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Summary: Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultrastructural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume‐EM techniques such as serial blockface scanning electron microscopy (SBF‐SEM) and Focussed Ion Beam‐SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. In addition, combining volume‐EM with high‐resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high‐resolution confocal microscopy with SBF‐SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D‐CLEM overlays of liver tissue and correlate the fluorescent signal to the ultrastructural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three‐dimensional correlative studies. Lay Description: As cells and tissues exist in three dimensions, microscopy techniques have been developed to image samples, in 3D, at the highest possible detail. In light microscopy, fluorescent probes are used to identify specific proteins or structures either in live samples, (providing dynamic information), or in fixed slices of tissue. A disadvantage of fluorescence microscopy is that only the labeled proteins/structures are visible, while their cellular context remains hidden. Electron microscopy is able to image biological samples at high resolution and has the advantage that all structures in the tissue are visible at nanometer (10−9 m) resolution. Disadvantages of this technique are that it is more difficult to label a single structure and that the samples must be imaged under high vacuum, so biological samples need to be fixed and embedded in a plastic resin to stay as close to their natural state as possible inside the microscope. Correlative Light and Electron Microscopy aims to combine the advantages of both light and electron microscopy on the same sample. This results in datasets where fluorescent labels can be combined with the high‐resolution contextual information provided by the electron microscope. In this study we present a workflow to guide a tissue sample from the light microscope to the electron microscope and image the ultra‐structure of a specific cell type in the liver. In particular we focus on the incorporation of fiducial markers during the sample preparation to help navigate through the tissue in 3D in both microscopes. One sample is followed throughout the workflow to visualize the important steps in the process, showing the final result; a dataset combining fluorescent labels with ultra‐structural detail. [ABSTRACT FROM AUTHOR]

Summary: The intercalated disc is an important structure in cardiomyocytes, as it is essential to maintain correct contraction and proper functioning of the heart. Adhesion and communication between cardiomyocytes are mediated by three main types of intercellular junctions, all residing in the intercalated disc: gap junctions, desmosomes and the areae compositae. Mutations in genes that encode junctional proteins, including αT‐catenin (encoded by CTNNA3), have been linked to arrhythmogenic cardiomyopathy and sudden cardiac death. In mice, the loss of αT‐catenin in cardiomyocytes leads to impaired heart function, fibrosis, changed expression of desmosomal proteins and increased risk for arrhythmias following ischemia‐reperfusion. Currently, it is unclear how the intercalated disc and the intercellular junctions are organised in 3D in the hearts of this αT‐catenin knockout (KO) mouse model. In order to scrutinise this, ventricular cardiac tissue of αT‐catenin KO mice was used for volume electron microscopy (VEM), making use of Focused Ion Beam Scanning Electron Microscopy (FIB‐SEM), allowing a careful 3D reconstruction of the intercalated disc, including gap junctions and desmosomes. Although αT‐catenin KO and control mice display a comparable organisation of the sarcomere and the different intercalated disc regions, the folds of the plicae region of the intercalated disc are longer and more narrow in the KO heart, and the pale region between the sarcomere and the intercalated disc is larger. In addition, αT‐catenin KO intercalated discs appear to have smaller gap junctions and desmosomes in the plicae region, while gap junctions are larger in the interplicae region of the intercalated disc. Although the reason for this remodelling of the ultrastructure after αT‐catenin deletion remains unclear, the excellent resolution of the FIB‐SEM technology allows us to reconstruct details that were not reported before. Lay Description: Cardiomyocytes are cells that make up the heart muscle. As the chief cell type of the heart, cardiomyocytes are primarily involved in the contractile function of the heart that enables the pumping of blood around the body. Cardiac muscle cells are connected to each other at their short end by numerous intercellular junctions forming together a structure called the intercalated disc. These intercellular junctions comprise specific protein complexes, which are crucial for both intercellular adhesion and correct contraction of the heart. Imaging by conventional electron microscopy (EM) revealed a heavily folded intercalated disc with apparently random organization of the intercellular junctions. However, this conclusion was based on analysis in two dimensions (2D). 3D information of these structures is needed to unravel their true organization and function. In the present study, we used a more contemporary technique, called volume EM, to image and reconstruct the intercalated discs in 3D. By this approach, EM images are made from a whole block of tissue what differs significantly from classical EM methods that uses only one very thin slice for imaging. Further, we analyzed in comparison to normal mice also a mouse model for cardiomyopathy in which a specific protein of the cardiac intercellular junctions, αT‐catenin, is absent. Volume EM revealed that in the hearts of these mice with cardiomyopathy, the finger‐like folds of the intercalated disc are longer and thinner compared to control hearts. Also the intercellular junctions on the folded parts of the intercalated disc are smaller and their connection to the striated cytoskeleton seems further away. In conclusion, our volume EM study has expanded our understanding of 3D structures at the intercalated discs and will pave the way for more detailed models of disturbed cell‐cell contacts associated with heart failure. [ABSTRACT FROM AUTHOR]

