MMP13 knockout alters Collagen I microstructure at the tumor-host interface. WT and MMP13 KO mice were implanted with E0771 mammary tumors. (A) Following excision of the primary tumor, tumor boundary regions were labeled with anti-collagen I. Collagen I immunofluorescence and BSHG signal were captured simultaneously in separate epidetection channels by two-photon excitation microscopy of fields of view (FOV) from random tumor boundary regions. Z-stacks from each FOV were maximum intensity projected and background subtracted, SHG and collagen I immunofluorescence signals masked to the same pixel areas, then from these masked images “normalized BSHG” (i.e. BSHG normalized to collagen I levels) was calculated as mean BSHG pixel value/mean collagen I immunofluorescence pixel value ± SEM for all images over the same XYZ pixels. This ratiometric normalized collagen I SHG value was calculated for 16 (WT) and 14 (MMP13 KO) tumor FOVs from the same cohort of animals, and was higher in MMP13 KO versus WT tumor boundaries (p < .03). (B) From the same cohort of animals, we also calculated FSHG/BSHG values, which provides insight into microstructural collagen changes, specifically the sub-resolution diameter and/or packing density/arrangement of collagen fibrils. FSHG/BSHG was significantly decreased in tumor boundary regions of MMP13 KO versus WT mice (p < .01).