Fluorescence microscopy is increasingly used to measure molecular organization at length scales where labeling, photophysics and sample preparation can dominate quantitative accuracy. Reference standards are therefore needed that combine defined nanoscale geometry with a protein-like environment and compatibility with biological imaging. Here we introduce circular tandem repeat protein as programmable protein standards for benchmarking sub-10-nm fluorescence microscopy. Using genetic code expansion and bioorthogonal labeling, we generated compact protein rings carrying up to six labeling sites. Photoswitching fingerprint analysis revealed geometry-dependent localization accumulation and blinking kinetics, demonstrating that short-range fluorophore interactions can be assessed as measurable benchmark parameters. DNA-PAINT confirmed accessible docking sites and programmed valency at the single-particle level. We further established recombinant tethering and genetically encoded membrane display, extending the standards to cellular environments. cTRP PicoRulers were also compatible with expansion microscopy. Together, cTRPs provide a modular protein-based platform for evaluating molecular-scale imaging performance in purified and cellular environments.

Expansion microscopy (ExM) enables nanoscale imaging on standard microscopes, but combining ExM with single-molecule localization microscopy (SMLM) remains difficult, owing to the incompatibility of expanded hydrogels with photoswitching buffers. Here, we introduce a single-step expansion microscopy method that allows SMLM with spontaneously blinking dyes in 6-14× expanded samples, without re-embedding. We demonstrate nanometer-resolution imaging by resolving the organization of the nuclear pore complex (NPC) and the molecular structure of recombinant homotrimeric proliferating cell nuclear antigen (PCNA).

Precise visualization of nanoparticles (NPs) at the subcellular level is crucial for targeted drug delivery. However, NPs functionalized with permanently fluorescent probes are challenging to detect when only a small fraction reaches the target compartment, as weak fluorescence signals are obscured by intense fluorescence from NPs accumulated in off-target subcellular compartments. Here, we introduce ‘turn on’ NPs that activate fluorescence via an inverse electron demand Diels–Alder (iEDDA) reaction only at their intracellular target. NPs were functionalized with coumarin-102-tetrazine (C102-Tet) turn-on fluorescence upon reaction with trans-cyclooctene (TCO), which was immobilized to microtubules via anchors of various geometry and spatial accessibility. We generated polymer NPs composed of poly(lactide-co-glycolide) (PLGA) and poly(ethylene glycol)-poly(lactic acid) (PEG–PLA), systematically varying PEG chain length (2k vs 5k) and C102-Tet surface density. Direct stochastic optical reconstruction microscopy (dSTORM) resolved individual NPs of ~80 nm and confirmed that C102-Tet presented on the NP surface remains reactive. Quantitatively, 15–45% of C102-Tet groups participated in the iEDDA reaction, depending on PEG chain length and surface density. To overcome insufficient endosomal escape, NPs were directly delivered into the cytosol to demonstrate the fluorogenic NP concept that becomes activated only upon bio-orthogonal reaction at the intracellular target. Confocal microscopy and super-resolution SIM2 imaging revealed NP fluorescence aligned along microtubules and support target-specific iEDDA activation on the surface of NPs. This strategy enables high-contrast tracking of nanocarriers at defined intracellular targets.

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) has become a widely adopted single-molecule localization microscopy (SMLM) technique owing to its high spatial resolution, versatile labeling strategies, and theoretically unlimited multiplexing capability. Recent developments in repetitive docking strand designs have enabled faster image acquisition by increasing the number of potential binding motifs per target. However, the effect of such architectural modifications on effective spatial resolution remains largely unexplored. Here, we systematically quantify how repetitive docking strands influence localization distributions and effective resolution using the well-defined geometry of the trimeric proliferating cell nuclear antigen (PCNA) as a model system. Whereas classical single-motif docking strands resolve the expected ∼6 nm spacing between PCNA subunits with high precision, repetitive docking motifs produce broadened localization distributions, despite comparable localization precision. Our results suggest that spatial blurring arises from a combination of variable binding site geometry, rotational flexibility of elongated multivalent DNA docking sequences, as well as the dynamic behavior of imager strands. This study provides a quantitative framework for understanding how docking strand architecture determines resolution limits in DNA-PAINT and underscores the need to balance multiplexing and imaging speed with structural fidelity. Our results thus offer guidance for the rational design of docking strands for high-precision DNA-PAINT imaging of protein complexes.

