1. Breast Cancer ECM Visualization using Multiphoton Imaging Jeremy Bredfeldt LOCI, Morgridge Institute for Research

2. H&E and SHG Protocol – Thaw & fix sample – Section sample to 200 microns on a vibratome – Image with SHG – Paraffin embed tissue wafer – Section and stain for H&E – Slide scan Green = SHG signal (Collagen Type I)

3. Normal Associated Tissue 11.6 mm H&E SHG

4. Invasive Ductal Carcinoma H&E SHG

5. Fibroadenoma H&ESHG

6. 7.5 mm Mucinous Carcinoma H&E SHG

7. Intrinsic Fluorescence Green = SHG Red = Autofluorescence from FAD or Collagen type III

8. FibroAdenoma

10. Extrinsic Fluorescence Green = SHG (Collagen type I) Red = E-Cadherin stained cells & Autofluorescence from Collagen type III

11. Terminal Ductal Lobular Unit (Red) Surrounded by Collagen (Green)

12. Duct in Cross Section, Surrounded by IDC cells IDC Duct

13. IDC Cells Near a Vessel with ECM IDC Cells Vessel