Correlative Light And Electron Microscopy Research Articles

Indirect Correlative Light and Electron Microscopy (iCLEM): A Novel Pipeline for Multiscale Quantification of Structure From Molecules to Organs.

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.

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Rapid in-EPON CLEM: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes and StayGold

Correlative light and electron microscopy (CLEM) combines light microscopy (LM) of fluorescent samples to ultrastructural analyses by electron microscopy (EM). Pre-embedding CLEM often suffers from inaccurate correlation between LM and EM modalities. Post-embedding CLEM enables precise registration of structures directly on EM sections, but requires fluorescent markers withstanding EM sample preparation, especially osmium tetroxide fixation, dehydration and EPON embedding. Most fluorescent proteins (FPs) lose their fluorescence during such conventional embedding (CE), but synthetic dyes represent promising alternatives as their stability exceeds those of FP. We analyzed various Janelia Fluor dyes and TMR conjugated to ligands for self-labeling enzymes, such as HaloTag, for fluorescence preservation after CE. We show that TMR, JF525, JF549, JFX549 and JFX554 retain fluorescence, with JFX549 and JFX554 yielding best results overall, also allowing integration of high-pressure freezing and freeze substitution. Furthermore, we found the recently published FP StayGold to resist CE, facilitating dual-fluorescence in-resin CLEM.

FLEX: genetically encodable enzymatic fluorescence signal amplification using engineered peroxidase

Fluorescent tagging of biomolecules enables their sensitive detection during separation and determining their subcellular location. In this context, peroxidase-based reactions are actively utilized for signal amplification. To harness this potential, we developed a genetically encodable enzymatic fluorescence signal amplification method using APEX (FLEX). We synthesized a fluorescent probe, Jenfluor triazole (JFT1), which effectively amplifies and restricts fluorescence signals under fixed conditions, enabling fluorescence-based detection of subcellularly localized electron-rich metabolites. Moreover, JFT1 exhibited stable fluorescence signals even under osmium-treated and polymer-embedded conditions, which supported findings from correlative light and electron microscopy (CLEM) using APEX. Using various APEX-conjugated proteins of interest (POIs) targeted to different organelles, we successfully visualized their localization through FLEX imaging while effectively preserving organelle ultrastructures. FLEX provides insights into dynamic lysosome-mitochondria interactions upon exposure to chemical stressors. Overall, FLEX holds significant promise as a sensitive and versatile system for fluorescently detecting APEX2-POIs in multiscale biological samples.

Imaging of Isolated Exosomes by Correlative Microscopy.

Correlative microscopy is a sophisticated imaging technique that combines optical and electron microscopes, with the most common approach being the integration of light microscopy and electron microscopy, known as correlative light and electron microscopy (CLEM). While CLEM provides a comprehensive view of biological samples, it presents a significant challenge in sample preparation due to the distinct processes involved in each technique. Striking a balance between these methods is crucial. Despite numerous approaches, achieving seamless imaging with CLEM remains a complex task. Exosomes, nanovesicles ranging from 30 to 150 nm in size, are enclosed by a lipid bilayer and released by various cell types. Visualizing exosomes poses difficulties due to their small size and minimal electric charge. However, imaging exosomes at high resolution offers a direct method to understand their morphology and functions. In this study, we evaluated exosome imaging with CLEM using a combination of confocal, transmission electron microscope, and scanning electron microscope (SEM). In addition, we conducted a comparative analysis of these two techniques, evaluating their suitability and efficiency in imaging nanoscale structures. In this study, we found that confocal-SEM correlation is more applicable for imaging exosomes. Moreover, we observed that exosomes were found in clusters in confocal-SEM correlation.

Safari with an Electron Gun: Visualization of Protein and Membrane Interactions in Mitochondria in Natural Environment.

This paper presents new structural data about mitochondria using correlative light and electron microscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them comparable to observing animals in their natural environment on a safari. The paper highlights two areas of research that can only be accomplished using these methods. The study visualized location of the Aβ42 amyloid aggregates in relation to mitochondria to test a hypothesis of development of mitochondrial dysfunction in Alzheimer's disease. The results showed that the Aβ42 aggregates do not interact with mitochondria, although some of them are closely located. Therefore, the study demonstrated that mitochondrial dysfunction is not directly associated with the effects of aggregates on mitochondrial structure. Other processes should be considered as sources of mitochondrial dysfunction. Second unique area presented in this work is high-resolution visualization of the mitochondrial membranes and proteins in them. Analysis of the cryo-ET data reveals toroidal holes in the lamellar structures of cardiac mitochondrial cristae, where ATP synthases are located. The study proposes a new mechanism for sorting and clustering protein complexes in the membrane based on topology. According to this suggestion, position of the OXPHOS system proteins in the membrane is determined by its curvature. High-resolution tomography expands and complements existing ideas about the structural and functional organization of mitochondria. This makes it possible to study the previously inaccessible structural interactions of proteins with each other and with membranes in vivo.