Receptor-interacting protein kinase 4 (RIPK4)-deficient mice have epidermal defects and fusion of all external orifices. These are similar to Bartsocas-Papas syndrome and popliteal pterygium syndrome (PPS) in humans, for which causative mutations have been documented in the RIPK4 and IRF6 (interferon regulatory factor 6) gene, respectively. Although genetically distinct, these syndromes share the anomalies of marked pterygia, syndactyly, clefting and hypoplastic genitalia. Despite the strong resemblance of these two syndromes, no molecular connection between the transcription factor IRF6 and the kinase RIPK4 was known and the mechanism underlying the phenotype was unclear. Here we describe that RIPK4 deficiency in mice causes epithelial fusions associated with abnormal periderm development and aberrant ectopic localization of E-cadherin on the apical membrane of the outer peridermal cell layers. In Xenopus, RIPK4 depletion causes the absence of ectodermal epiboly and concomitant gastrulation defects that phenocopy ectopic expression of dominant-negative IRF6. We found that IRF6 controls RIPK4 expression and that wild-type, but not kinase-dead, RIPK4 can complement the gastrulation defect in Xenopus caused by IRF6 malfunctioning. In contrast to the mouse, we observed only minor effects on cadherin membrane expression in Xenopus RIPK4 morphants. However, gastrulation defects were associated with a virtual absence of cortical actin in the ectodermal cells that face the blastocoel cavity and this was phenocopied in embryos expressing dominant-negative IRF6. A role for RIPK4 in actin cytoskeleton organization was also revealed in mouse epidermis and in human epithelial HaCaT cells. In conclusion, we showed that in mice RIPK4 is implicated in cortical actin organization and in E-cadherin localization or function, which can explain the characteristic epithelial fusions observed in PPSs. In addition, we provide a novel molecular link between IRF6 and RIPK4 that unifies the different PPSs to a common molecular pathway. [ABSTRACT FROM AUTHOR]

In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity. [ABSTRACT FROM AUTHOR]

