Ratiometric two-photon fluorescence probes for sensing, imaging and biomedicine applications at living cell and small animal levels

The unique two-photon excitable luminescence and dual-emission ratio output features endow organic molecule- based ratiometric two-photon fluorescence (RTPF) probes as powerful and versatile analysis tools with promising biological and biomedical applications, especially in target biosensing, bioimaging and biomedicine therapy in live cells, subcellular organelles, in-vivo deep tissues or organs. To further facilitate the ever-increasing studies on high- performance RTPF probes for important bio-applications in recent years, a timely and comprehensive review is still unavailable and it is highly expected for researches in multiple fields due to scientific urgency and necessity. In this context, this review systematically summarizes the recent advances in RTPF probes through thoroughly analyzing numerous studies published during the past decade. The review focuses on crucial aspects of RTPF probes, mainly referring to computational designs, construction principles, targets, bio-samples, sensing mechanisms, biosensing, bioimaging and biomedical applications, combination with other sensing methods, imaging modes and biomedicine therapy strategies at the living cell and small animal levels, biological toxicity, in-vivo distribution, etc. This review summarizes and discusses the state-of-the-art relevant studies about RTPF probes in various aspects, and rationally comments current states, probable challenges and perspectives in forthcoming studies. This present review is highly attractive for numerous researchers from multiple discipline fields and promotes the exploration of multifunctional and targeting therapeutic agents that have excellent performances in target biosensing, bioimaging and biomedicine applications in vitro and in vivo, and exhibit great prospects in early screening, precise diagnosis and clinic therapy of malignant diseases and cancers.