Fluorescence Amplification Tags for Single‐Molecule Imaging and Tracking of Proteins, RNA, and DNA in Live Cells

ABSTRACT Single‐molecule imaging and tracking of DNA, RNA, and proteins have revolutionized molecular and cellular biology by shifting the focus from population‐level analyses to the direct observation of individual molecular events. However, conventional fluorescent labeling strategies, which tag biomolecules with a single fluorophore, are often hindered by low brightness, suboptimal signal‐to‐noise ratio (SNR), and rapid photobleaching, limiting localization precision and tracking duration. To overcome these challenges in single‐molecule imaging, fluorescence amplification tags (FATs) have been developed to enable the multiplexed labeling of individual macromolecules with multiple fluorescent reporters, enhancing signal intensity and photostability. This review presents a comprehensive overview of FAT design principles, working mechanisms, and their applications in labeling DNA, RNA, and proteins. We highlight their impact on elucidating dynamic genetic processes, including chromatin remodeling, gene expression regulation, mRNA translation, and protein interactions. Finally, we discuss existing challenges and propose future directions to further optimize FATs for single‐molecule imaging and tracking.