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Prestress in tissue is currently detected through destructive methods which obviate both in vivo and longitudinal assessment. We hypothesized that prestress could be detected and quantified by analyzing the microstructure of the extracellular matrix at different spatial scales using non-invasive and non-destructive optical imaging. A simple model of tissue prestress was created using fibroblast-mediated contraction of collagen gels around a central mandrel. Using a quantitative, multiscale, image processing technique, termed generalized image correlation spectroscopy (GICS) of second harmonic images, collagen fiber number and alignment at three different length scales characteristic of the collagen fibril, collagen fiber, and cell were analyzed. GICS fiber alignment (σ_maj/min) was significantly different across load state, level of prestress, and length scale. The largest fiber ratio, and thus highest alignment, was …