Abstract
Genetically encoded far-red and near-infrared fluorescent proteins enable efficient imaging in studies of tumorigenesis, embryogenesis, and inflammation in model animals. Here we report comparative testing of available GFP-like far-red fluorescent proteins along with a modified protein, named Katushka2S, and near-infrared bacterial phytochrome-based markers. We compare fluorescence signal and signal-to-noise ratio at various excitation wavelength and emission filter combinations using transiently transfected cell implants in mice, providing a basis for rational choice of optimal marker(s) for in vivo imaging studies. We demonstrate that the signals of various far-red fluorescent proteins can be spectrally unmixed based on different signal-to-noise ratios in different channels, providing the straightforward possibility of multiplexed imaging with standard equipment. Katushka2S produced the brightest and fastest maturing fluorescence in all experimental setups. At the same time, signal-to-noise ratios for Katushka2S and near-infrared bacterial phytochrome, iRFP720 were comparable in their optimal channels. Distinct spectral and genetic characteristics suggest this pair of a far-red and a near-infrared fluorescent protein as an optimal combination for dual color, whole body imaging studies in model animals.
Highlights
Encoded far-red and near-infrared fluorescent proteins enable efficient imaging in studies of tumorigenesis, embryogenesis, and inflammation in model animals
Even more options exist as investigators have introduced a new generation of bacterial phytochrome-based fluorescent proteins that emit near-infrared light[7,8]
There has been a lack of accurate comparative analysis that would help investigators to select optimal fluorescent proteins as reporters for their current animal imaging studies
Summary
Encoded far-red and near-infrared fluorescent proteins enable efficient imaging in studies of tumorigenesis, embryogenesis, and inflammation in model animals. We report comparative testing of available GFP-like far-red fluorescent proteins along with a modified protein, named Katushka2S, and near-infrared bacterial phytochrome-based markers. Distinct spectral and genetic characteristics suggest this pair of a far-red and a near-infrared fluorescent protein as an optimal combination for dual color, whole body imaging studies in model animals. We used transfected cell implants in mice to compare previously published GFP-like far-red fluorescent proteins and a modified version of Katushka[1], named Katushka2S. The latter protein, representing a non-tandem version of td-Katushka[29] with elimination of a cryptic donor splice site[10], demonstrated advanced brightness in preliminary comparative tests in bacterial and eukaryotic cells (data not shown)
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