Expansion microscopy (ExM) enables nanoscale fluorescence imaging on standard microscopes, but its combination with single-molecule localization microscopy (SMLM) remains challenging due to the incompatibility of expanded hydrogels with photoswitching buffers. Here, we introduce a single-step expansion microscopy approach that enables SMLM using spontaneously blinking dyes in 6-14× expanded samples, without re-embedding or buffer exchange. Using this approach, we achieve nanometer-scale spatial resolution by resolving the organization of the nuclear pore complex and the molecular structure of recombinant homotrimeric proliferating cell nuclear antigen.

Efficient protein labeling with minimal linkage error is a key requirement for super-resolution fluorescence microscopy. Many commonly used labeling strategies, including antibodies, fluorescent proteins, and self-labeling enzymes are limited by steric hindrance, and therefore they limit labeling density or constrain labeling to protein termini, thereby restricting achievable resolution at the molecular scale. Here, we describe a practical and broadly applicable protocol for site-specific protein labeling based on genetic code expansion and bioorthogonal inverse electron-demand Diels-Alder click chemistry. The method relies on the incorporation of a strained alkene-modified noncanonical amino acid at a defined position within a protein of interest, followed by rapid and selective covalent labeling with tetrazine-conjugated organic fluorophores. This approach enables the attachment of small, bright dyes with minimal linkage error and is compatible with both live-cell and fixed-cell imaging. The protocol provides detailed guidance on the design of suitable click sites, expression of amber mutants in mammalian cells, selection of appropriate tetrazine dyes, and optimization of labeling conditions for super-resolution microscopy, including single-molecule localization microscopy. Critical parameters, common pitfalls, and limitations are discussed to facilitate robust implementation across different protein classes and experimental systems. This workflow supports high-density, stoichiometric labeling and enables molecular-scale imaging of proteins in their native cellular context.

