In vivo medical imaging has made great progress due to advances in the engineering of imaging devices and developments in the chemistry of imaging probes. Several modalities have been utilized for medical imaging, including X-ray radiography and computed tomography (x-ray CT), radionuclide imaging using single photons and positrons, magnetic resonance imaging (MRI), ultrasonography (US), and optical imaging. In order to extract more information from imaging, “contrast agents” have been employed. For example, organic iodine compounds have been used in X-ray radiography and computed tomography, superparamagnetic or paramagnetic metals have been used in MRI, and microbubbles have been used in ultrasonography. Most of these, however, are non-targeted reagents. Molecular imaging is widely considered the future for medical imaging. Molecular imaging has been defined as the in vivo characterization and measurement of biologic process at the cellular and molecular level1, or more broadly as a technique to directly or indirectly monitor and record the spatio-temporal distribution of molecular or cellular processes for biochemical, biologic, diagnostic, or therapeutic application2. Molecular imaging is the logical next step in the evolution of medical imaging after anatomic imaging (e.g. x-rays) and functional imaging (e.g. MRI). In order to attain truly targeted imaging of specific molecules which exist in relatively low concentrations in living tissues, the imaging techniques must be highly sensitive. Although MRI, US, and x-ray CT are often listed as molecular imaging modalities, in truth, radionuclide and optical imaging are the most practical modalities, for molecular imaging, because of their sensitivity and the specificity for target detection. Radionuclide imaging, including gamma scintigraphy and positron emission tomography (PET), are highly sensitive, quantitative, and offer the potential for whole body scanning. However, radionuclide imaging methods have the disadvantages of poor spatial and temporal resolution3. Additionally, they require radioactive compounds which have an intrinsically limited half life, and which expose the patient and practitioner to ionizing radiation and are therefore subject to a variety of stringent safety regulations which limit their repeated use4. Optical imaging, on the other hand, has comparable sensitivity to radionuclide imaging, and can be “targeted” if the emitting fluorophore is conjugated to a targeting ligand3. Optical imaging, by virtue of being “switchable”, can result in very high target to background ratios. “Switchable” or activatable optical probes are unique in the field of molecular imaging since these agents can be turned on in specific environments but otherwise remain undetectable. This improves the achievable target to background ratios, enabling the detection of small tumors against a dark background5,6. This advantage must be balanced against the lack of quantitation with optical imaging due to unpredictable light scattering and absorption, especially when the object of interest is deep within the tissue. Visualization through the skin is limited to superficial tissues such as the breast7-9 or lymph nodes10,11 The fluorescence signal from the bright GFP-expressing tumors can be seen in the deep organ only in the nude mice 12,13. However, optical molecular imaging can also be employed during endoscopy14 or surgery 15,16.