Advanced imaging and molecular labeling techniques for spatiotemporal protein analysis in single live cells are reviewed. This article assesses how these cutting-edge tools unveil protein localization, dynamics, and functional interactions. Technical challenges, future prospects, and transformative applications in precision medicine and systems biology are discussed.

The spatio-temporal detection of proteins in living cells has always been an ongoing challenge. This complexity stems from the dynamic nature of the cellular environment, in which proteins interact in a highly regulated manner. Some innovative methods collectively represent important advances in overcoming the challenges associated with spatiotemporal detection of proteins in living cells. They are essential for elucidating cell biology and advancing therapeutic diagnostic techniques. However, the spatio-temporal organization of living intracellular vessels still lacks sufficient characterization. At present, there is a lack of review articles that comprehensively review the progress in recent years. This review aims to fill this gap by providing a broad overview of protein analysis in single living cells. In this review, the recent technical advances in the spatio-temporal analysis of proteins in single living cells are investigated, and the prospects and challenges in this direction are briefly discussed.