ABSTRACT Redox biology has been increasingly recognized as one of the key themes in cell signaling. A major obstacle in this field is the lack of capabilities to monitor and measure clearly defined redox processes in live cells (including subcellular domains in live cells), tissues, or organisms. The goal of this 4-year project, submitted in response to PAR-17-045 “Focused Technology Research and Development”, is to expand the fluorescent protein toolbox for red and far-red fluorescent biosensors that can be used to image compartmentalized, thiol-based redox signaling in live cells and organisms. In this project, we aim to advance the fluorescent biosensor technology in the following aspects : (1) we will expand fluorescent-protein-based biosensors for additional redox parameters, including the redox of thioredoxin and protein disulfide isomerase (PDI) in various cell compartments, such as the nucleus, cytosol, mitochondria, and endoplasmic reticulum (ER); (2) we will expand the colors of fluorescent-protein-based redox biosensors to red and far-red; (3) we will validate these new biosensors and demonstrate the use of fluorescent-protein-based biosensors in compatible colors for multicompartment/multiplex imaging. Because UV or blue light, which can induce intracellular oxidation, is not needed to excite red and far- red fluorescent biosensors, our work will increase the biocompatibility of fluorescent-protein-based redox biosensors. Furthermore, red and far-red biosensors will assist in whole-animal imaging, since compared to green ones, they reduce tissue absorption and scattering. This project will lead to important imaging tools that may accelerate the understanding of redox regulation and signaling. As with the transformative impact of fluorescent-protein-based kinase sensors, we expect that these redox biosensors will enable and transform a large array of basic and translational studies.