (CLEM) methods are powerful methods that combine molecular organization (from light microscopy) with ultrastructure (from electron microscopy). However, CLEM methods pose high cost/difficulty barriers to entry and have very low experimental throughput. Therefore, we have developed an indirect correlative light and electron microscopy (iCLEM) pipeline to sidestep the rate-limiting steps of CLEM (i.e., preparing and imaging the same samples on multiple microscopes) and correlate multiscale structural data gleaned from separate samples imaged using different modalities by exploiting biological structures identifiable by both light and electron microscopy as intrinsic fiducials. We demonstrate here an application of iCLEM, where we utilized gap junctions and mechanical junctions between muscle cells in the heart as intrinsic fiducials to correlate ultrastructural measurements from transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) with molecular organization from confocal microscopy and single molecule localization microscopy (SMLM). We further demonstrate how iCLEM can be integrated with computational modeling to discover structure-function relationships. Thus, we present iCLEM as a novel approach that complements existing CLEM methods and provides a generalizable framework that can be applied to any set of imaging modalities, provided suitable intrinsic fiducials can be identified.