One of the most striking features of our immune system is its inherent ability to distinguish harmful from harmless based on the primary protein structure of antigens. T-cells embody this trait of adaptive immunity through their unique capability for detecting antigenic peptides. It is driven by αβT-cell receptors (TCRs) on the T-cell binding to particular antigenic peptide-loaded MHC molecules (pMHC) displayed by antigen-presenting cells. T-cells are exquisitely sensitive to antigen: they can detect even a single antigenic pMHC molecule among a great number of structurally similar yet non-stimulatory pMHCs. The molecular/cellular mechanisms underlying this remarkable quality are not at all understood, even though their relevance for both disease progression and intervention can hardly be overestimated.

The phenomenon of diffraction has long been thought to set an inevitable physical limit to resolution of light microscopy. According to Abbe’s diffraction limit approximation, structures smaller than half the wavelength of light cannot be resolved. The last decades however, have seen developments in fluorescence microscopy that enable to study cellular structures on the nanometer length scale.

During their random motion, biomolecules experience a manifold of interactions that transiently conjoin their paths. It is extremely difficult to measure such binding events directly in the context of a living cell: interactions may be short lived, they may affect only a minority fraction of molecules, or they may not lead to a macroscopically observable effect. We developed a single molecule imaging method that allows for detecting and quantifying associations of mobile molecules. By “thinning out clusters while conserving the stoichiometry of labeling” (TOCCSL) we can virtually dilute the probe directly in the cell, without affecting the fluorescence labeling of single clusters.