Fluorogenic Labeling Strategies for Biological Imaging

Abstract

The spatiotemporal fluorescence imaging of biological processes requires effective tools to label intracellular biomolecules in living systems. This review presents a brief overview of recent labeling strategies that permits one to make protein and RNA strongly fluorescent using synthetic fluorogenic probes. Genetically encoded tags selectively binding the exogenously applied molecules ensure high labeling selectivity, while high imaging contrast is achieved using fluorogenic chromophores that are fluorescent only when bound to their cognate tag, and are otherwise dark. Beyond avoiding the need for removal of unbound synthetic dyes, these approaches allow the development of sophisticated imaging assays, and open exciting prospects for advanced imaging, particularly for multiplexed imaging and super-resolution microscopy.

Keywords: RNA labeling; fluorogenic probes; protein labeling.

Figure 1

Fluorogenic labeling. (A) Selective fluorogenic labeling through genetic fusion to a tag (protein or RNA) able to bind a synthetic fluorogenic chromophore (so-called fluorogen) and activate its fluorescence; (B) Binding-induced fluorogenic response can result from various processes such as (i) unquenching of intramolecularly quenched fluorophores, (ii) fluorescence increase upon polarity change or (iii) conformational locking of molecular rotors or conjugated push-pull systems; (C) Main synthetic fluorogenic chromophores utilized for the development of fluorogenic labeling methods. The maximal emission wavelengths of the fluorogens bound to their cognate tag are given. Abbreviations: DFHBI = 3,5-difluoro-4-hydroxybenzylidene imidazolidinone; HBR = 4-hydroxybenzylidene rhodanine. The design of (B) was inspired from Reference [1].