All in vivo murine studies were performed under an approved protocol by the Emory University IACUC. The mouse mammary carcinoma cell line 4T1, which stably expresses a firefly luciferase gene, was obtained from Dr. Lily Yang at Emory University (Atlanta, GA). 4T1 cells were cultured in DMEM containing 10% FBS and 1X antibiotic/antimycotic agent. Prior to injection into mice, the cells were washed two times with PBS and diluted in sterile PBS to a final concentration of 2 × 107 cells/mL. Mammary tumors were inoculated into nude mice by the subcutaneous administration of 2 × 106 4T1 cells into the mouse flank. Once the tumors were approximately 4 mm in diameter, ICG was administered intravenously (i.v.) via a tail vein at a dose of 357 μg/kg. After 24 h, mice were anesthetized by intraperitoneal (i.p.) injection of a 2.5% solution of tribromoethanol (350 mg/kg). Tumor-bearing mice undergoing bioluminescence imaging were administered i.p. 100 uL of a luciferin solution (30 mg/ml). Bioluminescent images were acquired on a Kodak In-Vivo FX Imaging System (Carestream Molecular Imaging; Rochester, NY). Corresponding bright-field images were taken for anatomical reference of the bioluminescence signal. A series of spectra were acquired on tumor-bearing mice using the SpectroPen. First, the position of the SpectroPen was fixed to about 1–2 cm above the location of the acquisition area on the mouse. Spectra were collected in 1 s and were obtained from several locations, including directly over the center of the tumor and the peritumoral region. After the spectra were acquired, the integrated signal intensity was calculated. The signal intensity was compared to both the bright-field anatomical location and the bioluminescence signal.