Expanding Insights: Harnessing Expansion Microscopy for Super-Resolution Analysis of HIV-1-Cell Interactions.

Expansion microscopy has recently emerged as an alternative technique for achieving high-resolution imaging of biological structures. Improvements in resolution are achieved by physically expanding samples through embedding in a swellable hydrogel before microscopy. However, expansion microscopy has been rarely used in the field of virology. Here, we evaluate and characterize the ultrastructure expansion microscopy (U-ExM) protocol, which facilitates approximately four-fold sample expansion, enabling the visualization of different post-entry stages of the HIV-1 life cycle, focusing on nuclear events. Our findings demonstrate that U-ExM provides robust sample expansion and preservation across different cell types, including cell-culture-adapted and primary CD4+ T-cells as well as monocyte-derived macrophages, which are known HIV-1 reservoirs. Notably, cellular targets such as nuclear bodies and the chromatin landscape remain well preserved after expansion, allowing for detailed investigation of HIV-1-cell interactions at high resolution. Our data indicate that morphologically distinct HIV-1 capsid assemblies can be differentiated within the nuclei of infected cells and that U-ExM enables detection of targets that are masked in commonly used immunofluorescence protocols. In conclusion, we advocate for U-ExM as a valuable new tool for studying virus-host interactions with enhanced spatial resolution.

Genetically-encoded markers for confocal visualization of single dense core vesicles.

Neuronal dense core vesicles (DCVs) store and release a diverse array of neuromodulators, trophic factors and bioamines. The analysis of single DCVs has largely been possible only using electron microscopy, which makes understanding cargo segregation and DCV heterogeneity difficult. To address these limitations, we developed genetically-encoded markers for DCVs that can be used in combination with standard immunohistochemistry and expansion microscopy, to enable single-vesicle resolution with confocal microscopy.

Ex vivo nanoscale abluminal mapping of putative cargo receptors at the blood-brain barrier of expanded brain capillaries.

Receptor mediated transport of therapeutic antibodies through the blood-brain barrier (BBB) give promise for drug delivery to alleviate brain diseases. We developed a low-cost method to obtain nanoscale localization data of putative cargo receptors. We combine existing ex vivo isolation methods with expansion microscopy (ExM) to analyze receptor localizations in brain microcapillaries. Using this approach, we show how to analyze receptor localizations in endothelial cells of brain microcapillaries in relation to the abluminal marker collagen IV. By choosing the thinnest capillaries, microcapillaries for analysis, we ensure the validity of collagen IV as an abluminal marker. With this tool, we confirm transferrin receptors as well as sortilin to be both luminally and abluminally localized. Furthermore, we identify basigin to be an abluminal receptor. Our methodology can be adapted to analyze different types of isolated brain capillaries and we anticipate that this approach will be very useful for the research community to gain new insight into cargo receptor trafficking in the slim brain endothelial cells to elucidate novel paths for future drug design.

Iterative Immunostaining and NEDD Denoising for Improved Signal-To-Noise Ratio in ExM-LSCM.

Expansion microscopy (ExM) has significantly reformed the field of super-resolution imaging, emerging as a powerful tool for visualizing complex cellular structures with nanoscale precision. Despite its capabilities, the epitope accessibility, labeling density, and precision of individual molecule detection pose challenges. We recently developed an iterative indirect immunofluorescence (IT-IF) method to improve the epitope labeling density, improving the signal and total intensity. In our protocol, we iteratively apply immunostaining steps before the expansion and exploit signal processing through noise estimation, denoising, and deblurring (NEDD) to aid in quantitative image analyses. Herein, we describe the steps of the iterative staining procedure and provide instructions on how to perform NEDD-based signal processing. Overall, IT-IF in ExM-laser scanning confocal microscopy (LSCM) represents a significant advancement in the field of cellular imaging, offering researchers a versatile tool for unraveling the structural complexity of biological systems at the molecular level with an increased signal-to-noise ratio and fluorescence intensity. Key features • Builds upon the method developed by Mäntylä et al. [1] and introduces the IT-IF method and signal-processing platform for several nanoscopy imaging applications. • Retains signal-to-noise ratio and significantly enhances the fluorescence intensity of ExM-LSCM data. • Automatic estimation of noise, signal reconstruction, denoising, and deblurring for increased reliability in image quantifications. • Requires at least seven days to complete.

Visualizing highly bright and uniform cellular ultrastructure by expansion-microscopy with tetrahedral DNA nanostructures.

