Transient Fluorescence Labeling: Low Affinity—High Benefits

Abstract

Fluorescent labeling is an established method for visualizing cellular structures and dynamics. The fundamental diffraction limit in image resolution was recently bypassed with the development of super-resolution microscopy. Notably, both localization microscopy and stimulated emission depletion (STED) microscopy impose tight restrictions on the physico-chemical properties of labels. One of them—the requirement for high photostability—can be satisfied by transiently interacting labels: a constant supply of transient labels from a medium replenishes the loss in the signal caused by photobleaching. Moreover, exchangeable tags are less likely to hinder the intrinsic dynamics and cellular functions of labeled molecules. Low-affinity labels may be used both for fixed and living cells in a range of nanoscopy modalities. Nevertheless, the design of optimal labeling and imaging protocols with these novel tags remains tricky. In this review, we highlight the pros and cons of a wide variety of transiently interacting labels. We further discuss the state of the art and future perspectives of low-affinity labeling methods.

Versatile Near‐Infrared Super‐Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD‐2

“…This concept relies on the rapid replacement of photobleached fluorophores by active labels from the solution, thus enabling STED imaging over long acquisition times and accumulating multiple imaging frames. Generally, probes with binding affinity in the low micromolar range are preferred in exchange‐based imaging approaches, [27] especially when bulky exchangeable labels are used, [29] in order to minimize the interference with other dyes or the perturbation of the normal functioning of the target structures. Despite the relatively high affinity towards amyloids, the small size (410 Da) and the excellent fluorogenic properties of CRANAD‐2 encouraged us to assess its potential for exchange‐based STED (Figure 3A).…”

Synergizing exchangeable fluorophore labels for multi-target STED microscopy

“…Both challenges can be addressed with weak-affinity, non-covalent fluorophore labels that transiently bind to a target structure and are continuously replaced by intact fluorophores from a large buffer reservoir. These "exchangeable" labels provide an elegant way to bypass photobleaching and facilitate multiplexing and 3D imaging [6][7][8] . Originally introduced in the single-molecule imaging method termed point accumulation for imaging in nanoscale topography (PAINT 9 ) using the hydrophobic dye Nile Red, the concept was generalized by the application of specific fluorescent ligands (uPAINT 10 ) and was further extended to weak-affinity DNA-DNA hybridization (DNA-PAINT 11,12,13 ), peptide-fragments (IRIS 14 ), peptide-peptide interactions 15 or protein-targeting oligonucleotides (aptamers 16 ).…”

“…The approach is scalable, and labeling and imaging of even more targets is possible by implementing e.g. orthogonal xHTLs 18 , increasing the number of DNA-barcoded labels, by integrating orthogonal protein labels 8 , or by implementing other weak-affinity labels ( 28, 29, 30 ). Given the availability of highly specific target-ligand pairs in molecular biology, we envisage many more types of exchangeable labels that could turn compatible with this approach and further expand the multiplexing.…”

Synergizing Exchangeable Fluorophore Labels for Multitarget STED Microscopy

“…Both challenges can be addressed with weak-affinity, noncovalent fluorophore labels that transiently bind to a target structure and are continuously replaced by intact fluorophores from a large buffer reservoir. These “exchangeable” labels provide an elegant way to bypass photobleaching and facilitate multiplexing and 3D imaging. − Originally introduced in the single-molecule imaging method termed point accumulation for imaging in nanoscale topography (PAINT) using the hydrophobic dye Nile Red, the concept was generalized by the application of specific fluorescent ligands (uPAINT) and was further extended to weak-affinity DNA–DNA hybridization (DNA-PAINT − ), peptide fragments (IRIS), peptide–peptide interactions, or protein-targeting oligonucleotides (aptamers). HaloTag7 (HT7) is a self-labeling protein that covalently reacts with biorthogonal ligands (HTLs).…”