A female Foxn1 nude mouse was injected in the right flank with a mixture of HT-29 colorectal adenocarcinoma (5 x 10 6 cells) and matrigel (1:1) and 3 weeks later a subcutaneous tumor had developed. Five minutes before the fluorescent probe injection procedure, the mouse was placed under 2-3% isoflurane anesthesia and imaged using two fluorescence filter channels, the 700 nm filter for the ICB, ex: 640/20 nm, em: 710/20 nm, exposure: 3 s, percent power: 50%, F-stop: 2, FOV: 20, binning: low and 800 nm filter for targeted s775cRGD probe, ex: 745/20 nm, em: 850/20 nm, exposure: 3 s, percent power: 50%, F-stop: 2, FOV: 20 or 10, binning: low using an in vivo imaging station (Ami HT Spectral Imaging). After mouse retro-orbital injection with a 100 μL dose of the targeted probe s775cRGD (10 nmol/mouse) in saline, the mouse was imaged at 0, 1, 2 and 3 h time points. The tumor-to-background ratio for each living mouse image was calculated at each time point by analyzing the MPI of a region of interest (ROI) drawn around the tumor or the opposite flank in equal size. After three hours, a 100 μL dose of the ICB (100 nmol/mouse) was injected via the tail vein and the mouse was imaged at 0, 1 minute, 5 minutes, 10 minutes, 1 hour, 2 hours, 3 hours, and 21 hours post injection under 700 nm and 800 nm filter settings. At 24 hours post injection of s775cRGD, the mouse was anesthetized and sacrificed by cervical dislocation followed by immediate collection of blood from the heart. The whole mouse was imaged with the tumor exposed by removing the surrounding skin, and then after excising the tumor from the body. Next, the liver, lungs, heart, spleen, intestine, pancreas, kidneys, skin, and muscles were harvested. The excised tumors and organs were imaged on a transparent plastic tray. For image processing, the mouse body and the excised tumor images were false-colored "yellow" for 700 nm filter and "green" for 800 nm filter using ImageJ2 software. An arbitrary maximum fluorescence value was chosen for the analysis. For the biodistribution and ex vivo tumor analyses, the images of excised tumor and organs were imported to ImageJ2, and a manually drawn ROI was created around each fluorescent image. The MPI of each tumor or organ was divided by the MPI of the thigh muscle from the same mouse to give a normalized MPI.