Superoxide has been implicated in a variety of pathophysiological processes such as ischemia-reperfusion injury, neurodegenerative diseases, hypertension, aging, and cancer. Superoxide, when produced in low amounts, is involved in cell-signaling processes. However, large amounts produced during pathological processes may lead to cellular dysfunction and tissue damage. Hence, the detection of superoxide is critical to our understanding of its role in normal and pathophysiology. A variety of analytical techniques have been developed to quantify superoxide radicals in biological systems including spectrophotometric methods that use the reduction of ferricytochrome c or nitroblue tetrazolium by superoxide;chemiluminescence and fluorescence-based techniques;and electron paramagnetic resonance (EPR) spectroscopy. However, many of these methods have certain limitations for measuring superoxide, primarily lacking specificity or the necessary reaction rates. We have identified a new class of probes based on trityl radicals to detect and quantify superoxide produced in biological systems with greater sensitivity and specificity when compared to existing probes. One subset of these new probes is bi-modal - they can be detected by EPR as well as fluorescence microscopy techniques. Therefore, the goal of this proposal is to develop and characterize additional new trityl-based probes for detection and quantification of superoxide in biological systems, and to further develop and characterize the bi-modal subset of these probes for imaging applications. Accordingly, the following specific aims are proposed: (i) Synthesis, characterization, and validation testing of stable perchloro-triphenylmethyl (PTM)-derived paramagnetic spin probes for quantification of superoxide in biological systems and (ii) Synthesis, characterization, and validation testing of stable, bi-modal perchloro-triphenylmethyl (PTM)-derived paramagnetic spin probes for quantification and fluorescence imaging of superoxide distribution in biological systems. The goal of this proposal is to develop and characterize new trityl-based probes for detection, quantification, and imaging of superoxide in biological systems. Superoxide has been implicated in a variety of pathophysiological processes such as ischemia-reperfusion injury, neurodegenerative diseases, hypertension, aging, and cancer. Under normal circumstances, superoxide is produced in low amounts and is involved in cell-signaling processes. During pathological conditions, large amounts of superoxide may lead to cellular dysfunction and tissue damage. Hence, the detection of superoxide is critical to our understanding of its role in normal and pathophysiology.