Use of GFP for in vivo imaging: concepts and misconceptions

Although GFP and fluorescent proteins are used extensively for in vivo imaging, there are many misconceptions about GFP imaging especially compared to luciferase. GFP is not toxic, indeed, transgenic animals with GFP expressed in every cell (1) live as long as non-transgenic animals. Cancer cells with GFP are as aggressive and malignant as the cells without GFP (2-4). Cell lines can be made very bright with fluorescent proteins with no toxicity. The in vivo signal from fluorescent proteins is at least 1,000 times greater than luciferase (5). GFP is so bright that a single molecule of GFP can be seen in a bacterium (6). GFP can be observed through the skin on deep organs (7). Skin autofluorescence presents no problem for in vivo GFP imaging with proper filters (8). Fur can be rapidly clipped removing this autofluorescence (9). GFP is readily quantified by the image area which correlates to tumor volume (10). There are now numerous clones of GFP, RFP, YFP and proteins that change color (11) that can be used in vivo.