The current status of both technology and science allows for sensitive monitoring of fluorescence and bioluminescence signals from inside a live animal with high spatial and temporal resolution. These technologies provide a highly sensitive, lowbackground, noninvasive means of monitoring gene and protein expression, cell trafficking, protein-protein interactions, and other cellular events at a relatively low cost. Photon-based optical imaging has gained an indispensable position in multiple biomedical research fields such as oncology, gene therapy, drug development, and others. The major limitations of planar optical imaging are depth resolution and signal quantification. This could compromise the process of reconstructing a 3D image. Despite the success of NIR fluorescence tomography, and the commercialization of multiple instruments allowing 3D fluorescence measurements from small animals, both fluorescence and bioluminescence tomography based on genetically engineered probes is still in its infancy.
7.1 Fluorescence Molecular Tomography Based on Genetically Engineered Probes
The visualization of objects embedded deep in tissues via fluorescence can be problematic, depending to a great extent on light interaction-both excitation and emission-with the matter surrounding the object. As living tissue is a very complex material, the solution for reconstruction of a 3D fluorescence image is not a simple one. Penetration of excitation light through a living organism is affected by scattering and absorption. The less turbid and absorbent the tissue between the light source and the fluorescent probe is, the higher the fluorescence signal is expected to be. Photons in the UV-VIS range of the spectrum (<650 nm) are strongly absorbed by tissue, especially by deoxy- and oxyhemoglobin, thus making penetration depth limited by few a micrometers to a millimeter of tissue thickness. Near-infrared (NIR) light (700-900 nm) achieves the highest penetration, up to 10-12 cm, due to the minimal tissue absorbency in that spectral region. This explains in part the success of NIR fluorescence tomography. Most of the available genetically engineered fluorescent probes from the family of fluorescent proteins have excitation spectra in the range of 400-600 nm and therefore have limited depth resolution due to lower penetration of the excitation light.