Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins

Fluorescent proteins with long emission wavelengths are particularly attractive for deep tissue two-photon microscopy. Surprisingly, little is known about their two-photon absorption (2PA) properties. We present absolute 2PA spectra of a number of orange and red fluorescent proteins, including DsRed2, mRFP, TagRFP, and several mFruit proteins, in a wide range of excitation wavelengths (640-1400 nm). To evaluate 2PA cross section (sigma(2)), we use a new method relying only on the optical properties of the intact mature chromophore. In the tuning range of a mode-locked Ti:sapphire laser, 700-1000 nm, TagRFP possesses the highest two-photon cross section, sigma(2) = 315 GM, and brightness, sigma(2)phi = 130 GM, where phi is the fluorescence quantum yield. At longer wavelengths, 1000-1100 nm, tdTomato has the largest values, sigma(2) = 216 GM and sigma(2)phi = 120 GM, per protein chain. Compared to the benchmark EGFP, these proteins present 3-4 times improvement in two-photon brightness.