A few rules of thumb apply to choosing reporters for deep tissue in vivo imaging. Tissue is a turbid medium which absorbs and scatters light. The double bonds in the hemoglobin molecule structure significantly quench light <600nm (*). Therefore one preferentially chooses a reporter that emits >600nm. For fluorescence imaging we need an excitation light source to provide energy for subsequent light emission. Unfortunately, when applying an external light source, one will not only excite the source of interest but also proteins in the skin which autofluoresce at 400-750 nm (Troy et al., 2004). This skin autofluorescence generates copious non-specific background signal, masking the deeper tissue signal of the source to a large extent. Autofluoresence significantly drops off in the NIR. Therefore the best fluorescence reporters emit around 800nm. Unfortunately no one has yet identified fluorescent proteins that emit in the far red. However quite a few NIR fluorescent dyes have been generated. With Bioluminescence imaging the signal is created through interaction of the enzyme with a substrate. There is no need for an excitation light source and no background signal is generated. Therefore, bioluminescence is in general still the most sensitive method of detection for deep tissue imaging.

For reporter information see the Reporter Wizard

(*) http://www.biomedical-engineering-online.com/content/3/1/9/figure/F1