Brightening proteins: Fluorogenic probes with a chemical switch for protein tag labeling technology

Proteins govern cellular homeostasis through tightly regulated expression, localization, and interactions. Their dysregulation is the underlying cause of diseases ranging from cancer to neurodegeneration. Imaging of these dynamics in living systems therefore provides valuable information to understand not only biological phenomena but also pathogenesis. Although genetically encoded fluorescent proteins (FPs) enable live-cell imaging of proteins, they face several limitations, including susceptibility to photobleaching, scarcity of bright near-infrared (NIR) emitters, limited pulse–chase control, and bulky tags. To overcome these limitations, protein labeling techniques that use protein tags together with their specific fluorescent probes have been developed. These techniques afford superior photostability, near-infrared (NIR) compatibility, on-demand temporal labeling, and smaller tags. This review summarizes recent advances in fluorogenic probes for both covalent and noncovalent self-labeling tags, highlighting fluorescence regulation mechanisms that suppress background signals from free probes and promote emission upon protein labeling. The advantages and limitations of the probe design strategies are discussed, and then future perspectives on protein labeling techniques are described. • Protein imaging provides valuable biological and pathological information. • Fluorogenic probes for labeling protein tags are powerful tool for protein imaging. • Low background imaging is enabled by fluorogenic probes. • Probe design strategies for fluorescence regulation are highlighted in this review.