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Collagen fibrils contribute to the structural integrity and crack resistance of bone, but their response to stress while in bundles of fibrils is not well known. Digital image correlation (DIC) is a powerful technique for measuring strain by comparing images of deformed samples to non-deformed samples, while second harmonic generation microscopy (SHGM) captures bright, high contrast images of bone with strong signals from collagen fibrils. Combining both techniques allows the 3-dimensional strain environments of bundled collagen fibrils to be characterized. SHGM was performed using confocal microscopy, producing images of human femur bone at 5 MPa and 50 MPa of applied tensional stress with 0.5×0.5×1 μm voxels. A DIC algorithm generated preliminary displacement and strain maps from the images, with the average calculated strain varying from the theoretical strain. The local strain average in the direction of applied stress was 1.09%, compared to a global strain of 0.627%, which was attributed to the small volume of analysis compared to the global image, and the proximity of a lacuna to the analysis region. The strain response was also found to be highly heterogeneous. Future work will analyze larger regions of bone.