Abstract We have developed a one-day imaging acquisition and analysis protocol to quantify orientation of the collagen fibrils at the boundary of a mammary tumor lobule in a tissue biopsy. We reported that the degree of radially-aligned collagen fibrils in the tumor-stromal interface of rodent mammary tumor tissue is an indicator of chemotherapeutic potential of the EGFr inhibitor, gefitinib (Ericson et al. 2009 In: Proc Annu Meet AACR; Abstract nr 5002). The MNU-induced model of mammary carcinogenesis was used to develop image capture and analysis protocols. Small, 1-mm punch biopsies, were acquired from female rats with palpable (app. 250 mm2) tumor masses. Biopsy tissues were fixed and cut into 150-micron thick sections. No staining was necessary as fibrillar collagen, a hyperpolarizable structural protein, is readily detected using second harmonic generation imaging (SHG). Using this technique, the collagen fibrils can be imaged using fresh ex vivo, fixed, or paraffin-embedded tissue. Multiple SHG images of adjacent fields of view (FOV) were acquired at 200X magnification over the entire 1-mm biopsy section. Each FOV image is a stack of 14 five-micron optical sections, acquired in the Z-plane of the sample. This is an automated process using a programmable stage allowing for fast 3D data acquisition over the entire section. We developed Photoshop and Image J plug-ins to automate the projection of the individual optical Z-stacks and subsequent image stitching of all the projections. Tumor/stromal boundaries were then identified by the operator. The angle of divergence to the tangent at the tumor interface for all fibrils was quantified. Collagen fibrils running parallel to the tangent are at zero degrees while fibrils perpendicular to the tangent are at 90 degrees. This analysis can be completed in a single day. More perpendicular collagen orientation at the tumor/stromal interface may be indicative of a fast-growing aggressive tumor or enhanced ability of the tumor to invade surrounding tissue whereas predominant parallel collagen orientation could reflect increased encapsulation of the tumor mass which would then discourage further tumor growth or as our work has shown, the collagen orientation may serve as an indicator of responsiveness to chemotherapy. The development of an indicator/test for chemotherapeutic efficacy would be a useful tool for drug design or in creation of an individualized patient treatment plan. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4664.