Mechanism elucidation of cell‐selective fluorescent probes

Abstract Cell‐selective fluorescent probes have emerged as essential tools for live‐cell imaging, enabling the differentiation of specific cell types within complex biological systems. Unlike traditional antibody‐based methods that target extracellular proteins, small‐molecule probes can access intracellular environments and exploit diverse biochemical features for selective retention or activation. This perspective categorizes the mechanisms of cell selectivity into five principal strategies: Protein‐oriented, carbohydrate‐oriented, lipid‐oriented, gating‐oriented, and metabolism‐oriented live‐cell distinctions. Each class capitalizes on a unique cellular trait ranging from protein expression and membrane composition to transporter activity and metabolic enzyme presence. We discuss representative examples of each mechanism, outline a decision‐tree workflow for elucidating a new probe's mode of action, and highlight how understanding these mechanisms is critical for both basic biological research and therapeutic probe design. Looking ahead, the development of such mechanism‐informed cell‐specific probes holds promise for advancing precision cell targeting in biomedical applications.