Human myxovirus resistance 2 (MX2) can restrict HIV-1 and herpesviruses at a post-entry step through a process requiring an interaction between MX2 and the viral capsids. The involvement of other host cell factors, however, remains poorly understood. Here, we mapped the proximity interactome of MX2, revealing strong enrichment of phenylalanine-glycine (FG)-rich proteins related to the nuclear pore complex as well as proteins that are part of cytoplasmic ribonucleoprotein granules. MX2 interacted with these proteins to form multiprotein cytoplasmic biomolecular condensates that were essential for its anti-HIV-1 and anti-herpes simplex virus 1 (HSV-1) activity. MX2 condensate formation required the disordered N-terminal region and MX2 dimerization. Incoming HIV-1 and HSV-1 capsids associated with MX2 at these dynamic cytoplasmic biomolecular condensates, preventing nuclear entry of their viral genomes. Thus, MX2 forms cytoplasmic condensates that likely act as nuclear pore decoys, trapping capsids and inducing premature viral genome release to interfere with nuclear targeting of HIV-1 and HSV-1., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Modern life science research is a collaborative effort. Few research groups can single-handedly support the necessary equipment, expertise and personnel needed for the ever-expanding portfolio of technologies that are required across multiple disciplines in today's life science endeavours. Thus, research institutes are increasingly setting up scientific core facilities to provide access and specialised support for cutting-edge technologies. Maintaining the momentum needed to carry out leading research while ensuring high-quality daily operations is an ongoing challenge, regardless of the resources allocated to establish such facilities. Here, we outline and discuss the range of activities required to keep things running once a scientific imaging core facility has been established. These include managing a wide range of equipment and users, handling repairs and service contracts, planning for equipment upgrades, renewals, or decommissioning, and continuously upskilling while balancing innovation and consolidation., (© 2024 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Centralised core facilities have evolved into vital components of life science research, transitioning from a primary focus on centralising equipment to ensuring access to technology experts across all facets of an experimental workflow. Herein, we put forward a seven-pillar model to define what a core facility needs to meet its overarching goal of facilitating research. The seven equally weighted pillars are Technology, Core Facility Team, Training, Career Tracks, Technical Support, Community and Transparency. These seven pillars stand on a solid foundation of cultural, operational and framework policies including the elements of transparent and stable funding strategies, modern human resources support, progressive facility leadership and management as well as clear institute strategies and policies. This foundation, among other things, ensures a tight alignment of the core facilities to the vision and mission of the institute. To future-proof core facilities, it is crucial to foster all seven of these pillars, particularly focusing on newly identified pillars such as career tracks, thus enabling core facilities to continue supporting research and catalysing scientific advancement. Lay abstract: In research, there is a growing trend to bring advanced, high-performance equipment together into a centralised location. This is done to streamline how the equipment purchase is financed, how the equipment is maintained, and to enable an easier approach for research scientists to access these tools in a location that is supported by a team of technology experts who can help scientists use the equipment. These centralised equipment centres are called Core Facilities. The core facility model is relatively new in science and it requires an adapted approach to how core facilities are built and managed. In this paper, we put forward a seven-pillar model of the important supporting elements of core facilities. These supporting elements are: Technology (the instruments themselves), Core Facility Team (the technology experts who operate the instruments), Training (of the staff and research community), Career Tracks (for the core facility staff), Technical Support (the process of providing help to apply the technology to a scientific question), Community (of research scientist, technology experts and developers) and Transparency (of how the core facility works and the costs associated with using the service). These pillars stand on the bigger foundation of clear policies, guidelines, and leadership approaches at the institutional level. With a focus on these elements, the authors feel core facilities will be well positioned to support scientific discovery in the future., (© 2024 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Background: Alzheimer's disease (AD) is the most common neurodegenerative disorder affecting memory and cognition. The disease is accompanied by an abnormal deposition of ß-amyloid plaques in the brain that contributes to neurodegeneration and is known to induce glial inflammation. Studies in the APP/PS1 mouse model of ß-amyloid-induced neuropathology have suggested a role for inflammasome activation in ß-amyloid-induced neuroinflammation and neuropathology., Methods: Here, we evaluated the in vivo role of microglia-selective and full body inflammasome signalling in several mouse models of ß-amyloid-induced AD neuropathology., Results: Microglia-specific deletion of the inflammasome regulator A20 and inflammasome effector protease caspase-1 in the App NL-G-F and APP/PS1 models failed to identify a prominent role for microglial inflammasome signalling in ß-amyloid-induced neuropathology. Moreover, global inflammasome inactivation through respectively full body deletion of caspases 1 and 11 in App NL-G-F mice and Nlrp3 deletion in APP/PS1 mice also failed to modulate amyloid pathology and disease progression. In agreement, single-cell RNA sequencing did not reveal an important role for Nlrp3 signalling in driving microglial activation and the transition into disease-associated states, both during homeostasis and upon amyloid pathology., Conclusion: Collectively, these results question a generalizable role for inflammasome activation in preclinical amyloid-only models of neuroinflammation., Competing Interests: SD, IP, HM, and AB are employed by Janssen Pharmaceutica NV. ML serves as a consultant for Ventyx Biosciences and Novo Nordisk outside of the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Srinivasan, Kancheva, De Ren, Saito, Jans, Boone, Vandendriessche, Paesmans, Maurin, Vandenbroucke, Hoste, Voet, Scheyltjens, Pavie, Lippens, Schwabenland, Prinz, Saido, Bottelbergs, Movahedi, Lamkanfi and van Loo.)