Abstract Background Staphylococcus aureus ( S. aureus ) is an increasingly recognized intracellular pathogen, yet infection outcomes vary with bacterial isolate and host cell type. The mechanisms underlying these differences remain poorly understood. This study investigates how distinct intracellular S. aureus isolates influence host signaling programs and infection outcomes by modulating cell death pathways and TNF-R1 dependent regulation of host cell fates across different human cell lines. Methods Four S. aureus isolates were analyzed for intracellular localization using transmission electron microscopy (TEM), structured illumination microscopy (SIM), serial block-face scanning electron microscopy (SBF-SEM), and imaging flow cytometry. Transcriptional reprogramming of infected U937 monocytes was examined by mRNA sequencing. Infection outcomes were characterized and compared to A549 and SaOS-2 cell lines employing Luminex cytokine assays, flow cytometry and Western blot analysis to characterize host cell death mechanisms in both wild-type and TNF-R1 deficient backgrounds. Results All S. aureus isolates localized to endolysosomal and cytosolic compartments but also peri⍰ and putatively intranuclearly, revealing an unexpected intracellular niche. In U937 monocytes, infection induced a conserved stress signature alongside isolate⍰specific transcriptional programs divergently affecting inflammation, metabolism, and cell fate, which was markedly attenuated in response to the chronic⍰infection isolate EDCC 5464. Cell death outcomes were likewise isolate⍰dependent, involving intrinsic and extrinsic apoptosis, mitochondrial depolarization, and caspase-1 activation at distinct temporal dynamics. TNF⍰R1 loss initially delayed but exacerbated late, isolate-independent cytotoxicity, identifying TNF⍰R1 as a key regulator of U937 infection outcome. SaOS⍰2 and A549 cell death was far less affected by isolate or TNF-R1 deficiency. Conclusions These results highlight the multilayered determinants governing intracellular S. aureus survival, non-canonical intracellular localization, and host cell susceptibility. The TNF/TNF-R1 axis is identified to critically determine regulated host defense during early infection stages in a tissue-specific manner. Together with distinct isolate-driven gene expression profiles, infection risks under TNF-targeted therapies and the contribution of S. aureus heterogeneity should be considered in the design of future host-directed treatment strategies. Plain English summary The bacterium Staphylococcus aureus ( S. aureus ) often lives harmlessly in humans but can cause severe or recurrent infections when the skin barrier is broken or the immune system is weakened. A major reason for its persistence is its ability to hide inside human cells, where it is shielded from immune attacks and antibiotics. To effectively target such bacteria, it is crucial to understand that infections vary depending on both the bacterial strain and the infected cell type. Many reasons behind these differences are still puzzling. We explored how different types of S. aureus (collected from different disease types) change how human cells respond to infection. We focused on how the different strains influence the way immune cells adjust their gene activity during infection, and how a receptor called TNF-R1 is involved in managing cell death responses. Bacteria were found not only in compartments meant to destroy them but also near and even inside the cell nucleus, an unexpected location. All strains triggered a similar stress response but also distinct patterns influencing inflammation, metabolism, and cell survival. A strain linked to chronic infection caused weaker responses, suggesting greater stealth. Cells lacking TNF-R1 initially survived longer but later showed greater damage, indicating this receptor’s role in infection control. In lung and bone cells, these effects were less pronounced. Concludingly, S. aureus occupies unexpected niches inside human cells and uses varying survival strategies. TNF-R1 is a key regulator of host infection responses in the analyzed immune cells, highlighting that both bacterial diversity and host factors must be considered when developing targeted treatments. Graphical Abstract Peri- and intranuclear localization early after S. aureus uptake across host cell lines, with isolate-specific modulation of host fates and a critical role for TNF-R1 to mediate regulated death responses of U937 cells. At 2 hpi, intracellular S. aureus not only localizes in (LAMP-1 decorated) membrane-enclosed compartments or directly in the cytosol, but within invaginations of the nuclear surface and intranuclearly with or without being surrounded by a vesicular membrane in U937 wt , SaOS-2 wt , and A549 wt cells. At 4 hpi, S. aureus triggers differential gene expression in ( A ) U937 wt cells to an isolate-specific extent, with both unique and shared transcriptomic signatures across the four isolates, that is muted for the chronic infection isolate EDCC 5464. Apoptotic cell death is induced to an isolate-dependent extent involving extrinsic initiator caspase-8, intrinsic initiator caspase-9 (EDCC 5055 only), and variable effector caspase-3/-7 activity in the earlier stages of infection (6 hpi), which then barely increases (24 hpi) in U937 wt cells. S. aureus -induced cell death and caspase activation is abolished in ( B ) U937 ΔTNF-R1 at 6 hpi, but is significantly reinforced at 24 hpi with diminished isolate-specificity. Correspondingly, mitochondrial trans-membrane potential (ΔΨm) is disrupted for all isolates upon TNF-R1 knockout, as well as caspase-1 activity, suggesting pyroptotic pathway activation at later stages of infection. ( C ) SaOS-2 wt cells show moderate caspase-3/-7 and -1 activation, while infection induces detachment of ( D ) A549 wt cells with minimal caspase activation. Infection induces an isolate- and cell line-dependent cytokine release. Coloured arrows indicate the mean proportion of effector-positive cells (↑ ∼20-40%, ↑ ↑ 40-60%, ↑ ↑ ↑ >60%) representing each S. aureus isolate. Grayed signaling arrows indicate the hypothesis by which TNF-R1 activation and internalization is required to kill lysosomal S. aureus via activation of anti-microbial enzymes and downstream regulated death pathway activation. Created with BioRender.com .

Super-resolution microscopy (SRM) has transformed biological imaging by circumventing the diffraction limit of light and enabling the visualization of cellular structures and processes at the molecular level. Central to the capabilities of SRM is fluorescent labeling, which ensures the precise attachment of fluorophores to biomolecules and has direct impact on the accuracy and resolution of imaging. Continuous innovation and optimization in fluorescent labeling are essential for the successful application of SRM in cutting-edge biological research. In this review, we discuss recent advances in fluorescent labeling strategies for molecular bioimaging, with a special focus on protein labeling. We compare different approaches, highlight technological breakthroughs, and address challenges such as linkage error and labeling density. By evaluating both established and emerging methods, we aim to guide researchers through all aspects that should be considered before opting for any labeling technique.