5-ALA localises to the autophagy compartment and increases its fluorescence upon autophagy enhancement through caloric restriction and spermidine treatment in human glioblastoma

Glioblastoma Multiforme (GBM) is the most invasive and prevalent Central Nervous System (CNS) malignancy. It is characterised by diffuse infiltrative growth and metabolic dysregulation that impairs the extent of surgical resection (EoR), contributing to its poor prognosis. 5-Aminolevulinic acid (5-ALA) fluorescence-guided surgical resection (FGR) takes advantage of the preferential generation of 5-ALA-derived fluorescence signal in glioma cells, thereby improving visualisation and enhancing the EoR. However, despite 5-ALA FGR is a widely used technique in the surgical management of malignant gliomas, the infiltrative tumour margins usually show only vague or no visible fluorescence and thus a significant amount of residual tumour tissue may hence remain in the resection cavity, subsequently driving tumour recurrence. To investigate the molecular mechanisms that govern the preferential accumulation of 5-ALA in glioma cells, we investigated the precise subcellular localisation of 5-ALA signal using Correlative Light and Electron Microscopy (CLEM) and colocalisation analyses in U118MG glioma cells. Our results revealed strong 5-ALA signal localisation in the autophagy compartment – specifically autolysosomes and lysosomes. Flow cytometry was employed to investigate whether autophagy enhancement through spermidine treatment (SPD) or nutrient deprivation/caloric restriction (CR) would enhance 5-ALA fluorescence signal generation. Indeed, SPD, CR and a combination of SPD/CR treatment significantly increased 5-ALA signal intensity, with a most robust increase in signal intensity observed in the combination treatment of SPD/CR. When using 3-D glioma spheroids to assess the effect of 5-ALA on cellular ultrastructure, we demonstrate that 5-ALA exposure leads to cytoplasmic disruption, vacuolarisation and large-scale mitophagy induction. These findings not only suggest a critical role for the autophagy compartment in 5-ALA engagement and signal generation but also point towards a novel and practically feasible approach to enhance 5-ALA fluorescence signal intensity. The findings may highlight that indeed autophagy control may serve as a promising avenue to promote an improved resection and GBM prognosis.

How to apply the broad toolbox of correlative light and electron microscopy to address a specific biological question.

Correlative light and electron microscopy (CLEM) is an approach that combines the strength of multiple imaging techniques to obtain complementary information about a given specimen. The "toolbox" for CLEM is broad, making it sometimes difficult to choose an appropriate approach for a given biological question. In this chapter, we provide experimental details for three CLEM approaches that can help the interested reader in designing a personalized CLEM strategy for obtaining ultrastructural data by using transmission electron microscopy (TEM). First, we describe chemical fixation of cells grown on a solid support (broadest approach). Second, we apply high-pressure freezing/freeze substitution to describe cellular ultrastructure (cryo-immobilization approach). Third, we give a protocol for a ultrastructural labeling by immuno-electron microscopy (immuno-EM approach). In addition, we also describe how to overlay fluorescence and electron microscopy images, an approach that is applicable to each of the reported different CLEM strategies. Here we provide step-by step descriptions prior to discussing possible technical problems and variations of these three general schemes to suit different models or different biological questions. This chapter is written for electron microscopists that are new to CLEM and unsure how to begin. Therefore, our protocols are meant to provide basic information with further references that should help the reader get started with applying a tailored strategy for a specific CLEM experiment.

Some Guiding Principles for a "Simple" Correlative Light Electron Microscopy Experiment.

In recent years, Correlative Multimodal Imaging (CMI) has become an "en vogue" technique and a bit of a buzzword. It entails combining information from different imaging modalities to extract more information from a sample that would otherwise not be possible from each individual technique. The best established CMI technology is correlative light and electron microscopy (CLEM), which applies light and electron microscopy on the exact same sample/structure. In general, it entails the detection of fluorescently tagged proteins or structures by light microscopy and subsequently their relative intracellular localization is determined with nanometer resolution using transmission electron microscopy (TEM). Here, we describe the different steps involved in a "simple" CLEM approach. We describe the overall workflow, instrumentation, and basic principles of sample preparation for a CLEM experiment exploiting stable expression of fluorescent proteins.

