Two-photon absorption properties of fluorescent proteins

Abstract

Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest, and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right FP and excitation wavelength for two-photon applications.Two-photon laser scanning microscopy (2PLSM) 1,2 of cells and tissues expressing fluorescent proteins is becoming a powerful tool for biological studies at different levels of organization [2][3][4] . The advantages of two-photon excitation (2PE) include reduced out-offocus photobleaching, less autofluorescence, deeper tissue penetration, and intrinsically high three-dimensional resolution 1,2 . 2PLSM should make it possible to obtain even better optical recordings of ion concentration and cell signaling with genetically targeted sensors 5,6 . Twophoton excitation of fluorescent proteins can also be considered as potentially advantageous in the contexts of genetically targeted deep photodynamic therapy or chromophore-assisted light inactivation 6 , three-dimensional optical memory 7 , as well as superresolution (subdiffraction limited) imaging techniques, such as stimulated emission depletion 8 , photoactivated localization microscopy, and stochastic optical reconstruction microscopy 9 .To fully realize the potential of 2PE of the fluorescent proteins, it is important to know their two-photon absorption (2PA) spectra, cross sections, σ 2 , and 2PE action cross sections, or brightness, σ 2 ', (σ 2 ' = σ 2 × φ, where φ is the fluorescence quantum yield). The linear, onephoton absorption (1PA) spectra and extinction coefficients of many fluorescent proteins have been described and reviewed 5,6,10 (Supplementary Table 1 online), but the 1PA properties are not sufficient to predict the key 2PA properties such as optimum excitation wavelength and maximum brightness (Box 2).Correspondence should be addressed to M.D. (drobizhev@physics.montana.edu).. 4 Present address: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia. 5 Present address: Vollum Institute, Oregon Health and Science University, Portland, Oregon. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptHere we present a systematic characterization of the 2PA properties of 48 different fluorescent proteins using the same experimental setup, common 2PA reference standards, and an all-optical method for measuring mature chromophore concentration 11 (Supplementary Methods online). Briefly, we use a relative fluorescence method with femtosecond excitation and coumarin 485 (Exciton), coumarin 540A (Exciton), rhodamine 610 (Exciton), fluorescein (Aldrich), and styryl 9M (Aldrich) as 2PA standards 12 , which eliminates the necessity to calibrate the laser parameters. The power dependence of the fluorescence signal was quadratic for all data ...

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