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.
Highlights
Most fluorescent labeling methods are based on the permanent—high-affinity or covalent—attachment of dyes to the target molecules
Peptide-PAINT approach (C) E-coils are conjugated to target-specific antibodies and K-coils are Cy3B labeled
Transient labeling, which started with just a few low-affinity tags, has developed into a pleiad of methods compatible with most modern modalities of fluorescence microscopy (Table 2)
Summary
Most fluorescent labeling methods are based on the permanent—high-affinity or covalent—attachment of dyes to the target molecules. Antibodies-derived labels are highly specific to target structure [5,6] and allow using various organic dyes conjugated with antibodies Their field of application is usually limited to fixed cells. The size of antibodies limits the effective labeling density [8] Another covalent labeling technique is genetically fusing fluorescent proteins [9]. Silicon-rhodamine conjugates SiR-actin (with desbromo-desmethyljasplakinolide) and SiR-tubulin (with docetaxel) stain actin filaments and microtubules, respectively [19] These probes demonstrate bright and photostable labeling in various types of microscopies, both in vitro and in vivo, they slightly affect the polymerization and functionality of actin or tubulin, making the interpretation of live-cell imaging with these dyes more complicated [19,20]. In an attempt to make this task easier, we consider and compare low-affinity labeling techniques, highlight the general principles underlying such techniques, and suggest future directions of development
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