Endosomes are nanoscale intracellular compartments that sort and recycle cell-surface receptors such as epidermal growth factor receptor-1 (EGFR1). Nanometer-scale interactions and coclustering of signaling proteins, cargo, and the membrane are critical to this process, yet direct 3D visualization has been hindered by the limited resolution of conventional and super-resolution microscopies. Here, we adapt expansion microscopy (ExM) to visualize and quantify nanoclusters of endosomal proteins in human retinal pigment epithelial (RPE-1) cells. We developed a 3D distortion analysis leveraging the Farneback optical-flow principle to detect anisotropies in hydrogel expansion, revealing under-expansion of cytoplasmic regions within ExM hydrogels and overestimation of size and distance measurements of small compartments such as endosomes. To calibrate ExM images of cytoplasmic regions containing endosomes, we introduced a self-assembling protein nanocage that reports the true local nanoscale expansion factor. To stimulate and visualize EGFR1 internalization and sorting, we applied a pulse-chase protocol with fluorescently tagged epidermal growth factor (EGF), fixed cells at 15 and 30 min, and subjected samples to 10-fold ExM and multiplexed 3D Airyscan microscopy to map cargo and EGFR1 relative to other endosomal proteins. A volume tracing pipeline was developed to visualize the changes in the labeled EGF and EGFR1 densities at the limiting membrane of the endosomes. These changes included enrichment of EGF and EGFR1 in the endosomal interior and accumulation of Rab5a near the limiting membrane during early endosome maturation. Together, this multiplexed 3D ExM toolkit provides a quantitative framework for visualizing and measuring small subcellular organelles at true molecular-scale resolution.

Abstract The nanoscale organisation of ryanodine receptor type 2 (RyR2) channels and junctophilin-2 (JPH2) shapes cardiac Ca²⁺ release, but how this relationship is remodelled in right ventricular failure remains unclear. We developed an integrated analysis pipeline building on multiplexed DNA-PAINT data to quantify RyR2 and JPH2 abundance, stoichiometry of co-clustering, and spatial organisation within individual subsarcolemmal Ca²⁺-release nanodomains. Applied to cardiomyocytes from control rats with pulmonary hypertension-induced right ventricular failure, the approach revealed reduced co-localisation between RyR2 and JPH2 within peripheral junctions and greater variability in their co-clustering stoichiometry across the cell. A sub-variogram analysis further showed divergent remodelling of JPH2 expression patterns across subcellular length scales, indicating that disease alters both local molecular composition and cell-wide spatial heterogeneity. Experimentally derived RyR2/JPH2 maps were then used to model as two-dimensional templates for stochastic reaction-diffusion of Ca²⁺ release. Simulations of spontaneous Ca²⁺ waves from failing cells showed up to 25% wave propagation. Maps from failing cells supported faster transverse and longitudinal Ca²⁺ dependent release, consistent with the emergence, that support the likelihood of heterogeneous modulation and coupling of RyR resulting from the RyR redistribution and heterogeneous JPH2 expression in the failing cell. Together, these findings identify spatially heterogeneous RyR2-JPH2 remodelling as a potential substrate for dysregulated Ca²⁺ signalling in right ventricular failure and establish a transferable toolkit for linking molecular nanostructure to cellular function.

Expansion microscopy (ExM) enables superresolution imaging by embedding biological specimens in a swellable hydrogel, followed by optical clearing and physical expansion. Here we present a detailed protocol for applying ExM to embryonic stages of Paracentrotus lividus, a widely used model in developmental biology. Embryos at cleavage and gastrula stages, as well as pluteus larvae, were successfully expanded fourfold after proteinase digestion. Preexpansion Airyscan imaging provided only limited subcellular information due to autofluorescence, whereas post-expansion samples displayed markedly reduced background and resolved fine structures. To address distortions caused by the calcified skeleton in pluteus larvae, we incorporated a decalcification step with EDTA, which preserved morphology and enabled isotropic expansion. Distinct NHS ester dyes further allowed differential labelling of the fertilisation envelope and blastomeres, illustrating the versatility of this approach. Together, these adaptations establish a reproducible workflow for ExM in marine invertebrates, offering a valuable methodological resource and a foundation for future applications in developmental and environmental research.

