We have developed a spectral inversion method for three-dimensional tomography of far-red and near-infrared fluorescent proteins in animals. The method was developed in particular to address the steep light absorption transition of hemoglobin from the visible to the far-red occurring around 600 nm. Using an orthotopic mouse model of brain tumors expressing the red-shifted fluorescent protein mCherry, we demonstrate significant improvements in imaging accuracy over single-wavelength whole body reconstructions. Furthermore, we show an improvement in sensitivity of at least an order of magnitude over green fluorescent protein (GFP) for whole body imaging. We discuss how additional sensitivity gains are expected with the use of further red-shifted fluorescent proteins and we explain the differences and potential advantages of this approach over two-dimensional planar imaging methods.
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