Early-stage diagnosis of breast cancer is essential for improving the survival rate of women affected (1 in 8) in the United States. Near-infrared (NIR) optical imaging is a non-invasive and non-ionizing modality that is emerging as a diagnostic tool for breast cancer. In recent years, hand-held based optical imagers developed towards clinical translation of the technology are not capable of contouring along tissue curvatures, nor perform three-dimensional (3D) tomographic imaging to diagnose deep and/or early stage tumors. The overall objective of the proposed work is to implement the recently developed portable cart-based hand-held optical imager for tissue phantom and in-vivo breast imaging studies (in normal and breast cancer subjects), in order to demonstrate near-real time 2D tumor detection and 3D tomographic analysis, towards breast cancer diagnosis. The specific aims include: (i) Demonstrate real-time simultaneous bilateral breast imaging in tissue phantoms and in-vivo, using a novel hand-held optical imager, in order to detect tumor-mimicking targets in near real-time. (ii) Demonstrate the feasibility of 3D target localization in tissue phantoms and in-vivo, via 3D tomographic imaging studies using the hand-held optical imager; and (iii) Determine the effectiveness of tumor detection in breast cancer subjects using the hand-held optical imager, in comparison to the gold-standard x- ray mammography. The hypothesis of the application relies on a three-fold innovative implementation: (i) the forked probe design aids in performing simultaneous bilateral imaging of both the breast tissues, in order to improve the signal-to-noise (SNR) ratio between the normal and diseased tissue; (ii) co-registered measurements using a hand-held probe on any tissue volume and curvature aids in near real-time target detections as well as 3D tomographic imaging; and (iii) simultaneous illumination and detection at multiple locations on the tissue will allow rapid imaging time, appropriate for a clinical environment. The study design involves the following: (i) Perform simultaneous bilateral imaging on breast phantoms, in-vivo on healthy and breast cancer subjects to determine the effectiveness of 2D target (tumor) detectability with maximum SNR. (ii) Implement a coregistered imaging approach to obtain positional information of the probe(s) towards 3D tomographic analysis in phantom and in-vivo. (iii) Determine the 3D target (tumor) location(s) via tomographic analysis in phantoms and in-vivo; and (iv) Compare the optical images to the gold standard x-ray mammography images in breast cancer subject studies, in order to assess the feasibility of the hand-held optical imager in a clinical environment. The proposed research significantly impacts by accelerating the clinical translation of the emerging optical imaging technology towards early-stage breast cancer diagnosis, due to the unique and innovative method of 3D target localization using a portable and inexpensive hand-held optical imager. The technology also has potential towards prognostic breast imaging, image-guided breast cancer therapy, and volumetric analysis of tumors in response to chemotherapeutic trials.