Expansion microscopy (ExM) is a tissue-swelling technique that enables super-resolution imaging through a specialized preparation process that physically expands stained biomolecules within a fixed sample. Here, we detail a protocol to apply the 4× ExM technique to neuromuscular junctions (NMJs) from both human and mouse muscle preparations. We describe steps for muscle fixation, microdissection, staining, gelation, and digestion. We then detail procedures for expansion, mounting, imaging, analysis, and quantification. This protocol can be used to reveal nanoscale anatomical NMJ features. For complete details on the use and execution of this protocol, please refer to Ramadan et al.1.

The neuromuscular junction (NMJ) is a specialized synapse that relays signals from the lower motor neuron to the skeletal muscle. Here, we detail the development and application of expansion microscopy (ExM) as a highly accessible, relatively cheap, powerful, and reproducible tool with which to obtain high-resolution insights into the subcellular structure and function of NMJs from whole-mount preparations, previously only achievable using super-resolution microscopy. ExM is equally applicable to both mouse and human tissue samples, facilitating high-resolution comparative analyses. Qualitative and quantitative analysis of ExM images reveals significant differences in the distribution of acetylcholine receptors, synaptic vesicles, and voltage-gated Na+ 1.4 (NaV1.4) channels between human and mouse NMJs that are not readily observable using conventional confocal microscopy. We conclude that ExM offers a cost-effective and adaptable approach to facilitate nano-scale imaging of the NMJ.

Endosomes are nanoscale intracellular compartments that sort and recycle cell surface receptors such as epidermal growth factor receptor 1 (EGFR1). Nanometre-scale interactions and co-clustering of signalling proteins, cargo, and the membrane are critical to this process. Direct visualisation of these interactions has been hindered by the limited 3D resolution achievable with conventional and super-resolution microscopies. Here, we present the adaptation of expansion microscopy (ExM) to visualise, and quantify nanoclusters of endosomal proteins of human retinal pigment epithelial (RPE-1) cells. A 3D distortion analysis was developed leveraging the Farneback optical flow principle for detecting anisotropies in the hydrogel expansion. Analysis of pre- and post- ExM image volumes for 3D anisotropies revealed under-expansion of cytoplasmic regions within ExM hydrogels, often leading to over-estimation of size and distance measurements of small compartments such as endosomes. A self-assembling protein nanocage that reports the true local and nanoscale expansion factor was genetically introduced into the cells to calibrate ExM images of cytoplasmic regions containing endosomes. To stimulate and visualise the internalisation and sorting of EGFR1 in mammalian cells, a pulse-chase protocol was carried out with fluorescently-tagged EGF. The cells were subsequently fixed at 15- and 30- minute time points and subjected to 10-fold ExM and multiplexed 3D Airyscan microscopy to map cargo and EGFR1 vs other endosomal proteins. A volume tracing pipeline was developed to visualise the changes in the labelled EGF and EGFR1 densities at the limiting membrane of the endosomes. With multiplexed 3D ExM image volumes, we observed the enrichment of both EGF and EGFR1 in the endosomal interior and the accumulation of endosomal protein Rab5a near the limiting membrane during this early maturation of the endosomes. Taken together, the multiplexed 3D ExM toolkit offers a quantitative framework for visualising and measuring the intrinsic biology of small sub-cellular organelles like endosomes at true molecular-scale resolution. ### Competing Interest Statement The authors have declared no competing interest. UK Research and Innovation, MR/S03241X/1 Wellcome Trust, 212246/Z/18/Z

Visualising C. elegans germ granules using the super resolution technique expansion microscopy against the backdrop of proteome staining reveals granule ultrastructure. Light and electron microscopy techniques have been indispensable in the identification and characterization of liquid–liquid phase separation membraneless organelles. However, for complex membraneless organelles such as the perinuclear germ granule in C. elegans, our understanding of how the intact organelle is regulated is hampered by (1) technical limitations in confocal fluorescence imaging for the simultaneous examination of multiple granule protein markers and (2) inaccessibility of electron microscopy. We take advantage of the newly developed super resolution method of expansion microscopy (ExM) and in situ staining of the whole proteome to examine the C. elegans germ granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with WT animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, Mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. This study will serve as an important tool in our understanding of germ granule biology and the biological role of liquid–liquid phase separation.

