Redefining Molecular Probes for Monitoring Subcellular Environment: A Perspective

The development of small-molecule fluorescent probes has revolutionized the monitoring of in vivo physicochemical parameters, offering unprecedented insights into biological processes. In this perspective, we critically examine recent advances and trends in the design and application of fluorescent probes for real-time in vivo monitoring of subcellular environments. Traditional concepts such as membrane potential, microviscosity, and micropolarity have been superseded by more biologically relevant parameters like membrane voltage, tension, and hydration, enhancing the accuracy of physiological assessments. This redefinition not only presents an evolved concept with broader applications in monitoring subcellular dynamics but also addresses the unmet needs of subcellular biology more effectively. We also highlight the limitations of commonly used probes in providing specific information about the redox environment, noting their nonspecificity to oxidants and the influence of various chemical interactions. These probes typically rely on free radical mechanisms and require metal catalysts to react with hydrogen peroxide. They include naphthalimide, fluorescein, BODIPY, rhodamine, cyanine cores to cover the UV–vis–near-infrared window. The motif of this perspective is to provide critical insights into trending fluorescent-based systems employed in real-time or in vivo physicochemical-responsive monitoring, thus aiming to inform and inspire further research in creating robust and efficient fluorescent probes for comprehensive in vivo monitoring applications.