The contribution of deubiquitylating enzymes (DUBs) to β-Catenin stabilization in intestinal stem cells and colorectal cancer (CRC) is poorly understood. Here, and by using an unbiassed screen, we discovered that the DUB USP10 stabilizes β-Catenin specifically in APC-truncated CRC in vitro and in vivo. Mechanistic studies, including in vitro binding together with computational modelling, revealed that USP10 binding to β-Catenin is mediated via the unstructured N-terminus of USP10 and is outcompeted by intact APC, favouring β-catenin degradation. However, in APC-truncated cancer cells USP10 binds to β-catenin, increasing its stability which is critical for maintaining an undifferentiated tumour identity. Elimination of USP10 reduces the expression of WNT and stem cell signatures and induces the expression of differentiation genes. Remarkably, silencing of USP10 in murine and patient-derived CRC organoids established that it is essential for NOTUM signalling and the APC super competitor-phenotype, reducing tumorigenic properties of APC-truncated CRC. These findings are clinically relevant as patient-derived organoids are highly dependent on USP10, and abundance of USP10 correlates with poorer prognosis of CRC patients. Our findings reveal, therefore, a role for USP10 in CRC cell identity, stemness, and tumorigenic growth by stabilising β-Catenin, leading to aberrant WNT signalling and degradation resistant tumours. Thus, USP10 emerges as a unique therapeutic target in APC truncated CRC.

SNAP-tag is one of the most commonly used self-labeling protein tags for cell imaging studies. To achieve selective spatiotemporal imaging of cells, we set out to engineer a photoactivatable SNAP-tag. For this, we incorporated the well-established and readily available photocaged unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) into all three tyrosine positions of SNAP. In-gel imaging analysis and fluorescence polarization measurements revealed that placing ONBY in position Y114 of the SNAP-tag facilitates the most effective and most efficient photoactivation of the irreversible self-labeling reaction with (sulfonated) benzyl guanine substrates, which is why we dubbed this photoactivatable SNPA-tag variant "SNAPpa". To demonstrated its potential for live-cell imaging, we further tested SNAPpa in HEK293 cells, either fused to a nuclear localization domain for intracellular imaging or fused to either a transmembrane region or the glucagon-like peptide 1 receptor for extracellular imaging. Each SNAPpa construct produced no fluorescence signal when ONBY remained in its photocaged state by keeping the cells in the dark. However, a clear fluorescence signal appeared after light-induced decaging of ONBY. Applying a localized light beam thereby highlighted the precise spatiotemporal control of cell imaging. In conclusion, SNAPpa can be used for the efficient light-induced activation of fluorescence labeling and can be easily established, readily implemented and effectively combined with the broad repertoire of substrates that is already available for SNAP.

Super-resolution microscopy (SRM) has transformed our understanding of proteins' subcellular organization and revealed cellular details down to nanometers, far beyond conventional microscopy. While localization precision is independent of the number of fluorophores attached to a biomolecule, labeling density is a decisive factor for resolving complex biological structures. The average distance between adjacent fluorophores should be less than half the desired spatial resolution for optimal clarity. While this was not a major limitation in recent decades, the success of modern microscopy approaching molecular resolution down to the single-digit nanometer range will depend heavily on advancements in fluorescence labeling. This review highlights recent advances and challenges in labeling strategies for SRM, focusing on site-specific labeling technologies. These advancements are crucial for improving SRM precision and expanding our understanding of molecular interactions.

G protein-coupled receptors (GPCRs) are the most commonly targeted transmembrane proteins by approved drugs. GPCRs undergo conformational changes upon binding with ligands, which allow them to carry out their biological functions within living cells. Several factors affect or modulate the functionality of GPCRs, including molecular diffusion, kinetics, and ligand-dependent oligomerization; however, the tools and time-resolved methods to capture the underlying dynamics are still limited. Particularly crucial is the required wide temporal range when studying GPCR conformational changes which is flanked by other challenges related to data sparsity and data noise.

Zusammenfassung Mit Hilfe der hochauflösenden Fluoreszenzmikroskopie lassen sich Bilder mit einer räumlichen Auflösung von rund 20 nm, weit unterhalb der Beugungsgrenze, aufnehmen. Eine weitere Verbesserung der räumlichen Auflösung bis in den molekularen Bereich ist jedoch durch die Interaktion der Farbstoffe miteinander erschwert. Bei Abständen unterhalb von 10 nm führen verschiedene Energietransferprozesse zu einer schlechteren Lokalisationswahrscheinlichkeit, was die erreichbare strukturelle Auflösung stark einschränkt. Eine genaue zeitaufgelöste Analyse des Fluoreszenzsignals ermöglicht es, die Anzahl der interagierenden Farbstoffe und deren Abstände zu ermitteln. Durch die zeitaufgelöste Analyse des Photoswitching‐Fingerprints ist es uns gelungen, Abstände von 5 nm an Membranrezeptoren zu identifizieren.