Targeting of membrane proteins with fluoronanogold probes for high-resolution correlative microscopy.

Correlative light and electron microscopy (CLEM) can provide valuable information about a biological sample by giving information on the specific localization of a molecule of interest within an ultrastructural context. In this work, we describe a simple CLEM method to obtain high-resolution images of neurotransmitter receptor distribution in synapses by electron microscopy (EM). We use hippocampal organotypic slices from a previously reported mouse model expressing a modified AMPA receptor (AMPAR) subunit that binds biotin at the surface (Getz et al., 2022). This tag can be recognized by StreptAvidin-Fluoronanogold™ conjugates (SA-FNG), which reach receptors at synapses (synaptic cleft is 50-100nm thick). By using pre-embedding labeling, we found that SA-FNG reliably bind synaptic receptors and penetrate around 10-15μm in depth in live tissue. However, the silver enhancement was only reaching the surface of the slices. We show that permeabilization with triton is highly effective at increasing the in depth-gold amplification and that the membrane integrity is well preserved. Finally, we also apply high-resolution electron tomography, thus providing important information about the 3D organization of surface AMPA receptors in synapses at the nanoscale.

DeepCLEM: automated registration for correlative light and electron microscopy using deep learning

In correlative light and electron microscopy (CLEM), the fluorescent images must be registered to the EM images with high precision. Due to the different contrast of EM and fluorescence images, automated correlation-based alignment is not directly possible, and registration is often done by hand using a fluorescent stain, or semi-automatically with fiducial markers. We introduce “DeepCLEM”, a fully automated CLEM registration workflow. A convolutional neural network predicts the fluorescent signal from the EM images, which is then automatically registered to the experimentally measured chromatin signal from the sample using correlation-based alignment. The complete workflow is available as a Fiji plugin and could in principle be adapted for other imaging modalities as well as for 3D stacks.

Correlative Light and Electron Microscopy (CLEM): A Multifaceted Tool for the Study of Geological Specimens

Correlative light and electron microscopy (CLEM) is an advanced imaging approach that faces critical challenges in the analysis of both materials and biological specimens. CLEM integrates the strengths of both light and electron microscopy, in a hardware and software correlative environment, to produce a composite image that combines the high resolution of the electron microscope with the large field of view of the light microscope. It enables a more comprehensive understanding of a sample’s microstructure, texture, morphology, and elemental distribution, thereby facilitating the interpretation of its properties and characteristics. CLEM has diverse applications in the geoscience field, including mineralogy, petrography, and geochemistry. Despite its many advantages, CLEM has some limitations that need to be considered. One of its major limitations is the complexity of the imaging process. CLEM requires specialized equipment and expertise, and it can be challenging to obtain high-quality images that are suitable for analysis. In this study, we present a CLEM workflow based on an innovative sample holder design specially dedicated to the examination of thin sections and three-dimensional samples, with a particular emphasis on geosciences.

Pathogenesis of Reinke's Edema of the Vocal Fold.

The most frequent etiologic factor of Reinke's edema (RE) is considered to be smoking. However, the mechanism for the onset and development of the disease remains unclear. Hypoxia-inducible factor-1α (HIF-1α) is an oxygen-dependent transcriptional activator which plays crucial roles in angiogenesis in hypoxic microenvironments. HIF-1α induces the expression of vascular endothelial growth factor (VEGF) which involves angiogenesis and enhances vascular permeability. This study investigated the roles of HIF-1α in the pathogenesis of RE. Surgical specimens of RE from patients who underwent endolaryngeal microsurgery were used. Normal vocal folds were used as a control group. Expression of HIF-1α and VEGF was analyzed by immunohistochemistry. Three-dimensional fine structures of the vessels in RE were investigated using correlative light and electron microscopy (CLEM) technique. HIF-1α and VEGF were broadly expressed in the stromal, inflammatory, and endothelial cells in the lamina propria of the vocal fold of RE. The expression of HIF-1α and VEGF of RE were significantly higher than in the lamina propria of the normal vocal fold mucosa. CLEM showed vascularization and telangiectasia and there were many dilated capillaries with thin endothelium not covered with pericytes indicating the vessels were fragile. Transcription factor HIF-1α and induced VEGF likely play roles in the pathogenesis of RE. And increased vascular permeability with fragile vessels in angiogenesis is likely to be an etiology of RE. Transcription factor HIF-1α and induced VEGF are potential therapeutic targets for RE. NA Laryngoscope, 134:1785-1791, 2024.