Expansion Microscopy (ExM) is a widely used super-resolution technique that enables imaging of structures beyond the diffraction limit of light. However, ExM suffers from weak labeling signals and expansion distortions, limiting its applicability. Here, we present an innovative approach called Tetrahedral DNA nanostructure Expansion Microscopy (TDN-ExM), addressing these limitations by using tetrahedral DNA nanostructures (TDNs) for fluorescence labeling. Our approach demonstrates a 3- to 10-fold signal amplification due to the multivertex nature of TDNs, allowing the modification of multiple dyes. Previous studies have confirmed minimal distortion on a large scale, and our strategy can reduce the distortion at the ultrastructural level in samples because it does not rely on anchoring agents and is not affected by digestion. This results in a brighter fluorescence, better uniformity, and compatibility with different labeling strategies and optical super-resolution technologies. We validated the utility of TDN-ExM by imaging various biological structures with improved resolutions and signal-to-noise ratios.

Imaging Giardia intestinalis cellular organisation using expansion microscopy reveals atypical centrin localisation.

Advanced imaging of microorganisms, including protists, is challenging due to their small size. Specimen expansion prior to imaging is thus beneficial to increase resolution and cellular details. Here, we present a sample preparation workflow for improved observations of the single-celled eukaryotic pathogen Giardia intestinalis (Excavata, Metamonada). The binucleated trophozoites colonize the small intestine of humans and animals and cause a diarrhoeal disease. Their remarkable morphology includes two nuclei and a pronounced microtubular cytoskeleton enabling cell motility, attachment and proliferation. By use of expansion and confocal microscopy, we resolved in a great detail subcellular structures and organelles of the parasite cell. The acquired spatial resolution enabled novel observations of centrin localization at Giardia basal bodies. Interestingly, non-luminal centrin localization between the Giardia basal bodies was observed, which is an atypical eukaryotic arrangement. Our protocol includes antibody staining and can be used for the localization of epitope-tagged proteins, as well as for differential organelle labelling by amino reactive esters. This fast and simple technique is suitable for routine use without a superresolution microscopy equipment.

Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts.

The spindle is a key structure in cell division as it orchestrates the accurate segregation of genetic material. While its assembly and function are well-studied, the mechanisms regulating spindle architecture remain elusive. In this study, we investigate the differences in spindle organization between Xenopus laevis and Xenopus tropicalis, leveraging expansion microscopy (ExM) to overcome the limitations of conventional imaging techniques. We optimized an ExM protocol tailored for Xenopus egg extract spindles, improving upon fixation, denaturation and gelation methods to achieve higher resolution imaging of spindles. Our protocol preserves spindle integrity and allows effective pre-expansion immunofluorescence. This method enabled detailed analysis of the differences in microtubule organization between the two species. X. laevis spindles overall exhibited a broader range of bundle sizes, while X. tropicalis spindles contained mostly smaller bundles. Moreover, while both species exhibited larger bundle sizes near and at the spindle center, X. tropicalis spindles otherwise consisted of very small bundles, and X. laevis spindles medium-sized bundles. By enhancing resolution and minimizing distortions and fixation artifacts, our optimized ExM approach offers new insights into spindle morphology and provides a robust tool for studying the structural intricacies of these large cellular assemblies. This work advances our understanding of spindle architecture and opens up new avenues for exploring underlying mechanisms.

Enzymatically enhanced ultrastructure expansion microscopy unlocks expansion of in vitro Toxoplasma gondii cysts.