Serial Block Face Scanning Electron Microscopy (SBF-SEM) is one of several volume electron microscopy (vEM) techniques whose purpose is to reveal the nanostructure of cells and tissues in three dimensions. As one of the earliest, and possibly most widely adopted of the disruptive vEM techniques there have been hundreds of publications using the method, although very few comparative studies of specimen preparation parameters. While some studies have focused on staining and specimen acquisition no comparison of resin embedding has yet been conducted. To this end we have surveyed the SBF-SEM literature to determine which resins are commonly used and compared them in both cellular and fixed tissue samples in an attempt to optimize sample preparation for: effectiveness of resin infiltration, resistance to charging and beam damage and clarity of image in the resulting data set. Here we present the results and discuss the various factors that go into optimizing specimen preparation for SBF-SEM., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Core facilities have a different mission than academic research labs. Accordingly, they require different career paths and structures., (© 2022 The Authors.)

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Correlative light and electron microscopy is a valuable tool to image samples across resolution scales and link data on structure and function. While studies using this technique have been available since the 1960s, recent developments have enabled applying these workflows to large volumes of cells and tissues. Much of the development in this area has been facilitated through the collaborative efforts of microscopists and commercial companies to bring the methods, hardware and image processing technologies needed into laboratories and core imaging facilities. This is a prime example of how what was once a niche area can be brought into the mainstream of microscopy by the efforts of imaging pioneers who push the boundaries of possibility., (© 2021 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by the accumulation of amyloid β (Aβ) and neurofibrillary tangles. The last decade, it became increasingly clear that neuroinflammation plays a key role in both the initiation and progression of AD. Moreover, also the presence of peripheral inflammation has been extensively documented. However, it is still ambiguous whether this observed inflammation is cause or consequence of AD pathogenesis. Recently, this has been studied using amyloid precursor protein (APP) overexpression mouse models of AD. However, the findings might be confounded by APP-overexpression artifacts. Here, we investigated the effect of low-grade peripheral inflammation in the APP knock-in (App NL-G-F ) mouse model. This revealed that low-grade peripheral inflammation affects (1) microglia characteristics, (2) blood-cerebrospinal fluid barrier integrity, (3) peripheral immune cell infiltration and (4) Aβ deposition in the brain. Next, we identified mechanisms that might cause this effect on AD pathology, more precisely Aβ efflux, persistent microglial activation and insufficient Aβ clearance, neuronal dysfunction and promotion of Aβ aggregation. Our results further strengthen the believe that even low-grade peripheral inflammation has detrimental effects on AD progression and may further reinforce the idea to modulate peripheral inflammation as a therapeutic strategy for AD., (© 2021. The Author(s).)

