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Two-photon absorption properties of fluorescent proteins


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 fluorescent protein and excitation wavelength for two-photon applications.

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Figure 1: One- and two-photon absorption spectra of fluorescent proteins with different chromophores.
Figure 2: Structure of the two-photon absorption spectrum of a fluorescent protein.
Figure 3: One-photon absorption of the 'Fruit' proteins does not predict which is the brightest two-photon probe.
Figure 4: Two-photon absorption is highly sensitive to the electric field in the protein environment.
Figure 5: Matching of two-photon excitation spectra of red fluorescent proteins with the optimum tissue transparency and with the wavelengths of some short-pulse laser systems.


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This work was supported by the US National Institute of General Medical Sciences grant R01 GM086198. We thank B.H. Davis for technical help, and R. Campbell (University of Alberta, Edmonton, Canada), D.M. Chudakov (Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow), M. Lin (Stanford University) and R.Y. Tsien (University of California San Diego) for providing us cDNA of different fluorescent proteins.

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Correspondence to Mikhail Drobizhev.

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Supplementary Figures 1–3, Supplementary Tables 1–2, Supplementary Methods (PDF 1755 kb)

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Drobizhev, M., Makarov, N., Tillo, S. et al. Two-photon absorption properties of fluorescent proteins. Nat Methods 8, 393–399 (2011).

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