Simple, efficient transformations of fluorogenic nature that proceed under biocompatible conditions without the formation of byproducts are of high interest for in situ labeling and bioconjugation. Following these criteria, we describe in this work the discovery and optimization of imidazopyridinium dyes, obtained through in situ labeling of primary amines with pyridine, quinoline, and isoquinoline aldehydes. The so-generated dyes are excited with near-UV to violet light and emit in the orange region of the electromagnetic spectrum with Stokes shifts up to 12170 cm-1. We employed the reaction to obtain fluorescently labeled amino acids, lipids, and sugars; furthermore, we expanded the scope to proteins and tags for bioimaging. The robustness of the chemistry also allowed us to on-resin staple peptides, cleanly generating fluorescent, cyclic analogs, which showcase the broad future impact of our transformation.

Abstract Multiplexed fluorescence imaging is limited by spectral overlap, whereas fluorescence lifetime provides an orthogonal encoding dimension. We replace genetically fused HaloTags with recombinant nanobody-HaloTag constructs applied as immunofluorescence reagents and determine a lifetime palette. Integrating lifetime and spectral encoding enables up to eight targets in a single acquisition and supports multiplexed imaging in cells and tissue using a simple OneStep labeling strategy compatible with fluorescent proteins, STED nanoscopy, and any standard laboratory antibody.

Abstract mNeonGreen (mNG) is among the brightest and most photostable monomeric green fluorescent proteins and is widely used for protein tagging. Here, we present sdAb(mNG), a high-affinity single-domain antibody (sdAb) that enables biochemical capture, imaging, and manipulation of mNG-tagged proteins. A 1.26 Å crystal structure reveals an extensive interaction surface between mNG and sdAb(mNG), accounting for its high affinity (K D = 0.39 nM) and robust target recognition across diverse experimental conditions. This allows a single sdAb to support applications that typically require multiple specialized tools. We demonstrate the utility of sdAb(mNG) in several example applications including highly specific immunoprecipitation, direct immunofluorescence, and super-resolution imaging. Importantly, sdAb(mNG) retains high-performance target recognition even in intracellular environments. When expressed as an intrabody in living mammalian cells, sdAb(mNG) enables relocalization of mNG-tagged proteins to defined compartments or visualization of synaptic vesicle transport in primary neurons. In zebrafish, fusion of sdAb(mNG) to an F-box degradation domain induces cell-autonomous depletion of an endogenous mNG-tagged transcription factor and produces a clear developmental phenotype. These findings establish sdAb(mNG) as a versatile and robust affinity reagent that converts mNG from a passive fluorescent reporter into a multifunctional handle for imaging, proteomics, and programmable manipulation of endogenous and engineered proteins.

Fluorescence microscopy techniques rely strongly on revealing targets of interest using affinity binders, such as antibodies, through a process known as immunostaining or immunofluorescence. Immunostaining is typically achieved using a primary antibody against a specific target of interest, which is then detected by a fluorescently labeled secondary antibody that recognizes the primary antibody's species. This procedure, known as indirect immunofluorescence, constrains the detection of multiple targets by requiring primary antibodies from different species. Here, we describe NanoPlex, a methodology that allows multitarget detection in a species-independent fashion. NanoPlex employs secondary single-domain antibodies (sdAbs, or nanobodies) that enable the simultaneous use of multiple primary antibodies originating from the same species. In addition, these secondary sdAbs are engineered to allow for a mild and specific removal of the fluorescence signal after imaging, enabling a new round of immunostaining and imaging. NanoPlex includes secondary sdAbs with different functional groups, which support directed signal erasing upon application of either UV light (OptoPlex), proteolytic enzyme (EnzyPlex), or a reducing agent (ChemiPlex), fulfilling the demands of various experimental setups. This approach, which involves directed signal removal and re-staining of new targets on the same sample, permits the visualization of multiple targets under the same context in virtually any antibody-based application. Here, we present a detailed protocol for performing NanoPlex on cell cultures, including a practical example for 6-plex in ChemiPlex mode. In this practical example, three iterations are performed while employing dual-color confocal microscopy on each cycle. Notes have been included for step modifications required for EnzyPlex and OptoPlex.

DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is a powerful variant of single-molecule localization microscopy (SMLM) that overcomes the limitations of photobleaching, offers flexible fluorophore selection, and enables fine control of imaging parameters through tunable on- and off-binding kinetics. Its most distinctive feature is its capacity for multiplexing, typically implemented through sequential imaging of targets using an Exchange-PAINT. This technique involves assigning orthogonal DNA strands to different targets within a sample and then sequentially adding and removing complementary imager strands that are specific to only one target at a time. However, manual Exchange-PAINT workflows are often inefficient, prone to drift and variability, and lack reproducibility. Here, we introduce a custom compressed-air-driven microfluidics system specifically designed for multiplexed SMLM. Featuring a stackable and modular design that is, in principle, not limited by the number of channels, the system ensures robust, reproducible, and material-efficient buffer exchange with minimal dead volume. It operates in both manual and automated modes and can be readily adapted to a wide range of commercial and custom microscopes, including wide-field, confocal, STED, MINFLUX and other platforms. We demonstrate robust 5-plex Exchange-PAINT imaging in cancerous U2OS cells, and importantly, we establish multiplexed nanoscale imaging in fragile primary cardiomyocytes. These applications demonstrate that the platform enables reliable super-resolution multiplexing in physiologically relevant systems and supports detailed nanoscale analysis in complex primary cells.

Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes affected in neural diseases. Monitoring this process, particularly synaptic vesicle recycling, in living cells has been instrumental in revealing the mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from limitations, such as large probe sizes or limited compatibility for human neurons, hampering the quantitative analysis of synaptic pathophysiology. Here, we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes that target the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, noninvasive imaging and functional interrogation of Syt1 exo-endocytosis and trafficking in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed- and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for studying synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders.

ZUSAMMENFASSUNG Einfache, effiziente Transformationen fluorogenen Charakters, die unter biokompatiblen Bedingungen und ohne Bildung von Nebenprodukten ablaufen, sind für die In Situ Markierung und Biokonjugation von großem Interesse. Ausgehend von diesen Kriterien beschreiben wir hier die Entdeckung und Optimierung von Imidazopyridinium‐Farbstoffen, die durch In Situ Markierung primärer Amine mit Pyridin‐, Chinolin‐ und Isochinolinaldehyden erhalten werden. Die so erzeugten Farbstoffe werden mit nahem UV‐ bis violettem Licht angeregt und emittieren im orangefarbenen Bereich des elektromagnetischen Spektrums bei Stokes‐Verschiebungen von bis zu 12170 cm − 1 . Wir nutzten diese Reaktion zur Darstellung fluoreszenzmarkierter Aminosäuren, Lipide und Zucker; darüber hinaus erweiterten wir den Anwendungsbereich auf Proteine und Tags für die biologische Bildgebung. Die Robustheit dieser Chemie ermöglichte ferner das On‐Resin‐Stapling von Peptiden unter nebenproduktfreier Bildung fluoreszenter, cyclischer Analoga, was das breite, zukünftige Potenzial unserer Transformation unterstreicht.

Abstract Myelin is classically viewed as a uniform axon-insulating membrane, yet its molecular composition may differ between species and even within one species. Fatty acid binding protein-8 (FABP8/PMP2) was previously identified in CNS myelin of humans but not mice. Here we show that FABP8/PMP2 is a defining feature of CNS myelin in humans and old-world-monkeys, but absent from CNS myelin in other mammals, indicating evolutionary neofunctionalization of this lipid-binding protein in the primate lineage. In the human CNS, FABP8/PMP2 marks a subset of myelin sheaths that preferentially ensheath large-diameter axons, revealing sheath-to-sheath molecular heterogeneity correlated with axonal geometry. Chromatin is accessible at the PMP2/Pmp2 gene locus in oligodendrocytes of humans but not mice. Human oligodendrocytes intrinsically express FABP8/PMP2 when transplanted into mouse brains, demonstrating species-specific competence independent of environmental cues. ‘Humanized’ transgenic mice expressing FABP8/PMP2 in oligodendrocytes form morphologically normal but developmentally transiently thicker myelin sheaths, and show elevated cholesterol content in purified myelin. Because FABP8/PMP2 binds cholesterol, we propose that its emergence in primate CNS myelin contributes to the cholesterol enrichment of human myelin. Thus, CNS myelin protein composition is evolvable and modular, with relevance for myelin lipids and morphology, and previously unrecognized complexity in neuron–glia co-adaptation. Main points - Fatty acid binding protein 8 (FABP8/PMP2) is present in CNS myelin of humans and old-world monkeys - PMP2 defines sheath-to-sheath heterogeneity in the human CNS - PMP2-immunopositive myelin ensheaths large-diameter axons - Human oligodendrocytes intrinsically express PMP2 upon transplantation into mice - ‘Humanized’ PMP2-transgenic mice show thicker myelin and altered myelin lipid composition

Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes to be affected in neural diseases. Monitoring this process, and in particular synaptic vesicle recycling, in living cells has been instrumental in unraveling mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from major limitations such large probe size or lack of suitability for human neurons, hampering the understanding of human synaptic pathophysiology. Here we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes targeting the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, non-invasive imaging and functional interrogation of synaptic transmission in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed-and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for human synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders. ### Competing Interest Statement Silvio Rizzoli and Felipe Opazo are shareholders of NanoTag Biotechnology GmbH.

