Colocalization of differently labeled biomolecules is a valuable tool in fluorescence microscopy and can provide information on biomolecular interactions. With the advent of super-resolution microscopy, colocalization analysis is getting closer to molecular resolution, bridging the gap to other technologies such as fluorescence resonance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-resolution methods offer the advantage of providing single-molecule coordinates that, rather than intensity information, can be used for colocalization analysis. This requires adapting the existing mathematical algorithms for localization microscopy data. Here, we introduce an algorithm for coordinate-based colocalization analysis which is suited for single-molecule super-resolution data. In addition, we present an experimental configuration for simultaneous dual-color imaging together with a robust approach to correct for optical aberrations with an accuracy of a few nanometers. We demonstrate the potential of our approach for cellular structures and for two proteins binding actin filaments.

Super‐resolution fluorescence microscopy has enabled important breakthroughs in biology and materials science. Implementations such as single‐molecule localization microscopy (SMLM) and minimal emission fluxes (MINFLUX) microscopy in the localization mode exploit fluorophores that blink, i.e., switch on and off, stochastically. Here, we introduce nanographenes, namely large polycyclic aromatic hydrocarbons that can also be regarded as atomically precise graphene quantum dots, as a new class of fluorophores for super‐resolution fluorescence microscopy. Nanographenes exhibit outstanding photophysical properties: intrinsic blinking even in air, excellent fluorescence recovery, and stability over several months. As a proof of concept for super‐resolution applications, we use nanographenes in SMLM to generate 3D super‐resolution images of silica nanocracks. Our findings open the door for the widespread application of nanographenes in super‐resolution fluorescence microscopy.

Signaling from the epidermal growth factor receptor (EGFR) via phosphorylation on its C-terminal tyrosine residues requires self-association, which depends on the diffusional properties of the receptor and its density in the plasma membrane. Dimerization is a key event for EGFR activation, but the role of higher order clustering is unknown. We employed single particle tracking to relate the mobility and aggregation of EGFR to its signaling activity. EGFR mobility alternates between short-lived free, confined and immobile states. In the immobile state, EGFR tends to aggregate in clathrin-coated pits, which is further enhanced in a phosphorylation-dependent manner and does not require ligand binding. EGFR phosphorylation is further amplified by cross-phosphorylation in clathrin-coated pits. Because phosphorylated receptors can escape from the pits, local gradients of signaling active EGFR are formed. These results show that amplification of EGFR phosphorylation by receptor clustering in clathrin-coated pits supports signal activation at the plasma membrane.

Epidermal growth factor receptor (EGFR) activation by growth factors (GFs) relies on dimerization and allosteric activation of its intrinsic kinase activity, resulting in trans-phosphorylation of tyrosines on its C-terminal tail. While structural and biochemical studies identified this EGF-induced allosteric activation, imaging collective EGFR activation in cells and molecular dynamics simulations pointed at additional catalytic EGFR activation mechanisms. To gain more insight into EGFR activation mechanisms in living cells, we develop a Förster resonance energy transfer (FRET)-based conformational EGFR indicator (CONEGI) using genetic code expansion that reports on conformational transitions in the EGFR activation loop. Comparing conformational transitions, self-association and auto-phosphorylation of CONEGI and its Y845F mutant reveals that Y845 phosphorylation induces a catalytically active conformation in EGFR monomers. This conformational transition depends on EGFR kinase activity and auto-phosphorylation on its C-terminal tail, generating a looped causality that leads to autocatalytic amplification of EGFR phosphorylation at low EGF dose.