Effective investigation of murine femoral bone development utilizing correlative light and electron microscopy (CLEM)

BackgroundFor effective investigation of the developing structure and chemistry of bone, comprehensive studies including compositional analysis can be achieved through the gradual observation from the micro- to nanometer scale via correlative light and electron microscopy (CLEM). This technique is particularly useful considering the complex hierarchical arrangement of bioapatite and collagen fibrils which may vary according to specific bone tissue types (i.e., lamellar bone and woven bone) and different growth stages. Scanning electron microscopy (SEM) accompanied with the attachment of the scanning transmission electron microscopy (STEM) detector, referred to as the STEM-in-SEM can be utilized to produce high contrast images from materials composed of light elements, and efficiently allows the selection of suitable accelerating voltage for energy-dispersive spectroscopy (EDS). This study aims to emphasize the efficacy of CLEM techniques through applying STEM-in-SEM and EDS analyses, and its application to comparative murine bone investigation in differing ontogenetic stages.FindingsWe have designed a new grid-holder which can be used for both light and electron microscopy, and we presented an imaging technique for TEM specimens via reflective light microscopy (RLM). For performing CLEM, ultra-thin-sections (UTS) prepared from the femoral bones of 1- to 16-week old of Sprague-Dawley (SD) rats provided light and electron micrographs that can be correlated based on the regions of interest (ROIs). STEM-in-SEM micrographs revealed information not attainable by secondary electron (SE) and back-scattered electron (BSE) micrographs. In addition, for analyzing chemical variation according to growth and development of femoral bones from 1- to 16-week-old rats, comparative chemical analysis was performed through STEM-in-SEM EDS with two reference materials.ConclusionHerein, from femoral bones of SD rats, we have confirmed the rapid chemical and structural variations within the first 8 weeks after birth. STEM-in-SEM micrographs revealed the bone development process of the early stage porous bone matrix subsequently being filled with collagen fibrils and bioapatite. In addition, chemical analysis for carbon and oxygen showed the ratios of inorganic to organic phases according to growth and progress in bone mineralization. As a result, we were able to postulate the growth mechanism of murine femoral bone in the neonatal stages of development. We also anticipate that our CLEM techniques can be further utilized for more thorough investigation of bone structure and chemistry in diverse scales.

Correlative light-electron microscopy: integrating dynamics to structure

A challenge of classical electron microscopy (EM) modalities is the static and limited view they present of dynamic biological processes. Correlative light and EM (CLEM) pairs the precise imaging of the cellular location in light microscopy (LM) and EM, taking advantage of both methods. Recent advances in protein labeling, fluorescent markers, super-resolution microscopy, and EM technologies have allowed widespread application in modern biological research. Because of the wide versatility of CLEM, it is difficult to describe a single workflow. In general, samples are prepared and imaged by LM before EM preparation and imaging, with software utilized for the correlation of the two images. Fluorescent tags can help to select and localize structures for EM or add dynamics through live-cell imaging. Future developments in multimodal probes showing electron density and fluorescence activity will allow greater integration of the modalities and high-throughput workflows. Can be paired with focused ion beam-scanning EM (FIB-SEM), cryo-EM, transmission EM (TEM), and serial block-face SEM (SBF-SEM), as well as structured illumination microscopy (SIM), stimulated emission depletion (STED), and total internal reflection fluorescence (TIRF). Amenable to live-cell imaging, in part due to advances that can quickly freeze a sample for cryo-EM. Systems to pair with microscopic X-ray computed tomography to provide non-destructive forms of 3D imaging alongside high-resolution micrographs obtained by TEM and fluorescence. Sensitive enough for applications such as virus–host cell membrane interactions. Alongside other techniques, can localize the distribution of specific proteins or structures within a sample and correlate this information with the ultrastructural details provided by EM. Finding the appropriate FM and EM microscopes may be difficult and expensive. Preparing samples for both modalities requires careful planning and optimization so they are properly preserved, labeled, and processed for the chosen application. The growth of cells on the grid during the LM phase can make correlational positioning difficult. Aligning LM and EM micrographs can be challenging and time-consuming due to differences in resolution and sample shrinkage. Lower throughput and smaller region-of-interest size than traditional FM techniques due to being reliant on EM acquisition and additional sample preparation times. We thank Kit Neikirk for his thoughtful suggestions and edits to the manuscript. Funding by the UNCF/Bristol-Myers Squibb E.E. Just Faculty Fund, BWF Career Awards at the Scientific Interface Award, BWF Ad-hoc Award, National Institutes of Health (NIH) Small Research Pilot Subaward to 5R25HL106365-12 from the NIH PRIDE Program, DK020593, Vanderbilt Diabetes and Research Training Center for DRTC Alzheimer’s Disease Pilot & Feasibility Program. CZI Science Diversity Leadership grant number 2022- 253529 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (to A.H.). The images were arranged using BioRender and all representative images are original. The authors have no interests to declare.

