Résumé
Significance: Redox reactions play important roles in both physiological and pathological processes, highlighting the importance of quantifying and localizing intracellular redox-active components. Most research has focused on direct investigation of reactive oxygen species (ROS). Intensity-based fluorescent methods are very sensitive and easy to use, but they lack specificity and can produce artifacts. In this article, we focus on synthetic sensors, describing experimental pitfalls associated with their use. We also present alternative methods for the detection of free radicals. Recent Advances: New approaches have been developed to overcome the main artifact of intensity-based methods: spurious changes in fluorescence intensity caused by oxidation. These new approaches are based on analytical measurements of the oxidized sensors or techniques that are not susceptible to oxidation, such as electron spin resonance and fluorescence lifetime-based methods. Regardless of the approach, the need for detection of ROS on the subcellular level, especially in the mitochondria, has motivated the development of new probes. Critical Issues: Flow cytometry systems and confocal microscopes are now available to the majority of biologists, and commercially available probes are, therefore, more widely used. The fact that these new applications are cited in thousands of publications makes these sensors even more attractive. Future Directions: The field of ROS detection by synthetic sensors continues to expand, bringing needed additional research to the development of robust techniques that are applicable both in vitro and in vivo. Antioxid. Redox Signal. 25, 520–533.