Female C57Bl/6 mice (Mus musculus) of varying ages (6–22mo) (n=12, Jackson Laboratories, Bar Harbor, ME) were injected intraperitoneally with 1–10×106 ID8-RFP murine EOC cells as previously described (49 (link)). Normal collagen was also examined in tumor free mice (n=30) as a component of a larger study. No randomization method was used. Animal procedures were carried out according to the regulations of the University of Notre Dame Animal Care and Use Committee. Mice were sacrificed by CO2 inhalation followed by cervical dislocation at 8–10 weeks post-injection. The parietal peritoneum was dissected, rinsed with PBS and placed between coverslips for imaging with the mesothelium side facing the objective (25X XLPlanN, 1.05na WATER) of the 2-Photon confocal microscope (Olympus FV1000, software FLUOVIEW FV1000). Using a Mai Tai DeepSee titanium-sapphire 690–1040nm laser, ID8 metastatic implants and peritoneal collagen were visualized by the RFP and SHG signals, respectively. The investigator was not blinded during animal study. Areas of peritoneum with collagen-tumor interactions present were specifically imaged. At 12% laser power, the 2-photon laser was set to 860 nm and emission was simultaneously collected at 425–465 nm and 575–625 nm for SHG and RFP, respectively.