2 ',3 '-Cyclic nucleotide 3 '-phosphodiesterase (CNPase) is an abundant constituent of central nervous system non-compact myelin, and its loss in mice and humans causes neurodegeneration. Additionally, CNPase is frequently used as a marker antigen for myelinating cells. The catalytic activity of CNPase, the 3 '-hydrolysis of 2 ',3 '-cyclic nucleotides, is well characterised in vitro, but the in vivo function of CNPase remains unclear. CNPase interacts with the actin cytoskeleton to counteract the developmental closure of cytoplasmic channels that travel through compact myelin; its enzymatic activity may be involved in adenosine metabolism and RNA degradation. We developed a set of high-affinity nanobodies recognising the phosphodiesterase domain of CNPase, and the crystal structures of each complex show that the five nanobodies have distinct epitopes. One of the nanobodies bound deep into the CNPase active site and acted as an inhibitor. Moreover, the nanobodies were characterised in imaging applications and as intrabodies, expressed in mammalian cells, such as primary oligodendrocytes. Fluorescently labelled nanobodies functioned in imaging of teased nerve fibres and whole brain tissue sections, as well as super-resolution microscopy. These anti-CNPase nanobodies provide new tools for structural and functional studies on myelin formation, dynamics, and disease, including high-resolution imaging of nerve tissue.image

Direct STORM relies on robust fluorophore blinking; observations suggest that AlexaFluor647 underperforms when coupled to nanobodies, thereby undermining the advantages of nanobodies. We show that the self-blinking dye JF635b preserves excellent blinking behavior upon nanobody conjugation, enabling sparse, stable localizations in saline buffers. Using these self-blinking nanobodies, we obtained robust dSTORM, FL-SMLM, and MINFLUX images, achieving localization precision of ∼1 nm. Its simple implementation improves reproducibility and makes single-molecule localization microscopy more accessible.

The attainable resolution of fluorescence microscopy has reached the subnanometer range, but this technique still fails to image the morphology of single proteins or small molecular complexes. Here, we expand the specimens at least tenfold, label them with conventional fluorophores and image them with conventional light microscopes, acquiring videos in which we analyze fluorescence fluctuations. One-step nanoscale expansion (ONE) microscopy enables the visualization of the shapes of individual membrane and soluble proteins, achieving around 1-nm resolution. We show that conformational changes are readily observable, such as those undergone by the ~17-kDa protein calmodulin upon Ca2+ binding. ONE is also applied to clinical samples, analyzing the morphology of protein aggregates in cerebrospinal fluid from persons with Parkinson disease, potentially aiding disease diagnosis. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, providing new avenues for discoveries in biology and medicine.

To unravel the complexity of biological processes, it is necessary to resolve the underlying protein organization down to single proteins. Here, we present a protocol for secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a DNA-PAINT-based super-resolution microscopy technique that is capable of resolving virtually unlimited protein species with single-protein resolution. We describe the steps to prepare neuronal cultures, troubleshoot and conduct SUM-PAINT experiments, and analyze the resulting feature-rich neuronal cell atlases using unsupervised machine learning approaches. For complete details on the use and execution of this protocol, please refer to Unterauer et al.1.

Deuterium (2H) MRI is an emerging tool for noninvasive imaging. We explore the integration of 2H MRI with deuterated multifunctional nanopolymers for deuterated particle imaging (DPI). To this end, amine-terminated G5-polyamidoamine (PAMAM) dendrimers were labeled with deuterated acetyl surface groups, leading to highly 2H-loaded bioparticles, making them ideal for imaging studies. The accumulation of ∼5 nm PAMAM dendrimers in the kidneys could then be seen by 2H MRI with high submillimeter resolution. The natural abundance HDO signal provided an internal concentration reference to these measurements, leading to quantitative dynamic maps showing distinct nanopolymer uptakes within the renal compartments. Further, these nanopolymers allowed us to obtain in vivo maps of activity in the lymph nodes in an inflammatory rodent leg model, demonstrating these deuterated nanopolymers' potential as a novel class of contrast agents for the quantitative mapping of physiological processes.

Expansion microscopy (ExM) is continually improving, and new ExM variants need to be validated on well-defined biological structures. There is no consensus on validation structures for ExM, especially as nuclear pore complexes or DNA nanorulers are not popular for ExM studies. Here we propose that microtubules should be used for ExM validation. The diameter of microtubules immunostained using primary and secondary antibodies is sufficiently large for the validation of techniques with resolutions better than 50 nm. For techniques with higher precision (up to ~10 nm), microtubules can be assembled and imaged in vitro, using a protocol that we introduce here. Alternatively, a cellular extraction procedure can be employed, followed by labeling the peptide chains of the tubulin molecules with NHS-ester fluorophores. Finally, for nanometer-scale techniques, single tubulin molecules can be analyzed. We conclude that microtubules are valuable structures for the validation of ExM and related technologies.