Caveolae are small flask-shaped invaginations of the surface membrane which are proposed to recruit and co-localise signalling molecules. The distinctive caveolar shape is achieved by the oligomeric structural protein caveolin, of which three isoforms exist. Aside from the finding that caveolin-3 is specifically expressed in muscle, functional differences between the caveolin isoforms have not been rigorously investigated. Caveolin-3 is relatively cysteine-rich compared to caveolins 1 and 2, so we investigated its cysteine post-translational modifications. We find that caveolin-3 is palmitoylated at 6 cysteines and becomes glutathiolated following redox stress. We map the caveolin-3 palmitoylation sites to a cluster of cysteines in its C terminal membrane domain, and the glutathiolation site to an N terminal cysteine close to the region of caveolin-3 proposed to engage in protein interactions. Glutathiolation abolishes caveolin-3 interaction with heterotrimeric G protein alpha subunits. Our results indicate that a caveolin-3 oligomer contains up to 66 palmitates, compared to up to 33 for caveolin-1. The additional palmitoylation sites in caveolin-3 therefore provide a mechanistic basis by which caveolae in smooth and striated muscle can possess unique phospholipid and protein cargoes. These unique adaptations of the muscle-specific caveolin isoform have important implications for caveolar assembly and signalling.

Super-resolution fluorescence microscopy (SRM), including expansion microscopy (ExM), enables imaging of cellular structures and also locating proteins in a cellular context with nanometer resolution. Expansion microscopy is a method that physically magnifies samples, thus allowing the obtention of super-resolution images by using a conventional microscope. SRM, when combined with novel image analysis, provides a powerful tool to investigate the complex cellular environment in order to study the mechanisms by which angiotensin type 1 receptor AT1Rs are distributed in the plasma membrane.

To build a just, equitable, and diverse academy, scientists and institutions must address systemic barriers that sex and gender minorities face. This Commentary summarizes (1) critical context informing the contemporary oppression of transgender people, (2) how this shapes extant research on sex and gender, and (3) actions to build an inclusive and rigorous academy for all.

Correction to Materials and Methods.

Amine-reactive esters of aromatic fluorescent dyes are emerging as imaging probes for nondescript staining of cellular and tissue architectures. We characterised the staining patterns of 14 fluorescent dye ester species with varying physical and spectral properties in the broadly studied human HeLa cell line. When combined with the super-resolution technique expansion microscopy (ExM) involving swellable acrylamide hydrogels, fluorescent esters reveal nanoscale features including cytoplasmic membrane-bound compartments and nucleolar densities. We observe differential labelling patterns linked to the biochemical properties of the conjugated dye. Alterations in staining density and compartment specificity were seen depending on the timepoint of application in the ExM protocol. Additional complexity in labelling patterns was detected arising from inter-ester interactions. Our findings raise a number of considerations for the use of fluorescent esters. We demonstrate esters as a useful addition to the repertoire of stains of the cellular proteome, whether applied either on their own to visualise overall cellular morphology, or as counterstains providing ultrastructural context alongside specific target markers like antibodies.

Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, 'calcium sparks', determine the contraction of the myocardium. We adapted a correlative super-resolution microscopy protocol to correlate sub-plasmalemmal spontaneous calcium sparks in rat right ventricular myocytes with the local nanoscale RyR2 positions. This revealed a steep relationship between the integral of a calcium spark and the sum of the local RyR2s. Segmentation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR2 clusters. In myocytes taken from failing right ventricles, RyR2 clusters themselves showed a dissipated morphology and fragmented (smaller) clusters. They also featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyR2s. While fragmented (smaller) RyR2 clusters were rarely observed directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight, non-random clustering of the local RyR2 in both healthy and failing ventricles.