Expansion microscopy (ExM) is an innovative approach to achieve super-resolution images without using super-resolution microscopes, based on the physical expansion of the sample. The advent of ExM has unlocked the detail of super-resolution images for a broader scientific circle, lowering the cost and entry skill requirements for the field. One of its branches, ultrastructure expansion microscopy (U-ExM), has become popular among research groups studying apicomplexan parasites, including the acute stage of Toxoplasma gondii infection. Here, we show that the chronic cyst-forming stage of Toxoplasma, however, resists U-ExM expansion, impeding precise protein localization. We then solve the in vitro cyst's resistance to denaturation required for successful U-ExM. As the cyst's main structural protein CST1 contains a mucin domain, we added an enzymatic digestion step using the pan-mucinase StcE prior to the expansion protocol. This allowed full expansion of the cysts in fibroblasts and primary neuronal cell culture without disrupting immunofluorescence analysis of parasite proteins. Using StcE-enhanced U-ExM, we clarified the localization of the GRA2 protein, which is important for establishing a normal cyst, observing GRA2 granules spanning across the CST1 cyst wall. The StcE-U-ExM protocol allows accurate pinpointing of proteins in the bradyzoite cyst, which will greatly facilitate investigation of the underlying biology of cyst formation and its vulnerabilities. IMPORTANCE: Toxoplasma gondii is an intracellular parasite capable of establishing long-term chronic infection in nearly all warm-blooded animals. During the chronic stage, parasites encapsulate to form cysts predominantly in neurons and skeletal muscle. Current anti-Toxoplasma drugs do not eradicate chronic parasites, leaving a reservoir of infection. The cyst is critical for disease transmission and pathology, yet it is harder to study, with the function of many chronic-stage proteins still unknown. Ultrastructure expansion microscopy, a new method to overcome the light microscopy's diffraction limit by physically expanding the sample, allowed in-depth studies of acute Toxoplasma infection. We show that Toxoplasma cysts resist expansion using standard protocol, but an additional enzymatic digestion with the mucinase StcE allows full expansion. This protocol offers new avenues for examining the chronic stage, including precise spatial organization of cyst-specific proteins, linking these locations to morphological structures, and detailed investigations of components of the durable cyst wall.

Multiplexed spatial transcriptomics methods and the application of expansion microscopy.

While spatial transcriptomics has undeniably revolutionized our ability to study cellular organization, it has driven the development of a great number of innovative transcriptomics methods, which can be classified into in situ sequencing (ISS) methods, in situ hybridization (ISH) techniques, and next-generation sequencing (NGS)-based sequencing with region capture. These technologies not only refine our understanding of cellular processes, but also open up new possibilities for breakthroughs in various research domains. One challenge of spatial transcriptomics experiments is the limitation of RNA detection due to optical crowding of RNA in the cells. Expansion microscopy (ExM), characterized by the controlled enlargement of biological specimens, offers a means to achieve super-resolution imaging, overcoming the diffraction limit inherent in conventional microscopy and enabling precise visualization of RNA in spatial transcriptomics methods. In this review, we elaborate on ISS, ISH and NGS-based spatial transcriptomic protocols and on how performance of these techniques can be extended by the combination of these protocols with ExM. Moving beyond the techniques and procedures, we highlight the broader implications of transcriptomics in biology and medicine. These include valuable insight into the spatial organization of gene expression in cells within tissues, aid in the identification and the distinction of cell types and subpopulations and understanding of molecular mechanisms and intercellular changes driving disease development.

Multiplexed Nanoscopy via Buffer Exchange.

Understanding cellular functions, particularly in their intricate complexity, can greatly benefit from the spatial mapping of diverse molecules through multitarget single-molecule localization microscopy (SMLM). Existing methodologies, primarily restricting the encoding dimensions to color and lifetime or requiring cyclic staining, often involve broad chromatic detection, specialized optical configurations, or sophisticated labeling techniques. Here, we propose a simple approach called buffer-exchange stochastic optical reconstruction microscopy (beSTORM), which introduces an additional dimension to differentiate between single molecules irrespective of their spectral properties. This method leverages the distinguishable photoblinking responses to distinct buffer conditions, offering a straightforward yet effective means of fluorophore discrimination. Through buffer exchanges, beSTORM achieves multitarget SMLM imaging with minimal crosstalk. Direct integration with expansion microscopy (ExM) demonstrates its capability to resolve up to six proteins at the molecular level within a single emission color without chromatic aberration. Overall, beSTORM presents a highly compatible imaging platform, promising significant advancements in highly multiplexed nanoscopy for exploring multiple targets in biological systems with nanoscale precision.

Expansion microscopy reveals characteristic ultrastructural features of pathogenic budding yeast species.

Candida albicans is the most prevalent fungal pathogen associated with candidemia. Similar to other fungi, the complex life cycle of C. albicans has been challenging to study with high-resolution microscopy due to its small size. Here, we employed ultrastructure expansion microscopy (U-ExM) to directly visualise subcellular structures at high resolution in the yeast and during its transition to hyphal growth. N-hydroxysuccinimide (NHS)-ester pan-labelling in combination with immunofluorescence via snapshots of various mitotic stages provided a comprehensive map of nucleolar and mitochondrial segregation dynamics and enabled the resolution of the inner and outer plaque of spindle pole bodies (SPBs). Analyses of microtubules (MTs) and SPBs suggest that C. albicans displays a side-by-side SPB arrangement with a short mitotic spindle and longer astral MTs (aMTs) at the pre-anaphase stage. Modifications to the established U-ExM protocol enabled the expansion of six other human fungal pathogens, revealing that the side-by-side SPB configuration is a plausibly conserved feature shared by many fungal species. We highlight the power of U-ExM to investigate subcellular organisation at high resolution and low cost in poorly studied and medically relevant microbial pathogens.

