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Super‐resolution techniques like single‐molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non‐covalent, weak affinity‐based transient labelling systems. DNA‐based hybrid systems are a prominent example among these transient labelling systems, offering excellent opportunities for multi‐target fluorescence imaging. However, these techniques suffer from higher background relative to covalently bound fluorophores, originating from unbound fluorophore‐labelled single‐stranded oligonucleotides. Here, we introduce short‐distance self‐quenching in fluorophore dimers as an efficient mechanism to reduce background fluorescence signal, while at the same time increasing the photon budget in the bound state by almost 2‐fold. We characterise the optical and thermodynamic properties of fluorophore‐dimer single‐stranded DNA, and show super‐resolution imaging applications with STED and SMLM with increased spatial resolution and reduced background. [ABSTRACT FROM AUTHOR]

Super‐resolution techniques like single‐molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non‐covalent, weak affinity‐based transient labelling systems. DNA‐based hybrid systems are a prominent example among these transient labelling systems, offering excellent opportunities for multi‐target fluorescence imaging. However, these techniques suffer from higher background relative to covalently bound fluorophores, originating from unbound fluorophore‐labelled single‐stranded oligonucleotides. Here, we introduce short‐distance self‐quenching in fluorophore dimers as an efficient mechanism to reduce background fluorescence signal, while at the same time increasing the photon budget in the bound state by almost 2‐fold. We characterise the optical and thermodynamic properties of fluorophore‐dimer single‐stranded DNA, and show super‐resolution imaging applications with STED and SMLM with increased spatial resolution and reduced background. [ABSTRACT FROM AUTHOR]

[Display omitted] • DNA nanotechnology was correlated with multiplexed single-molecule super-resolution imaging. • A procedure for near-molecular visualization and analysis of membrane receptor networks was established. • This approach enabled quantitative microscopy of the FGFR1-4 receptor network at the native cellular expression level. • Information on receptor clustering and distance relationships was extracted. • The imaging and analysis pipeline can be easily adapted to other protein networks. Fibroblast growth factor receptors (FGFRs) are a subfamily of receptor tyrosine kinases and central players in health and disease. Following ligand binding and the formation of homo- and heteromeric complexes, FGFRs initiate a cellular response. Challenges in studying FGFR activation are inner-subfamily interactions and a complex heterogeneity of these in the cell membrane, which demand for observation techniques that can resolve individual protein complexes and that are compatible with endogenous protein levels. Here, we established an imaging and analysis pipeline for multiplexed single-molecule localization microscopy (SMLM) of the FGFR network at the plasma membrane. Using DNA-labeled primary antibodies, we visualize all four FGFRs in the same cell with near-molecular spatial resolution. From the super-resolution imaging data, we extract information on FGFR density, spatial distribution, and inner-subfamily colocalization. Our approach is straightforward and easily adaptable to other multiplexed SMLM data of membrane proteins. [ABSTRACT FROM AUTHOR]

Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors. [ABSTRACT FROM AUTHOR]