Application of immuno- and affinity labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells

In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same Epon-embedded ultrathin section. This method offers the advantage of high positional accuracy compared to standard CLEM. However, it requires the expression of recombinant proteins. In order to detect the localization of endogenous target(s) and their localized ultrastructures of Epon-embedded samples using in-resin CLEM, we investigated whether immunological and affinity-labeling using fluorescent dyes applied to in-resin CLEM of Epon-embedded cells. The orange fluorescent (λem ∼550 nm) and far-red (λem ∼650 nm) fluorescent dyes examined maintained a sufficient level of fluorescent intensity after staining with osmium tetroxide and subsequent dehydration treatment with ethanol. Immunological in-resin CLEM of mitochondria and the Golgi apparatus was achieved using anti-TOM20, anti-GM130 antibodies, and fluorescent dyes. Two-color in-resin CLEM revealed that wheat germ agglutinin-puncta showed the ultrastructures of multivesicular body-like structures. Finally, taking the advantage of high positional accuracy, volume in-resin CLEM of mitochondria in the semi-thin section (2 μm thick) of Epon-embedded cells was performed by focused ion beam scanning electron microscopy. These results suggested that the application of immunological reaction and affinity-labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells is suitable for analyzing the localization of endogenous targets and their ultrastructures by scanning and transmission electron microscopy.

Modular characterization of SARS-CoV-2 nucleocapsid protein domain functions in nucleocapsid-like assembly

SARS-CoV-2 and its variants, with the Omicron subvariant XBB currently prevailing the global infections, continue to pose threats on public health worldwide. This non-segmented positive-stranded RNA virus encodes the multi-functional nucleocapsid protein (N) that plays key roles in viral infection, replication, genome packaging and budding. N protein consists of two structural domains, NTD and CTD, and three intrinsically disordered regions (IDRs) including the NIDR, the serine/arginine rich motif (SRIDR), and the CIDR. Previous studies revealed functions of N protein in RNA binding, oligomerization, and liquid–liquid phase separation (LLPS), however, characterizations of individual domains and their dissected contributions to N protein functions remain incomplete. In particular, little is known about N protein assembly that may play essential roles in viral replication and genome packing. Here, we present a modular approach to dissect functional roles of individual domains in SARS-CoV-2 N protein that reveals inhibitory or augmented modulations of protein assembly and LLPS in the presence of viral RNAs. Intriguingly, full-length N protein (NFL) assembles into ring-like architecture whereas the truncated SRIDR-CTD-CIDR (N182-419) promotes filamentous assembly. Moreover, LLPS droplets of NFL and N182-419 are significantly enlarged in the presence of viral RNAs, and we observed filamentous structures in the N182-419 droplets using correlative light and electron microscopy (CLEM), suggesting that the formation of LLPS droplets may promote higher-order assembly of N protein for transcription, replication and packaging. Together this study expands our understanding of the multiple functions of N protein in SARS-CoV-2.

Recent advances inin-resin correlative light andelectron microscopy ofEpon-embedded cells.

Correlative fluorescent and electron microscopic images of the same section of epoxy (or other polymer)-embedded samples, hereafter referred to as 'in-resin CLEM', have been developed to improve the positional accuracy and Z-axis resolution limitations of conventional correlative light and electron microscopy (CLEM). High-pressure freezing and quick-freezing substitution result in in-resin CLEM of acrylic-based resin-embedded cells expressing green fluorescent protein, yellow fluorescent protein, mVenus and mCherry, which are sensitive to osmium tetroxide. The identification of osmium-resistant fluorescent proteins leads to the development of in-resin CLEM of Epon-embedded cells. Using subtraction-based fluorescence microscopy with a photoconvertible fluorescent protein, mEosEM-E, its green fluorescence can be observed in thin sections of Epon-embedded cells, and two-color in-resin CLEM using mEosEM-E and mScarlet-H can be performed. Green fluorescent proteins, CoGFP variant 0 and mWasabi, and far-red fluorescent proteins, mCherry2 and mKate2, are available for in-resin CLEM of Epon-embedded cells using the standard procedure for Epon-embedding with additional incubation. Proximity labeling is applied to in-resin CLEM to overcome the limitations of fluorescent proteins in epoxy resin. These approaches will contribute significantly to the future of CLEM analysis.

