Clinical tissue samples were obtained through the University of Wisconsin Hospital as described previously.3 (link) This study was approved by the University of Wisconsin Human Subjects Committee Institutional Review Board in compliance with the 1975 Declaration of Helsinki. Briefly, samples were obtained from patients with indeterminate depth burn injury at the time of surgery, 6–13 days after burn injury following a period of observation to assess healing trajectory. Intraoperatively, burn wound depth was determined by the burn surgeon using visual assessment prior to and during excision.3 (link) Normal skin tissue used as a control was obtained from non-burn related elective reconstructive surgery. Control non-burned and burned tissue samples were stored for up to 1 hour in normal saline (Baxter) prior to further processing. A 6-mm biopsy punch was taken from the skin samples and 100 μL of 100 μM Cy5-CMP or 100 μL of 100 μM Cy5-CI was applied directly onto the superficial side of the biopsy, fully submerged in dye solution, and incubated in a petri dish covered with aluminum foil at room temperature for 1 hour. The samples (n=6 burn pairs) were washed three times in rapid succession with 1× PBS (Sigma) prior to imaging and kept covered in 1× PBS. Images were collected from both superficial and deep surfaces of the biopsies immediately following rinsing.