Bioluminescence

The development of the first transgenic tobacco plant expressing the firefly luciferase (luc) gene [45] demonstrated the feasibility of using bioluminescent reporter genes to monitor gene expression in vivo. Bioluminescence refers to the generation of (visible) light by living organisms, commonly due to an enzymatic reaction [46,47]. Reporter genes are used to study the expression of a gene of interest. This is achieved by inserting into the host cell genome a gene cassette containing the reporter gene construct under the control of the target gene. Bioluminescent reporters yield exquisite sensitivity as there is no endogenous background signal in mammalian cells, resulting in high signal-to-background ratios. Using sensitive detection devices, such as photomultiplier tubes or cooled charge-coupled devices (CCD), sensitivity is sufficient to count only a few emitted photons.

A prerequisite for bioluminescence imaging is genetic engineering of the tissue cells of interest, i.e., the incorporation of an exogenous reporter gene. The most common bioluminescent reporter is luciferase from the North American firefly. Luciferases are oxygenases that catalyze the transformation of D-luciferin (injected, for example, intraperitoneally) into oxyluciferin in the presence of both 02 and Mg2+-ATP. Significant portions of the emission spectrum of firefly luciferase are at wavelengths larger than 600 nm [48], i.e., they fall into the window of reduced tissue absorption, thereby increasing detectability under in vivo conditions. Reporter gene assays have been demonstrated to yield fundamental biological information on, for example, transcriptional regulation, signal transduction, protein-protein interactions, cell trafficking, and targeted drug action [49,50].

A difficulty of bioluminescence imaging and optical imaging in general is spatial resolution: the light intensity distribution measured at the surface critically depends on the depth of the light source within the tissue. A population of luciferase-expressing cells near the surface of the skin will appear both brighter and more focused than the same number of cells growing at deeper tissue sites. This drawback can be accounted for by devising tomographic approaches that are critical to improve data quantification [51].

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