Low-affinity exchangeable probes for super-resolution and long-term fluorescence imaging.
- 1Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, ITI SysChem-Chimie des Systèmes Complexes, Université de Strasbourg 74 route du Rhin 67401 Illkirch France [email protected].
Abstract
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.
Related
- Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes
- A computational super-resolution framework for multidimensional fluorescence imaging
- Fluorescence imaging-induced phospholipid oxidation drives membrane phase separation by shifting miscibility boundaries
- Multi-immersion oblique plane microscope (miOPM): a reconfigurable platform for high-resolution light-sheet fluorescence microscopy
- Application of a light emitting diode based autofluorescence removal system to improve fluorescence imaging in plant tissues
BibTeX
@article{klymchenko2026lowaffinity,
title = {{Low-affinity exchangeable probes for super-resolution and long-term fluorescence imaging.}},
author = {Andrey S Klymchenko and Remi Pelletier and Pascal Didier},
journal = {Chemical science},
year = {2026},
month = oct,
doi = {10.1039/d6sc05417j},
url = {https://europepmc.org/article/MED/42953157},
note = {CC BY},
}
RIS
TY - JOUR
TI - Low-affinity exchangeable probes for super-resolution and long-term fluorescence imaging.
AU - Andrey S Klymchenko
AU - Remi Pelletier
AU - Pascal Didier
PY - 2026
DA - 2026/10/22
JO - Chemical science
DO - 10.1039/d6sc05417j
UR - https://europepmc.org/article/MED/42953157
AB - 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.
SN - 2041-6520
SN - 2041-6539
ER -