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.

] Shemiakin Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Science Miklukho Maklaya 16 10 117997 Moscow Russia [2] CEITEC MU Masaryk University Brno Czech republic [3] Pirogov Russian National Research Medical University 117997 Moscow Russia

] Shemiakin Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Science Miklukho Maklaya 16 10 117997 Moscow Russia [2] Evrogen JSC Miklukho Maklaya 16 10 117997 Moscow Russia

] Shemiakin Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Science Miklukho Maklaya 16 10 117997 Moscow Russia [2] Nizhny Novgorod State Medical Academy Nizhny Novgorod Russia

Department of Radiology University of Michigan Medical School Ann Arbor MI 48109 2200 USA

Shemiakin Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Science Miklukho Maklaya 16 10 117997 Moscow Russia

Tallinn University of Technology Department of Gene Technology 15 Akadeemia St Tallinn 12618 Estonia

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