Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial phytochromes but were not systematically compared in neurons. To fluoresce, NIR FPs utilize an enzymatic derivative of heme, the linear tetrapyrrole biliverdin, as a chromophore whose level in neurons is poorly studied. Here, we evaluated NIR FPs of the iRFP protein family, which were reported to be the brightest in non-neuronal mammalian cells, in primary neuronal culture, in brain slices of mouse and monkey, and in mouse brain in vivo. We applied several fluorescence imaging modes, such as wide-field and confocal one-photon and two-photon microscopy, to compare photochemical and biophysical properties of various iRFPs. The iRFP682 and iRFP670 proteins exhibited the highest brightness and photostability under one-photon and two-photon excitation modes, respectively. All studied iRFPs exhibited efficient binding of the endogenous biliverdin chromophore in cultured neurons and in the mammalian brain and can be readily applied to neuroimaging.
Bibliographical noteFunding Information:
We thank Nikita Pak (Massachusetts Institute of Technology (MIT)) for help with mouse perfusion and slice preparation, Siranush Babakhanova (MIT) for help with statistical analysis, Demian Park (MIT) for preparation of the primary neuron culture; Rosana Molina (Montana State University) for help with two-photon spectra measurements; and Dave Entenberg (Albert Einstein College of Medicine) for two-photon imaging of iRFP702 in brain slices. This work was supported by National Institutes of Health grants GM122567 , NS099573 , and NS103573 (all to V.V.V.), NS094246 (to M.D. and T.E.H.), DA029639 , GM104948 , and EY023173 (all to E.S.B.), and by a New York Stem Cell Foundation Robertson Award and a Howard Hughes Medical Institute Simons Faculty Scholars grant (both to E.S.B.). H-J.S. was supported by a Samsung Fellowship .
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