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One-Photon and Two-Photon Excitation of Fluorescent Proteins

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Fluorescent Proteins I

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 11))

Abstract

Fluorescent proteins (FPs) offer a wide palette of colors for imaging applications. One purpose of this chapter is to review the variety of FP spectral properties, with a focus on their structural basis. Fluorescence in FPs originates from the autocatalytically formed chromophore. Several studies exist on synthetic chromophore analogs in gas phase and in solution. Together with the X-ray structures of many FPs, these studies help to understand how excitation and emission energies are tuned by chromophore structure, protonation state, and interactions with the surrounding environment, either solvent molecules or amino acids residues. The increasing use of FPs in two-photon microscopy also prompted detailed investigations of their two-photon excitation properties. The comparison with one-photon excitation reveals nontrivial features, which are relevant both for their implications in understanding multiphoton properties of fluorophores and for application purposes.

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Notes

  1. 1.

    AsFP595 chromophore structure has been subject to debate, regarding the possible presence of an imino (NH) group in place of the keto group at position 2 of the imidazolinone (see discussion in [50]).

  2. 2.

    The early study by Niwa et al. used ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxo-1-imidazolacetate, a model chromophore with the methyl group in p-HBDI substituted with CO2C2H5.

  3. 3.

    Somewhat upsetting this picture, Wachter and coworkers pointed out that a very similar arrangement of three H-bond donors would also be present in the immature form of the DsRed protein, which, however, emits green, not cyan, light [86]. However, no isolated structure of DsRed with immature green chromophore is available, the X-ray structures being a mixture of mature and immature structures.

  4. 4.

    In avGFP mutants, such as EYFP, featuring the anionic band only, the neutral band emerges by decreasing the pH below the protein pKa.

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    R. Nifosì & V. Tozzini

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  1. R. Nifosì
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  2. V. Tozzini
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    Gregor Jung

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Nifosì, R., Tozzini, V. (2011). One-Photon and Two-Photon Excitation of Fluorescent Proteins. In: Jung, G. (eds) Fluorescent Proteins I. Springer Series on Fluorescence, vol 11. Springer, Berlin, Heidelberg. https://doi.org/10.1007/4243_2011_26

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