New Far-Red Fluorescent Proteins for Non-Invasive Imaging of Stem Cell Differentiation

Non-invasive high-resolution imaging of cell populations in animal disease models would greatly facilitate the development of cell-based therapies. Optical imaging of fluorescent proteins (FPs) that can be excited by red light in the “optical window” above 600 nm, such as the monomeric far-red FP mNeptune, is one potential method for tracking implanted cells. However the utility of red-absorbing FPs for tracking the fates of implanted cells has not yet been explored. Here we report three new red-absorbing monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence, while the third, mCardinal, has an additionally red-shifted absorbance spectrum. We show that mCardinal can be used to non-invasively visualize muscle regeneration from myoblasts and stem cells in a mouse injury model with high anatomical detail.