Diabetic wounds exhibit distinct microstructural and metabolic heterogeneity through label-free multiphoton microscopy

Animal model All animal studies were conducted in accordance with NIH guidelines and approved Beth Israel Deaconess Medical Center (BIDMC) Institutional Animal Care and Use Committee IACUC protocol #072-2012. Diabetes was induced in C57BL/6J mice with low doses of streptozotocin (STZ, 50 mg/kg, intraperitoneally) administered for 5 consecutive days, 8 weeks prior to wound healing experiments. Hyperglycemia was confirmed by a fasting blood glucose level exceeding 250 mg/dL. Mice were anesthetized (100 mg/kg ketamine, 5 mg/kg xylazine, intraperitoneally), and full-thickness excisional wounds (6-mm diameter) were produced on the shaved dorsum of the mice. At Days 3 or 10 post-wounding, the surrounding wounded tissue was excised for imaging and stored at-80°C prior to imaging. Multiphoton microscopy Unfixed, unstained frozen sections (10µm thickness) from both diabetic and non-diabetic mice were thawed prior to TPEF/SHG imaging. Sections from Day 0 (excised tissue from the punch biopsy), as well as Days 3 and 10 post-wounding, were imaged (512 x 512 pixels; 12-bit depth) with a particular emphasis along the wound edge using a 20x objective (0.7 NA) and a laser-scanning confocal microscope (Leica TCS SP2) equipped with a tunable (710-920 nm) titanium-sapphire laser (Mai Tai; Spectra Physics; Mountain View, CA). To identify collagen fiber deposition, SHG signal using 800nm excitation light was collected in the forward direction by a non-descanned photomultiplier tube (PMT) detector with a 400(±10) nm filter. TPEF signal in the epi-direction was also simultaneously measured by two non-descanned PMTs outfitted with a filter cube that included a 700nm short pass filter and a 495nm dichroic mirror. To isolate