Engineered vesicular stomatitis virus (VSV) pseudotyping offers an essential method for exploring virus-cell interactions, particularly for viruses that require high biosafety levels. Although this approach has been employed effectively, the current methodologies for virus visualization and labeling can interfere with infectivity and lead to misinterpretation of results. In this study, we introduce an innovative approach combining genetic code expansion (GCE) and click chemistry with pseudotyped VSV to produce highly fluorescent and infectious pseudoviruses (clickVSVs). These clickVSVs enable robust and precise virus-cell interaction studies without compromising the biological function of the viral surface proteins. We evaluated this approach by generating VSVs bearing a unique chemical handle for click labeling and assessing the infectivity in relevant cell lines. Our results demonstrate that clickVSVs maintain their infectivity post-labeling and present an efficiency about two times higher in detecting surface proteins compared to classical immunolabeling. The utilization of clickVSVs further allowed us to visualize and track 3D virus binding and infection in living cells, offering enhanced observation of virus-host interactions. Thus, clickVSVs provide an efficient alternative for virus-associated research under the standard biosafety levels.

Super-resolution microscopy has revolutionized biological imaging enabling direct insight into cellular structures and protein arrangements with so far unmatched spatial resolution. Today, refined single-molecule localization microscopy methods achieve spatial resolutions in the one-digit nanometer range. As the race for molecular resolution fluorescence imaging with visible light continues, reliable biologically compatible reference structures will become essential to validate the resolution power. Here, PicoRulers (protein-based imaging calibration optical rulers), multilabeled oligomeric proteins designed as advanced molecular nanorulers for super-resolution fluorescence imaging are introduced. Genetic code expansion (GCE) is used to site-specifically incorporate three noncanonical amino acids (ncAAs) into the homotrimeric proliferating cell nuclear antigen (PCNA) at 6 nm distances. Bioorthogonal click labeling with tetrazine-dyes and tetrazine-functionalized oligonucleotides allows efficient labeling of the PicoRuler with minimal linkage error. Time-resolved photoswitching fingerprint analysis is used to demonstrate the successful synthesis and DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is used to resolve 6 nm PCNA PicoRulers. Since PicoRulers maintain their structural integrity under cellular conditions they represent ideal molecular nanorulers for benchmarking the performance of super-resolution imaging techniques, particularly in complex biological environments.

The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. Here we used direct stochastic optical reconstruction microscopy ( d STORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1–2 receptors μm −2 . In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection, which provides SARS-CoV-2 a high infectivity.

The introduction of an engineered aminoacyl-tRNA synthetase/tRNA pair enables site-specific incorporation of unnatural amino acids (uAAs) with functionalized side chains into proteins of interest. Genetic Code Expansion (GCE) via amber codon suppression confers functionalities to proteins but can also be used to temporally control the incorporation of genetically encoded elements into proteins. Here, we report an optimized GCE system (GCEXpress) for efficient and fast uAA incorporation. We demonstrate that GCEXpress can be used to efficiently alter the subcellular localization of proteins within living cells. We show that click labeling can resolve co-labeling problems of intercellular adhesive protein complexes. We apply this strategy to study the adhesion G protein-coupled receptor (aGPCR) ADGRE5/CD97 and its ligand CD55/DAF that play central roles in immune functions and oncological processes. Furthermore, we use GCEXpress to analyze the time course of ADGRE5-CD55 ligation and replenishment of mature receptor-ligand complexes. Supported by fluorescence recovery after photobleaching (FRAP) experiments our results show that ADGRE5 and CD55 form stable intercellular contacts that may support transmission of mechanical forces onto ADGRE5 in a ligand-dependent manner. We conclude that GCE in combination with biophysical measurements can be a useful approach to analyze the adhesive, mechanical and signaling properties of aGPCRs and their ligand interactions.

A coronavirus (round particles) carrying trimeric spike proteins infects a host cell that expresses monomeric ACE2 receptors. The low density of ACE2 receptors on the cell membrane demonstrates that on average only one spike protein per virus particle can bind to an ACE2 receptor simultaneously, which provides SARS-COV-2 a high infectivity. After binding, the membranes fuse and viral components are released into the cell, as reported by Gerti Beliu, Markus Sauer et al. in their Research Article (e202300821).