With the advantage of inherent responsiveness that can change the spectroscopic signals from “off” to “on” state in responding to targets (e. g. biological analytes/microenvironmental factors), activatable fluorescent probes have attracted extensive attention and made significant progress in the field of bioimaging and biosensing. Due to the high depth of tissue penetration, minimal tissue damage and negligible background signal at longer wavelengths, the development of second near‐infrared window (NIR‐II) fluorescent materials provides a new opportunity to develop activable fluorescent probes. Here, we summarized properties, advantages and disadvantages of mainly NIR‐II fluorophores (such as rare earth‐doped nanoparticles, quantum dots, single‐walled carbon nanotubes, small molecule dyes, conjugated polymers and gold nanoclusters), then overviewed current role and development of activatable NIR‐II fluorescent probes (AFPs) for biomedical applications including biosensing, bioimaging and therapeutic. The potential challenges and perspectives of AFPs in deep‐tissue imaging and clinical application are also discussed.

Disease signaling molecules and biomarkers are important in biosensing and biomedicine, and their ultra‐sensitive detection and imaging are critical in medical diagnosis. However, most biomarkers have low levels of expression in cells and are easily degraded; therefore, they cannot be accurately detected in real time by conventional amplification methods, which are typically confined to cell lysates. To achieve highly sensitive detection of intracellular low‐abundance molecules, extensive efforts have been devoted to the development of in situ signal amplification techniques. This review tracks the development of recent advances in in situ signal amplification strategies, and systematically summarizes the advantages and disadvantages of enzymatic and non‐enzymatic amplification techniques for detecting and tracing intracellular targets. Further, perspectives, challenges, and opportunities for intracellular signal amplification are discussed to promote its adoption for in situ signal amplification in clinical settings and to provide new insights into signaling molecules and biomarkers in cellular functions and associated diseases.