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Searching for the two-photon brightest red fluorescent protein and its optimum excitation wavelength

We study 2PA spectra of red fluorescent proteins (FPs), including DsRed2, mRFP, tagRFP, and mFruits in a wide range of excitation wavelengths, 600 - 1200 nm. For evaluation of mature FP extinction coefficient and concentration we propose a pure optical method which is based on Strickler - Berg equation, relating fluorescence radiative lifetime with molecular extinction coefficient. 2PA spectra and maximum cross sections are very sensitive to either changes in the chromophore structure (mOrange vs mRFP) or mutations in chromophore surrounding (DsRed and mFruits). All red FPs show two pronounced 2PA transitions, the first peaking in the 1000 - 1100-nm region, and the second - near 700 - 760 nm. For each region we have found a mutant, which is 3 - 4 times two-photon brighter than the benchmark EGFP.