Expansion of metabolically labelled endocytic organelles and cytoskeletal cell structures in Giardia lamblia using optimised U-ExM protocols.

Understanding cellular ultrastructure is tightly bound to microscopic resolution and the ability to identify individual components at that resolution. Expansion microscopy has revolutionised this topic. Here we present and compare two protocols of ultrastructure expansion microscopy that allow for 4.5-fold mostly isotropic expansion and the use of antibodies, metabolic labelling, and DNA stains to demarcate individual regions such as the endoplasmic reticulum, the nuclei, the peripheral endocytic compartments as well as the ventral disc and the cytoskeleton in Giardia lamblia. We present an optimised, shortened, and modular protocol that can be swiftly adjusted to the investigators needs in this important protozoan model organism.

Superresolution Expansion Microscopy in Dictyostelium Amoebae.

Expansion microscopy (ExM) is a superresolution technique for fixed specimens that improves resolution of a given microscopy system approximately fourfold. The gain in resolution in ExM is not achieved by improvement of the resolution of the microscope itself but by isotropic expansion of the sample. To achieve this, the sample is cross-linked to an expandable gel matrix that swells approximately fourfold by incubation in water. We have applied the method to Dictyostelium amoebae and discuss the pros and cons of different labeling techniques in combination with pre- and post-expansion staining protocols.

Inefficient tissue immune response against MPXV in an immunocompromised mpox patient.

The recent outbreak of monkeypox virus (MPXV) was unprecedented in its size and distribution. Those living with uncontrolled HIV and low CD4 T cell counts might develop a fulminant clinical mpox course with increased mortality, secondary infections, and necrotizing lesions. Fatal cases display a high and widespread MPXV tissue burden. The underlying pathomechanisms are not fully understood. We report here the pathological findings of an MPXV-driven abscess in gastrocnemius muscle requiring surgery in an immunocompromised patient with severe mpox. Presence of virus particles and infectivity were confirmed by electron microscopy, expansion microscopy, and virus culture, respectively. MPXV tissue distribution by immunohistochemistry (IHC) showed a necrotic core with infection of different cell types. In contrast, at the lesion rim fibroblasts were mainly infected. Immune cells were almost absent in the necrotic core, but were abundant at the infection rim and predominantly macrophages. Further, we detected high amounts of alternatively activated GPNMB+-macrophages at the lesion border. Of note, macrophages only rarely colocalized with virus-infected cells. Insufficient clearance of infected cells and infection of lesion-associated fibroblasts sustained by the abundance of profibrotic macrophages might lead to the coalescing of lesions and the severe and persistent clinical mpox course observed in immunocompromised patients.

Orthogonal DNA Self-Assembly-Based Expansion Microscopy Platform for Amplified, Multiplexed Biomarker Imaging.

Expansion microscopy (ExM) facilitates nanoscale imaging under conventional microscopes, but it frequently encounters challenges such as fluorescence losses, low signal-to-noise ratio (SNR), and limited detection throughput. To address these issues, a method of orthogonal DNA self-assembly-based ExM (o-DAExM) platform is developed, which employs hybridization chain reaction instead of conventional fluorescence labeling units, showcasing signal amplification efficacy, enhancement of SNR, and expandable multiplexing capability at any stage of the ExM process. In this work, o-DAExM has been applied to compare with immunofluorescence-based ExM for cellular cytoskeleton imaging, and the resolved nanoscale spatial distributions of cytoskeleton show outstanding performance and reliability of o-DAExM. Furthermore, the study demonstrates the utility of o-DAExM in accurately revealing exosome heterogeneous information and multiplexed analysis of protein targets in single cells, which provides infinite possibilities in super-resolution imaging of cells and other samples. Therefore, o-DAExM offers a straightforward expansion and signal labeling method, highlighting future prospects to study nanoscale structures and functional networks in biological systems.

Coaching ribosome biogenesis from the nuclear periphery.