Light and electron microscopy techniques have been indispensable in the identification and characterization of liquid–liquid phase separation membraneless organelles. However, for complex membraneless organelles such as the perinuclear germ granule inC. elegans, our understanding of how the intact organelle is regulated is hampered by (1) technical limitations in confocal fluorescence imaging for the simultaneous examination of multiple granule protein markers and (2) inaccessibility of electron microscopy. We take advantage of the newly developed super resolution method of expansion microscopy (ExM) and in situ staining of the whole proteome to examine theC. elegansgerm granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with WT animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, Mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. This study will serve as an important tool in our understanding of germ granule biology and the biological role of liquid–liquid phase separation.

Over the course of almost a decade, electron microscopy has been peerless in its ability to visualise nanoscale cellular ultrastructure. Recent innovations in fluorescence microscopy have pushed the resolution closer to electron microscopy levels, with the added benefit of labelling specific protein targets with antibodies.

Expansion microscopy (ExM) is a versatile super-resolution microscopy pipeline, leveraging nanoscale biomolecular crosslinking and osmotically driven swelling of hydrogels. Currently, ExM is a laborious and skill-intensive technique, involving manual handling of the hydrogels that can compromise the integrity of the gels and capacity to track gel isotropy, hence diminishing reproducibility. We have developed a 3D-printable microplate system to contain the entire ExM workflow within each well, enabling in situ image acquisition and eliminating the need for direct handling of the hydrogels. The preservation of the gel geometry and orientation of the microplate wells enables convenient tracking of gel expansion, pre-and post-ExM image acquisition, and distortion mapping of every cell or region of interest. We demonstrate the utility of this approach with both single-color and multiplexed ExM of cultured HeLa cells and dissected pupal Drosophila melanogasterwing tissue to reveal distortion-prone structures ranging from sub-cellular organelles to micron-scale tissue regions.

Light and electron microscopy techniques have been indispensable in the identification and characterization of liquid-liquid phase separation membraneless organelles. However, for complex membraneless organelles such as the perinuclear germ granule in C. elegans, our understanding of how the intact organelle is regulated is hampered by (1) technical limitations in confocal fluorescence imaging for the simultaneous examination of multiple granule protein markers and (2) inaccessibility of electron microscopy. We take advantage of the newly developed super resolution method of expansion microscopy (ExM) and in situ staining of the whole proteome to examine the C. elegans germ granule, the P granule. We show that in small RNA pathway mutants, the P granule is smaller compared with WT animals. Furthermore, we investigate the relationship between the P granule and two other germ granules, Mutator foci and Z granule, and show that they are located within the same protein-dense regions while occupying distinct subdomains within this ultrastructure. This study will serve as an important tool in our understanding of germ granule biology and the biological role of liquid-liquid phase separation.

Coordinated events of calcium (Ca2+) released from the endoplasmic reticulum (ER) are key second messengers in excitable cells. In pain-sensing dorsal root ganglion (DRG) neurons, these events can be observed as Ca2+ sparks, produced by a combination of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R1). These microscopic signals offer the neuronal cells with a possible means of modulating the subplasmalemmal Ca2+ handling, initiating vesicular exocytosis. With super-resolution dSTORM and expansion microscopies, we visualised the nanoscale distributions of both RyR and IP3R1 that featured loosely organised clusters in the subplasmalemmal regions of cultured rat DRG somata. We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of RyR and IP3R1 in the locality of each Ca2+ spark. We found that most subplasmalemmal sparks correlated with relatively small groups of RyR whilst larger sparks were often associated with larger groups of IP3R1. These data also showed spontaneous Ca2+ sparks in <30% of the subplasmalemmal cell area but consisted of both these channel species at a 3.8–5 times higher density than in nonactive regions of the cell. Taken together, these observations reveal distinct patterns and length scales of RyR and IP3R1 co-clustering at contact sites between the ER and the surface plasmalemma that encode the positions and the quantity of Ca2+ released at each Ca2+ spark.