Successful monitoring tumor response to neo-adjuvant chemotherapy (NAC) could offer the opportunity to tightly- tailored individualized therapy in patients with breast cancer. Current treatment of breast cancer generally applies a “one-size fits all” regardless of treatment success. The ability to monitor “on-treatment” response is critical for both 1) the patients that have complete response and could benefit from reduced NAC to reduce morbidity and 2) to the patients who are not responding to current NAC to suggest a change in treatments which induce greater anti-tumor response. Thus, the ability to reliably monitor tumor response to NAC treatment via an imaging-based system is a vital step toward realizing patient-tailored therapy and would enable us to further move away from a “one size fits all” paradigm in breast oncology. Therefore, we propose to develop a new Squaraine-based biosensor (SAB) to identify apoptosis detectable using a new imaging technology, Multispectral Optoacoustic Tomography (MSOT). As MSOT is a new imaging technology, exogenous reporter dyes are limited to 2 FDA approved dyes, isosulfane blue and indocynaine green, neither of which can be conjugated to peptides. Our objective is to: (1) develop a new optoacoustically optimized conjugatable reporter dye as part of the apoptosis biosensor and (2) test the Squaraine apoptosis biosensor (SAB) in vivo to identify apoptosis following chemotherapy treatment in vivo. We propose to build upon our recent success of identifying tumors using multispectral optoacoustic tomography combined with our expertise in NIR dye chemistry and molecular biology. To improve specificity of cell uptake of the prototype SAB, we have included a portion of a cell penetrating peptide that is responsive to extracellular acidic pH. We hypothesize that our lead prototype for the Squaraine Apoptosis Biosensor (SAB) will have improved cell penetrating peptide (TS-CPP) and “turn on” at the apoptosis sequence, DEVD, to separate the Squaraine dye from the QC1 dye, ultimately identifying apoptosis. To test our hypothesis, we propose the following aims: 1) develop and characterize derivatives of prototype Squaraine to serve as the reporter for the Squaraine apoptosis biosensor (SAB); 2) assess performance of optimized Squaraine and SAB as optoacoustic agents in vitro; and 3) assess optimized SAB to facilitate detection of apoptosis in breast tumors in vivo using multispectral optoacoustic tomography. Successful completion of these specific aims will develop an apoptosis biosensor that ultimately could be used to identify apoptosis in vivo and clinically to monitor tumor response and a new reporter dye detectable using optoacoustic imaging. Ultimately, this apoptosis biosensor would be well suited for use as part of a multispectral contrast agent cocktail for identifying molecular features of disease.