For this study, 16 male Golden Syrian hamsters were evaluated (). This study was approved by the Vanderbilt University Animal Care and Use Committee and meets the National Institutes of Health guidelines for animal welfare. Hamsters were anesthetized with a mixture of ketamine and xylazine with an intraperitoneal injection. The mucosa of the cheek pouch was exposed and secured against a coverslip for imaging. All images were acquired from the stratum spinosum or basal epithelial layers at depths of approximately 20 or 30 μm, respectively. In vivo measurements of the cheek pouch were obtained from the cheek epithelium. Then, two 6-mm biopsies of the cheek pouch were acquired immediately after the in vivo measurements from the same region that was imaged in vivo. For 10 hamsters, one biopsy per hamster was flash-frozen in liquid nitrogen (frozen-thawed sample). This sample was placed immediately in a freezer. The second 6-mm biopsy (live tissue culture sample) was immediately placed in a glass-bottom petri dish with chilled (3°C), sterile tissue culture media [Dulbecco’s Modified Eagle Medium (DMEM) without phenol red; GIBCO, Grand Island, New York). The stratum spinosum layer of the epithelium was immediately measured and sequentially measured every 30 min for 4 h after the time of the biopsy from the live tissue culture sample. For this high time-resolution protocol, a single tissue layer was interrogated to allow for comparisons between time points and to minimize tissue warming at room temperature during imaging. This high time-resolution, short time-frame protocol was chosen to investigate acute changes in the metabolism of the tissue immediately after excision. For the remaining six hamsters, after in vivo imaging, two biopsies were obtained and both were placed in chilled tissue media for the long-term (low time-resolution) imaging studies. This low time-resolution, long time-frame protocol investigates the maximum duration over which optical metabolic measurements in live culture correspond with in vivo measurements. One of the biopsies was imaged after 4, 8, 12, 24, and 48 h at two different depths corresponding to the spinous and basal regions. The second biopsy was fixed in formalin for histological analysis after 4, 8, 12, 24, or 48 h in live culture. Each measurement in live tissue culture over this time course was conducted within 5 min at room temperature (22°C), and the sample was refrigerated (3°C) between measurements. Prior to measurement of the frozen biopsy, the specimen was thawed in chilled tissue culture media for 15 to 20 min.25 Each animal was sacrificed after both biopsies had been obtained.