Low-affinity exchangeable probes for super-resolution and long-term fluorescence imaging

To overcome the limitations of conventional fluorescence labelling based on covalent bonds or strong probe-target interactions, low-affinity labelling has recently emerged as an attractive alternative. Owing to the rapid exchange of probes at the labelling site, it circumvents two major challenges: it enables virtually unlimited labelling with minimal photobleaching and provides a simple mechanism for probe ON/OFF switching, which is essential for super-resolution imaging. This review covers the main classes of low-affinity probes for biological targets. The first major class is based on lock-and-key interactions between fluorophores and their targets, such as biomembranes, RNA aptamers, protein tags and endogenous biomolecules. In these systems, the formation of dye-target complexes generally induces a fluorogenic response, enabling imaging with low background despite the presence of a large pool of unbound probes. The second class relies on fluorescently labelled ligands that specifically recognize biological targets. The primary example is DNA-PAINT, which exploits the reversible hybridization of a short fluorescently labelled oligonucleotide with its complementary counterpart attached to a target-specific vector (e.g., antibodies, nanobodies, or affimers). Emerging approaches also employ peptides, proteins, nucleic acid aptamers, and small molecules as low-affinity ligands. Low-affinity labelling already enables imaging with resolution down to ∼1 nm, but it remains rather slow and mainly compatible with fixed cells. We anticipate that this field will continue to expand rapidly, driven on the one hand by new concepts in fluorogenic dye design and reversible labelling strategies and, on the other hand, by the growing range of applications in advanced fluorescence microscopy.