Tumor slices were stained for collagen by standard immunohistochemical techniques and imaged to detect immunofluorescent and SHG emission by multiphoton microscopy. Image analysis was conducted with Image J as described in materials and methods. (A) Two representative pseudo-colored images of SHG (blue) versus collagen type I (green) from a 4T1 tumor. Scale bars = 100 μm. (B, E, H) SHG pixel intensity above theshold; (C, F, I) Percentage of SHG pixels above threshold; (D, G, J) Anti-collagen immunohistochemical analysis. Results shown are mean ± SEM, n= 8–9 mice per group for both DMI and DEX experiments; n=6 per groups for ISO experiment. Asterisk indicates significant differences based on student's t-test, p<0.05 versus placebo or saline by student's t-test; M-W = Mann-Whitney non-parametric U-test.