Antibodies, also known as immunoglobulins, share an evolutionarily conserved dimeric core structure with two antigen binding sites. However, recognition of foreign molecules can be achieved by monovalent binding domains, as evidenced by the T-cell antigen receptor and various innate immune receptors. Thus, the reason for the strict evolutionary conservation of immunoglobulin divalence remains unclear. In addition to being soluble immune effector molecules, each immunoglobulin is also expressed as a membrane-bound isoform in the context of the B-cell antigen receptor (BCR). Here, we generated monovalent BCRs and found that their signaling and antigen internalization capabilities were strongly impaired. By using advanced superresolution imaging of BCRs following stimulation with antigens of distinct valences, we showed that the receptor cluster scale in the plasma membrane determines the magnitude of intracellular signaling. The incorporation of additional ITAMs into single BCRs did not increase receptor sensitivity but caused cellular desensitization. Our results demonstrate that the BCR-controlled signaling machinery senses the clustering status of the BCR and that subtle changes in cluster sizes are translated into cellular responses. These findings improve our knowledge of adaptive immune receptor function and will aid in the design of synthetic chimeric antigen receptors.

Multiplexed super-resolution microscopy enables spatial proteomics at single-protein resolution, but current methods often depend on secondary labels, complicating implementation and limiting throughput. We introduce a streamlined approach that combines speed-optimized DNA-PAINT sequences with their mirror-image analogs (left-handed DNA), enabling rapid and efficient 12-plex imaging. Validated on synthetic and cellular benchmarks, our method maps dense neuronal interactomes in 3D with 15 nm spatial resolution across a 200 × 200 µm2 field of view.

The synaptic vesicle cluster (SVC) is an essential component of chemical synapses, which provides neurotransmitter-loaded vesicles during synaptic activity, at the same time as also controlling the local concentrations of numerous exo- and endocytosis cofactors. In addition, the SVC hosts molecules that participate in other aspects of synaptic function, from cytoskeletal components to adhesion proteins, and affects the location and function of organelles such as mitochondria and the endoplasmic reticulum. We argue here that these features extend the functional involvement of the SVC in synapse formation, signalling and plasticity, as well as synapse stabilization and metabolism. We also propose that changes in the size of the SVC coalesce with changes in the postsynaptic compartment, supporting the interplay between pre- and postsynaptic dynamics. Thereby, the SVC could be seen as an 'all-in-one' regulator of synaptic structure and function, which should be investigated in more detail, to reveal molecular mechanisms that control synaptic function and heterogeneity.

Neurexins are key adhesion proteins that coordinate extracellular and intracellular synaptic components. Nonetheless, the low abundance of these multidomain proteins has complicated any localization and structure-function studies. Here we combine an ALFA tag (AT)/nanobody (NbALFA) tool with classic genetics, cell biology and electrophysiology to examine the distribution and function of the Drosophila Nrx-1 in vivo. We generate full-length and ΔPDZ ALFA-tagged Nrx-1 variants and find that the PDZ binding motif is key to Nrx-1 surface expression. A PDZ binding motif provided in trans, via genetically encoded cytosolic NbALFA-PDZ chimera, fully restores the synaptic localization and function of NrxΔPDZ-AT. Using cytosolic NbALFA-mScarlet intrabody, we achieve compartment-specific detection of endogenous Nrx-1, track live Nrx-1 transport along the motor neuron axons, and demonstrate that Nrx-1 co-migrates with Rab2-positive vesicles. Our findings illustrate the versatility of the ALFA system and pave the way towards dissecting functional domains of complex proteins in vivo.

AbstractFluorescence microscopy has long been a transformative technique in biological sciences. Nevertheless, most implementations are limited to a few targets, which have been revealed using primary antibodies and fluorescently conjugated secondary antibodies. Super-resolution techniques such as Exchange-PAINT and, more recently, SUM-PAINT have increased multiplexing capabilities, but they require specialized equipment, software, and knowledge. To enable multiplexing for any imaging technique in any laboratory, we developed NanoPlex, a streamlined method based on conventional antibodies revealed by engineered secondary nanobodies that allow the selective removal of fluorescence signals. We develop three complementary signal removal strategies: OptoPlex (light-induced), EnzyPlex (enzymatic), and ChemiPlex (chemical). We showcase NanoPlex reaching 21 targets for 3D confocal analyses and 5–8 targets for dSTORM and STED super-resolution imaging. NanoPlex has the potential to revolutionize multi-target fluorescent imaging methods, potentially redefining the multiplexing capabilities of antibody-based assays.