Pericytes and endothelial cells share membranous interdigitations called "peg-and-socket" interactions that facilitate their adhesion and biochemical crosstalk during vascular homeostasis. However, the morphology and distribution of these ultrastructures have remained elusive. Using a combination of 3D electron microscopy techniques, we examined peg-and-socket interactions in mouse brain capillaries. We found that pegs extending from pericytes to endothelial cells were morphologically diverse, exhibiting claw-like morphologies at the edge of the cell and bouton-shaped swellings away from the edge. Reciprocal endothelial pegs projecting into pericytes were less abundant and appeared as larger columnar protuberances. A large-scale 3D EM data set revealed enrichment of both pericyte and endothelial pegs around pericyte somata. The ratio of pericyte versus endothelial pegs was conserved among the pericytes examined, but total peg abundance was heterogeneous across cells. These data show considerable investment between pericytes and endothelial cells, and provide morphological evidence for pericyte somata as sites of enriched physical and biochemical interaction.

Core facilities (CFs) provide a centralised access to costly equipment, scientific expertise, experimental design, day-to-day technical support and training of users. CFs have a tremendous impact on research outputs, skills and educational agendas, increasing the competencies of staff, researchers and students. However, the rapid development of new technologies and methodologies for the life sciences requires fast adaptation and development of existing core facilities and their technical and scientific staff. Given the scarcity of well-defined CF career paths, CF staff positions are typically filled by people having followed either academic or technical tracks. Each academic institution follows different policies and often fails to adequately recognize the merits of CF personnel and to support their training efficiently. Thus, the Core Technologies for Life Science association (CTLS), through the Training working group, has conducted an anonymous online survey to assess the training needs of CF personnel, as well as to identify common characteristics and challenges in this relatively new and dynamic career type. 275 individuals, including core managers and directors, technicians, technologists and administrators, participated in the survey. The survey was divided into 2 sections; the first, applied to all respondents, and the second, specifically targeted core management issues. Training needs in technological areas, financial and soft skills, management and administrative issues were surveyed as well. The lack of clarity and consistency regarding established career paths for CF professionals was evident from the second part of the survey, highlighting geographical or cultural differences. Gender balance was achieved and the distribution was always taken into account. The results of this survey highlight a need to develop better training resources for CF staff, to improve their recognition within academic institutions, and to establish a recognized career pathway., (© Association of Biomolecular Resource Facilities.)

While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive for reasons that remain unclear. Here, we show that Linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of T helper-17 cell effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromised the integrity of the electron transport chain. Ultimately, this led to deficient oxidative phosphorylation, diminishing nicotinamide adenine dinucleotide concentrations and impairing cytokine production in differentiating T cells. In accordance, mice lacking mEF-G1 in T cells were protected from experimental autoimmune encephalomyelitis, demonstrating that this pathway is crucial in maintaining T cell function and pathogenicity., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow. Recruited macrophages existed in two subsets with distinct activation states, either closely resembling homeostatic KCs or lipid-associated macrophages (LAMs) from obese adipose tissue. Hepatic LAMs expressed Osteopontin, a biomarker for patients with NASH, linked with the development of fibrosis. Fitting with this, LAMs were found in regions of the liver with reduced numbers of KCs, characterized by increased Desmin expression. Together, our data highlight considerable heterogeneity within the macrophage pool and suggest a need for more specific macrophage targeting strategies in MAFLD., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The recent advent of 3D in electron microscopy (EM) has allowed for detection of nanometer resolution structures. This has caused an explosion in dataset size, necessitating the development of automated workflows. Moreover, large 3D EM datasets typically require hours to days to be acquired and accelerated imaging typically results in noisy data. Advanced denoising techniques can alleviate this, but tend to be less accessible to the community due to low-level programming environments, complex parameter tuning or a computational bottleneck. We present DenoisEM: an interactive and GPU accelerated denoising plugin for ImageJ that ensures fast parameter tuning and processing through parallel computing. Experimental results show that DenoisEM is one order of magnitude faster than related software and can accelerate data acquisition by a factor of 4 without significantly affecting data quality. Lastly, we show that image denoising benefits visualization and (semi-)automated segmentation and analysis of ultrastructure in various volume EM datasets.

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., (Copyright © 2020 Kerstens et al.)