Severe invagination of the nuclear envelope is a hallmark of cancers, aging, neurodegeneration, and infections. However, the outcomes of nuclear invagination remain unclear. This work identified a new function of nuclear invagination: regulating ribosome biogenesis. With expansion microscopy, we observed frequent physical contact between nuclear invaginations and nucleoli. Surprisingly, the higher the invagination curvature, the more ribosomal RNA and pre-ribosomes are made in the contacted nucleolus. By growing cells on nanopillars that generate nuclear invaginations with desired curvatures, we can increase and decrease ribosome biogenesis. Based on this causation, we repressed the ribosome levels in breast cancer and progeria cells by growing cells on low-curvature nanopillars, indicating that overactivated ribosome biogenesis can be rescued by reshaping nuclei. Mechanistically, high-curvature nuclear invaginations reduce heterochromatin and enrich nuclear pore complexes, which promote ribosome biogenesis. We anticipate that our findings will serve as a foundation for further studies on nuclear deformation.

Adhesive interactions within microbial consortia can be differentiated at the single-cell level through expansion microscopy.

Investigating microbe-microbe interactions at the single-cell level is critical to unraveling the ecology and dynamics of microbial communities. In many situations, microbes assemble themselves into densely packed multi-species biofilms. The density and complexity pose acute difficulties for visualizing individual cells and analyzing their interactions. Here, we address this problem through an unconventional application of expansion microscopy, which allows for the 'decrowding' of individual bacterial cells within a multispecies community. Expansion microscopy generally has been carried out under isotropic expansion conditions and used as a resolution-enhancing method. In our variation of expansion microscopy, we carry out expansion under heterotropic conditions; that is, we expand the space between bacterial cells but not the space within individual cells. The separation of individual bacterial cells from each other reflects the competition between the expansion force pulling them apart and the adhesion force holding them together. We employed heterotropic expansion microscopy to study the relative strength of adhesion in model biofilm communities. These included mono and dual-species Streptococcus biofilms, and a three-species synthetic community (Fusobacterium nucleatum, Streptococcus mutans, and Streptococcus sanguinis) under conditions that facilitated interspecies coaggregation. Using adhesion mutants, we investigated the interplay between F. nucleatum outer membrane protein RadD and different Streptococcus species. We also examined the Schaalia-TM7 epibiont association. Quantitative proximity analysis was used to evaluate the separation of individual microbial members. Our study demonstrates that heterotropic expansion microscopy can 'decrowd' dense biofilm communities, improve visualization of individual bacterial members, and enable analysis of microbe-microbe adhesive interactions at the single-cell level.

Mapping protein distribution in the canine photoreceptor sensory cilium and calyceal processes by ultrastructure expansion microscopy.

Photoreceptors are highly polarized sensory neurons, possessing a unique ciliary structure known as the photoreceptor sensory cilium (PSC). Vertebrates have two subtypes of photoreceptors: rods, which are responsible for night vision, and cones, which support daylight vision and color perception. Despite identifying functional and morphological differences between these subtypes, ultrastructural analyses of the PSC molecular architecture in rods and cones are still lacking. In this study, we employed ultrastructure expansion microscopy (U-ExM) to characterize the molecular architecture of the PSC in canine retina. We demonstrated that U-ExM is applicable to both non-frozen and cryopreserved retinal tissues with standard paraformaldehyde fixation. Using this validated U-ExM protocol, we revealed the molecular localization of numerous ciliopathy-related proteins in canine photoreceptors. Furthermore, we identified significant architectural differences in the PSC, ciliary rootlet, and calyceal processes between canine rods and cones. These findings pave the way for a better understanding of alterations in the molecular architecture of the PSC in canine models of retinal ciliopathies.

On-demand expansion fluorescence and photoacoustic microscopy (ExFLPAM).

Expansion microscopy (ExM) is a promising technology that enables nanoscale imaging on conventional optical microscopes by physically magnifying the specimens. Here, we report the development of a strategy that enables i) on-demand labeling of subcellular organelles in live cells for ExM through transfection of fluorescent proteins that are well-retained during the expansion procedure; and ii) non-fluorescent chromogenic color-development towards efficient bright-field and photoacoustic imaging in both planar and volumetric formats, which is applicable to both cultured cells and biological tissues. Compared to the conventional ExM methods, our strategy provides an expanded toolkit, which we term as expansion fluorescence and photoacoustic microscopy (ExFLPAM), by allowing on-demand fluorescent protein labeling of cultured cells, as well as non-fluorescent absorption contrast-imaging of biological samples.