Absolute Two-photon Absorption Spectra Of Orange And Red Fluorescent Proteins

Abstract

Two-Photon laser scanning microscopy, which makes use of genetically encoded fluorescent protein (FP) probes, is becoming a method of choice for studying biological systems from sub-cellular to the whole body level. However, reliable information on two-photon absorption (2PA) properties of FPs, specially for the more popular orange and red variants, is still very fragmentary. 2PA spectra, measured in absolute cross section values, will allow us to select the two-photon brightest FP variant with desired fluorescence properties and also to choose the optimum laser system and excitation wavelength. Here we study 2PA spectra of a large set of orange and red FPs, including DsRed2, mRFP, TagRFP, and mFruits series in a wide range of excitation wavelengths, 600 -1200 nm. We have found the the 2PA spectra and maximum cross sections are very sensitive to either changes in chromophore structure (mOrange vs mRFP) or to mutations in chromophore surrounding (DsRed and mFruits series). All red FPs show two pronounced 2PA transitions, the first peaking in the 1000 - 1100 nm region, and second - near 700 - 760 nm. We quantitatively describe the first transition within the framework of two-level model, and the second - within three-level model with strong resonance enhancement. Excitation in the longer wavelength region, accessible for Nd- and Yb-doped short-pulse lasers, has advantages of producing less two-photon autofluorescence and deeper penetration. For each region of wavelengths, we have found a mutant, which is 3-4 times two-photon brighter than the benchmark EGFP.