Abstract

Imaging proteins with high resolution is crucial for studying cellular physiology and pathology. Fluorescence imaging is a privileged method to visualize proteins with subcellular precision in live cells. In recent years, there has been a tremendous advance in the field of fluorescent dyes that are optically more sophisticated than genetically‐encodable fluorescent proteins. In this review, we aim to discuss modern bioconjugation methods to specifically incorporate these dyes into protein‐of‐interests. We focus on advances in live‐cell labeling strategies and fluorescent probes, especially the HaloTag, SNAP‐tag, TMP‐tag, and unnatural amino acid systems and their applications. These protein labeling methods, along with cutting‐edge dyes and novel microscopy methods, have become the infrastructure for biological research in the era of super‐resolution imaging.