Ultima 4

Multiphoton microscopy for patient specimens was performed at the University of Wisconsin Laboratory for Optical and Computational Instrumentation (Conklin et al, 2011). Imaging was done on a Prairie Technologies Ultima IV multiphoton microscope at an excitation wavelength of 890 with a Spectra Physics Insight laser. All images were collected with 20× 1.0 NA Objective (Olympus) and using a 445‐nm bandpass filter to discriminate for SHG collagen. SHG images at a resolution of 1,024 × 1,024 pixels were taken in triplicates, and two tumor border ROIs per image were analyzed using the CurveAlign (version 2.2 R2012a) software, covering 700–1,000 collagen fibers (curvelets) analyzed over 6 ROIs per sample. Curvelets with an angle against the border below 40° were assigned a score of 1, between 40° and 60° a score of 2, and above 60° a score of 3. The fraction of curvelets belonging to each of these three angle ranges was multiplied by the respective scores and added to obtain the final TACS‐3 score per patient. n = 18 for nulliparous, n = 28 for parous. For PAPP‐A and IGFBP‐5 immunohistochemistry, a score of 0 was assigned to an internal negative control stain in invasive regions, score 1 to regions with weak staining, and score 2 to regions with strong staining. A section was considered positive if more than 20% of cells displayed a score 2 stain.

We imaged parietal cortex in KA treated mice with a custom Prairie Technologies (Madison, WI) Ultima IV in vivo two-photon microscope powered by a Spectra-physics DeepSee Mai Tai HP (Mountain View, CA) Titanium: Sapphire laser. The microscope was developed with an integrated intrinsic signal optical recording setup (Optical Imaging Inc.), using epi-illumination powered by a Till Photonics (GmbH, Germany) Polychrome 5000 monochromator. Image analysis followed the analysis described for fiber-optic confocal recordings and was carried out with ImageJ38 (link).