We retrospectively analyzed 14 breast cancer tissues, all of which were invasive ductal carcinomas (IDC). Eight of these breast cancers were and six were . All cancers were tumor grade 2 to 3, estrogen receptor positive (), and progesterone receptor positive (). All of our samples were obtained from hospitals in Maryland and were graded by board-certified breast pathologists including P. Argani. Snap-frozen primary breast tumor specimens were placed in Tissue Tek OCT freezing compound (Sakura Finetek USA), cryosectioned with a microcryotome (Microm International) at 100-μm thickness for whole-mount sections and 5-μm thickness for adjacent sections, fixed with 4% paraformaldehyde (Sigma-Aldrich) solution, stained for nuclei with Hoechst 33342 (Invitrogen, Carlsbad, CA), and mounted with Faramount aqueous mounting medium (DakoCytomation) as previously described.19 For testing the within-tumor consistency of SHG-detected Col1 inter-fiber distances and Col1 fiber volume, we obtained FFPE samples from four different biopsy passes of the same breast cancer for four human specimens, which were grade 3, , , and ; grade 2, , , ; grade 2, , , ; and grade 3, , , . These FFPE samples were embedded in paraffin blocks, within which they were marked with the pink dye phloxine, which visualizes the shape of the biopsy specimen within the white paraffin block. We also obtained the adjacent hematoxylin and eosin (H&E) slide. In our pathology lab, the H&E section is always cut as the last slide in the series from a given FFPE block. Therefore it was possible to spatially correlate the shape of the H&E section with the specimen in the FFPE block.