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The necessity for a non-invasive and inexpensive imaging modality to both diagnose and monitor treatment response has lead to renewed interest in the potential of optical imaging. The aim of this study was to investigate the potential of diffuse optical spectroscopy for monitoring of patients with locally advanced breast cancer undergoing neoadjuvant chemotherapy. Fifteen women receiving neoadjuvant treatment for breast cancer had the affected breast scanned 5 times: before, 1 week, 4 weeks, and 8 weeks following initiation of the treatment and prior to surgery. Data was collected using a commercial optical system at four different wavelengths (690 nm, 730 nm, 780 nm, and 830 nm) and used to create three dimensional tomographic images. Mean measured values of deoxyhemoglobin (Hb), oxyhemoglobin (HbO2), and water in the entire breast were obtained and integrated over the entire breast volume to calculate the integrated optical index for each parameter. Volume-of-interest weighted tissue Hb, HbO2, and water corresponding to the tumor were also calculated. Patient response to the treatment was evaluated from clinical and pathological response using whole-mount pathology after mastectomy.