Imaging Receptomics as a tool for biomedical discovery

Project Information

ReceptorPAINT

Grant agreement ID: 101003275

DOI

10.3030/101003275

EC signature date 30 April 2021

Start date 1 April 2022

End date 31 March 2027

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 348 025,00

EU contribution

€ 2 348 025,00

Coordinated by

LUDWIG-MAXIMILIANS-UNIVERSITAET MUENCHEN
Germany

Periodic Reporting for period 2 - ReceptorPAINT (Imaging Receptomics as a tool for biomedical discovery)

Reporting period: 2023-10-01 to 2025-03-31

Summary of the context and overall objectives of the project

The central core of this ERC project is to push the limits of what can be visualized and quantified on cell surfaces at the single-molecule level. Building on DNA-PAINT, our group is creating a suite of technological advances designed to overcome major bottlenecks of current super-resolution microscopy techniques.

First, we aim to extend DNA-based microscopy to achieve isotropic ∼1 nm localization precision across large fields of view, providing a practical route to molecular-scale imaging with relatively simple instrumentation. Second, we are devising proximity-PAINT, a DNA-nanotechnology-based readout that reports binary “in-proximity” events with tunable distance thresholds, enabling the detection of nanoscale interactions even in very dense clusters where resolution alone is insufficient. Third, we are accelerating image acquisition by rational sequence design and buffer optimization, aiming for 100-fold faster throughput than classical DNA-PAINT so that hundreds of cells and hundreds of targets can be imaged in hours rather than weeks. Fourth, we are systematically assaying and optimizing small, efficient DNA-conjugated binders (aptamers, nanobodies, Fab fragments) for virtually all cell-surface proteins to make labelling quantitative at the molecular scale. Finally, we are developing combinatorial labeling and imaging, a multiplexing strategy inspired by MERFISH, which combines combinatorial barcoding with strand-displacement cycles to visualize hundreds of targets at true single-protein resolution without sacrificing speed or precision.

Together these innovations will establish a complete “Imaging Receptomics” platform poised to be capable of mapping the entire surfaceome of a cell with molecular precision and high throughput. Once these enabling technologies are in place, we will apply them to important biological cases with societal impact: the nanoscale organization and interaction motifs of receptor tyrosine kinases, immune checkpoint receptors and their ligands on dendritic cells, tumor cells and T cells in different activation states. This unprecedented view of cell-surface patterning will close long-standing gaps in our understanding of how immune cells communicate and how tumors evade immunity, providing a rational foundation for the design of more precise and less toxic immunotherapies.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Toward our ultimate goal of establishing a comprehensive platform for Imaging Receptomics, we have already achieved several major milestones. We developed DNA-PAINT imaging that is up to 100-fold faster through optimized sequence design and the incorporation of repetitive binding motifs. In parallel, we devised site-specific, highly efficient labeling strategies for small, monovalent reagents such as nanobodies, Fabs, and scFvs. To quantitatively benchmark these affinity probes at the single-protein level, we created a new methodology that measures labeling efficiency of those binders in engineered cell lines equipped with genetically encoded tags on target proteins of interest.

To push spatial resolution beyond current limits, we recently surpassed our initial target of isotropic 1-nm resolution by introducing Resolution Enhancement by Sequential Imaging (RESI), enabling – for the first time – Ångström-resolution fluorescence microscopy. For multiplexing, we established SUM-PAINT, which combines our speed-enhanced DNA-PAINT sequences with barcoded identifiers to (in principle) encode the entire proteome.

We have begun applying these advances to map the nanoscale arrangement and interaction networks of receptor tyrosine kinases (RTKs) on the cell surface together with their ligands and downstream effectors. Finally, we initiated studies of surface-protein architectures in dendritic and cancer cells, focusing on immune-checkpoint molecules, thereby laying the groundwork for comprehensive receptor-pattern analysis in health and disease.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Our work has pushed the boundaries of super-resolution microscopy far beyond what was previously possible. We achieved two orders of magnitude faster DNA-PAINT imaging by combining optimized sequence design with repetitive binding motifs, setting a new benchmark for acquisition speed in single-molecule localization microscopy. We also introduced novel site-specific, efficient labeling strategies for small, monovalent binders such as nanobodies, Fabs and scFvs – tools that were previously difficult to implement at high density and with defined stoichiometry. To rigorously assess performance, we developed the first quantitative framework for single-protein labeling efficiency using engineered cell lines with genetically encoded tags, filling a critical gap in benchmarking affinity reagents.
In terms of resolution, we created Resolution Enhancement by Sequential Imaging (RESI). This breakthrough provided the first experimental demonstration of Ångström-level fluorescence microscopy, transforming the conceptual limit of SMLM from the nanometer to the sub-nanometer scale. For multiplexing, our SUM-PAINT approach unites speed-enhanced DNA-PAINT sequences with barcoded identifiers, offering – at least in theory – the ability to image the entire surface proteome within a single experiment. Together, these developments establish a platform that transcends the traditional trade-offs between speed, resolution and multiplexing that have constrained super-resolution imaging for over a decade.

Building on this foundation, the remaining project period will focus on translating these technological advances into a functional “Imaging Receptomics” platform. Specifically, we aim to (i) complete and validate a full workflow for simultaneous mapping of hundreds to thousands of surface receptors, ligands and downstream effectors at single-protein resolution; (ii) expand our library of engineered cell lines and genetically encoded tags to cover key receptor classes relevant to immunity and cancer biology; and (iii) develop automated data-analysis pipelines for high-throughput spatial pattern discovery and receptor interactome mapping.
On the application side, we will systematically chart the nanoscale organization and interaction networks of receptor tyrosine kinases (RTKs) under different ligand and drug conditions and extend these studies to immune-checkpoint proteins in dendritic and cancer cells, thereby uncovering receptor architectures that govern immune evasion and therapeutic response. These data sets will provide an unprecedented “molecular atlas” of the cell surface and will enable us to derive quantitative, pattern-based signatures of receptor function.

By the end of the project, we expect to deliver a validated end-to-end Imaging Receptomics platform – comprising high-speed, sub-nanometer, massively multiplexed imaging, robust labeling strategies, and scalable analysis tools – ready for adoption by the broader scientific community. This will not only redefine what is technically achievable in fluorescence microscopy but also open the door to systematic, single-protein-resolved studies of cell-surface organization in health and disease, creating a resource and methodology that goes far beyond the current state of the art.