NanoPlex: a universal strategy for fluorescence microscopy multiplexing using nanobodies with erasable signals

Fluorescence microscopy is a cornerstone technique in biological research, but its multiplexing capabilities are often limited. Traditional methods using primary and fluorescently conjugated secondary antibodies typically allow the visualization of only a few targets simultaneously. While super-resolution techniques like Exchange-PAINT and SUM-PAINT offer improved multiplexing, they require specialized equipment and expertise. The need for a simple, universally applicable method for multiplexing across various imaging techniques and laboratories drove the development of NanoPlex. This method leverages the versatility of conventional antibodies and employs engineered secondary nanobodies (2.Nbs) modified for selective fluorescence signal removal. The use of 2.Nbs provides significant advantages, including one-step multi-target staining and species-independent capabilities. Existing iterative multiplexing approaches often involve harsh chemical treatments (detergents, chaotropic salts, extreme pH), microwave heating, or high-energy photobleaching, all of which can compromise cellular structures and epitopes. NanoPlex avoids these issues by employing mild and specific signal removal strategies. The goal is to establish a simple and robust method that can be adapted to any laboratory setting without requiring specialized equipment or extensive expertise, expanding the multiplexing capabilities of antibody-based assays across various microscopy techniques and applications like single-cell proteomics.

Early attempts at multiplexing involved chemical stripping or photobleaching of antibodies, enabling sequential immunofluorescence cycles. However, these approaches often damaged cellular structures. Other methods utilize single-stranded DNA (ssDNA) barcoding (immuno-SABER, CODEX), which are well-suited for large-area histological imaging but may lack resolution and compromise subcellular ultrastructure. The advent of super-resolution microscopy led to new strategies, with Exchange-PAINT and SUM-PAINT achieving impressive multiplexing (up to 30-plex). These DNA-PAINT methods, while powerful, remain technically challenging to implement in laboratories without prior experience in the technique, thus limiting wider adoption. This study aims to overcome these limitations by providing a more accessible and versatile approach.

NanoPlex uses engineered secondary nanobodies (2.Nbs) for iterative imaging cycles. Three distinct signal removal strategies were developed: **OptoPlex (light-induced):** 2.Nbs are conjugated to a light-responsive tag (LRT) containing an ortho-nitrobenzene (ONB) photolabile group. Irradiation with 365 nm light cleaves the LRT, releasing the fluorophore. This was tested on U2OS-Nup96-GFP cells, demonstrating target-specific signal removal and achieving 6-plex confocal imaging. **EnzyPlex (enzymatic):** 2.Nbs are fused to a small ubiquitin-related modifier (SUMO) substrate from *Brachypodium distachyon* and an ALFA-tag. The *B. distachyon* protease bdSENP1 specifically cleaves the SUMO, releasing the fluorophore. This method achieved 6-plex confocal imaging and 5-plex dSTORM imaging (with an average localization precision of 12.9 ± 2.5 nm). **ChemiPlex (chemical):** A disulfide bond links the 2.Nb to the ALFA-tag. Treatment with a reducing agent (TCEP) cleaves the disulfide, releasing the fluorophore. This method provided efficient and homogeneous signal removal, achieving 6-plex confocal and 8-plex STED imaging. The STED experiments included additional steps to minimize ROS damage from high-energy excitation, including antioxidants and thiol quenchers. To enhance the overall efficiency of the labeling, the authors used a cocktail of unlabeled nanobodies after each cleaving to block potentially available epitopes from previous cycles. Detailed descriptions of the synthesis of the LRT, cell culture procedures, nanobody engineering and conjugation, protease expression and purification, immunofluorescence protocols, microscopy techniques (confocal, STED, dSTORM), image analysis (colocalization, localization precision), statistical methods, and data availability are included in the supplementary information.

NanoPlex successfully demonstrated high-multiplex imaging using three distinct signal-removal strategies: OptoPlex, EnzyPlex, and ChemiPlex. * **OptoPlex:** Achieved 6-plex confocal imaging in U2OS cells, showing region-specific signal removal. Signal removal was not uniform across all targets, with the poorest removal observed for membrane-associated proteins. * **EnzyPlex:** Showed superior signal removal compared to OptoPlex. Achieved 6-plex confocal imaging and 5-plex dSTORM super-resolution imaging with an average localization precision of 12.9 ± 2.5 nm. Cleavage efficiency varied across targets, potentially due to steric hindrance or limited diffusion. * **ChemiPlex:** Demonstrated highly efficient and homogeneous signal removal, outperforming the other methods. Achieved 6-plex confocal imaging and 8-plex STED super-resolution imaging. The STED experiments successfully imaged various subcellular structures. In primary hippocampal neurons, ChemiPlex enabled 21-plex 3D confocal imaging of various neuronal components and synaptic proteins. A further investigation focusing on nine synaptic proteins using ChemiPlex provided insights into the roles of liquid-liquid phase separation (LLPS) in synapse function. Treatment with 1,6-hexanediol (HEX), which disrupts LLPS, revealed changes in protein correlations that align with previous findings and interpretations from different studies, suggesting that NanoPlex accurately captures and reveals the expected correlation among synaptic proteins. The study showed that HEX treatment impacted protein correlations and distributions, indicating that LLPS plays a significant role in synaptic organization and function.

NanoPlex addresses the limitations of current multiplexing techniques by offering a simple, versatile, and broadly applicable method for multi-target imaging. The use of engineered secondary nanobodies with mild, selective signal removal strategies preserves cellular structure and epitopes, unlike harsh chemical or photobleaching methods. The three distinct strategies (OptoPlex, EnzyPlex, ChemiPlex) offer flexibility for diverse applications and microscopy techniques. The high-multiplex imaging achieved in primary hippocampal neurons demonstrates the power of NanoPlex in revealing complex cellular interactions and responses to perturbations. The findings confirm the utility of NanoPlex for addressing research questions in single-cell proteomics and beyond. The simplicity and broad applicability of NanoPlex make it a valuable tool for researchers across various fields, democratizing access to high-content, multiplexed imaging.

NanoPlex presents a significant advancement in fluorescence microscopy multiplexing. Its simplicity, versatility, and compatibility with various microscopy techniques make it a powerful tool for diverse biological applications. Future research could focus on optimizing the signal removal efficiency across all targets, expanding the range of compatible antibodies, and integrating automation for high-throughput applications. NanoPlex's ability to achieve high-multiplexing imaging in various microscopy methods, particularly in challenging systems such as primary neuronal cultures, positions it as a valuable technique for exploring complex biological questions.

While NanoPlex offers significant advantages, some limitations exist. Signal removal efficiency varied slightly among targets, potentially due to differences in protein abundance, accessibility, or local chemical environment. The number of cycles in the STED experiments was limited by the availability of suitable primary antibodies for each cycle. Moreover, the study primarily focused on fixed samples; further investigation is needed to fully explore its applicability to live-cell imaging.