Three-dimensional correlative light and focused ion beam scanning electron microscopy reveals the distribution and ultrastructure of lanceolate nerve endings surrounding terminal hair follicles in human scalp skin.

Lanceolate nerve endings (LNEs) surrounding hair follicles (HFs) play an important role in detecting hair deflection. Complexes of the LNEs form a palisade-like structure along the longitudinal axis of hair roots in which axons are sandwiched between two processes of terminal Schwann cells (tSCs) at the isthmus of HFs. The structure and molecular mechanism of LNEs in animal sinus hair, pelage, and human vellus hairs have been investigated. Despite the high density of HFs in human scalp skin, the LNEs in human terminal HFs have not been investigated. In this study, we aimed to reveal the distribution and ultrastructure of LNEs in terminal HFs of human scalp skin. Using light-sheet microscopy and immunostaining, the LNEs were observed at one terminal HF but not at the other terminal HFs in the same follicular unit. The ultrastructure of the LNEs of terminal HFs in human scalp skin was characterized using correlated light and electron microscopy (CLEM). Confocal laser microscopy and transmission electron microscopy of serial transverse sections of HFs revealed that LNEs were aligned adjacent to the basal lamina outside the outer root sheath (ORS), at the isthmus of terminal HFs, and adjacent to CD200-positive ORS cells in the upper bulge region. Moreover, axons with abundant mitochondria were sandwiched between tSCs. Three-dimensional CLEM, specifically confocal laser microscopy and focused ion beam scanning electron microscopy, of stained serial transverse sections revealed that LNEs were wrapped with type I and type II tSCs, with the processes protruding from the space between the Schwann cells. Moreover, the ultrastructures of LNEs at miniaturized HFs were similar to those of LNEs at terminal HFs. Preembedding immunoelectron microscopy revealed that Piezo-type mechanosensitive ion channel component 2 (Piezo2), a gated ion channel, was in axons and tSCs and adjacent to the cell membrane of axons and tSCs, suggesting that LNEs function as mechanosensors. The number of LNEs increased as the diameter of the ORS decreased, suggesting that LNEs dynamically adapt to the HF environment as terminal HFs miniaturize into vellus-like hair. These findings will provide insights for investigations of mechanosensory organs, aging, and re-innervation during wound healing.

Indirect correlative light and electron microscopy an integrated rule-based imaging approach with robust quantitative capabilities.

Although powerful, the utility of correlative light and electron microscopy (CLEM) is severely restricted by lack of experimental throughput and quantitative capability. We present indirect CLEM (iCLEM), a rule-based imaging (RUBI) pipeline for low-cost, high throughput assessment of multiscale structure. iCLEM correlates multiscale morphometric measurements independently obtained from each technique by exploiting structural landmarks identifiable via both light and electron microscopy, sidestepping CLEM's rate-limiting steps. Exploiting landmarks robust to perturbation enables comparison between health and disease. As proof of principle, we have used iCLEM to compile a cohesive and comprehensive quantitative description of cell-cell contact structures in cardiac muscle known as intercalated discs (ID). For this, we exploited ID components, such as gap junctions, adherens junctions and desmosomes, as structural landmarks. Confocal microscopy enabled assessments on organ through cellular scales, such as cell geometry, the sizes of IDs in different parts of the heart, and the distribution of ion transport proteins between IDs and other parts of heart muscle cells. Paired sets of transmission electron micrographs at different magnifications enable quantification of ultrastructure from micro through nanoscales ranging from the size and tortuosity of the whole ID to intermembrane spacing within its different nanodomains. Next, we assessed the spatial distribution of ion transport proteins within the ID, relative to structural landmarks and each other, using super-resolution imaging (STORM single molecule localization microscopy). Molecular organization was assessed by spatial analysis of fluorescence images and machine learning-based cluster analysis of single molecule localizations. These data enabled construction of 3D finite element models of IDs populated with ion transport proteins mimicking experimental observations. Integration of these models into physiological models is revealing previously unappreciated structure-function relationships underlying health and disease.