Fluorescence Lifetime of Fluorescent Proteins

  • Gregor Jung
  • Andreas Brockhinke
  • Thomas Gensch
  • Benjamin Hötzer
  • Stefanie Schwedler
  • Seena Koyadan Veettil
Chapter
Part of the Springer Series on Fluorescence book series (SS FLUOR, volume 11)

Abstract

Abstract

Fluorescence is a photophysical phenomenon, which obeys basic physical laws. The fluorescence of the autofluorescent proteins arises on the molecular level from chromophores, which are buried in the protein matrix. The three-dimensional, well-defined architecture of the surrounding is a prerequisite for their function. Excitation of the isolated chromophores leads only to a negligible light emission at room temperature. Several processes competing with the radiative decay are responsible for the quenching. To understand how nature has learned to suppress these alternative pathways from the excited state in autofluorescent proteins, the molecular dynamics as well as the influence of several amino acids in the interior of the protein has to be analysed. We review the current status of the understanding of the non-radiative decay mechanisms for the different fluorescent protein classes, i.e., colours. Furthermore, we address what can be learned from fluorescence lifetime measurements and how they can be exploited for analytical purposes such as fluorescence lifetime imaging microscopy. Finally, we sketch the needs of increased fluorescence quantum yields and present strategies to prolong the fluorescence lifetimes.

Graphical Abstract

Keywords

FLIM Internal conversion Photophysics Protein dynamics TCSPC 

References

  1. 1.
    Kummer AD, Kompa C, Niwa H, Hirano T, Kojima S, Michel-Beyerle ME (2002) Viscosity-dependent fluorescence decay of the GFP chromophore in solution due to fast internal conversion. J Phys Chem B 106:7557–7559Google Scholar
  2. 2.
    Lammich L, Petersen MA, Brøndsted NM, Andersen LH (2007) The gas-phase absorption spectrum of a neutral GFP model chromophore. Biophys J 92:201–207Google Scholar
  3. 3.
    Litvinenko KL, Webber NM, Meech SR (2003) Internal conversion in the chromophore of the green fluorescent protein: temperature dependence and isoviscosity analysis. J Phys Chem A 107:2616–2623Google Scholar
  4. 4.
    Kojima S, Hirano T, Niwa H, Ohashi M, Inouye S, Tsuji FI (1997) Mechanism of the redox reaction of the Aequorea green fluorescent protein (GFP). Tetrahedron Lett 38:2875–2878Google Scholar
  5. 5.
    Inouye S, Tsuji FI (1994) Evidence for redox forms of the Aequorea green fluorescent protein. FEBS Lett 351:211–214Google Scholar
  6. 6.
    Drobizhev M, Tillo S, Makarov NS, Hughes TE, Rebane A (2009) Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins. J Phys Chem 113:855–859Google Scholar
  7. 7.
    Nifosi R, Luo Y (2007) Predictions of novel two-photon absorption bands in fluorescent proteins. J Phys Chem B 111:14043–14050Google Scholar
  8. 8.
    Tillo SE, Hughes TE, Makarov NS, Rebane A, Drobizhev M (2010) A new approach to dual-color two-photon microscopy with fluorescent proteins. BMC Biotechnol 10:6Google Scholar
  9. 9.
    Langhojer F, Dimler F, Jung G, Brixner T (2009) Ultrafast photoconversion of the green fluorescent protein studied by accumulative femtosecond spectroscopy. Biophys J 96:2763–2770Google Scholar
  10. 10.
    van Thor JJ, Gensch T, Hellingwerf KJ, Johnson LN (2002) Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222. Nat Struct Biol 9:37–41Google Scholar
  11. 11.
    Hell SW (2007) Far-field optical nanoscopy. Science 316:1153–1158Google Scholar
  12. 12.
    Klar TA, Jakobs S, Dyba M, Egner A, Hell SW (2000) Fluorescence microscopy with diffraction resolution limit broken by stimulated emission. Proc Natl Acad Sci USA 97:8206–8210Google Scholar
  13. 13.
    Widengren J, Mets Ü, Rigler R (1999) Photodynamic properties of green fluorescent proteins investigated by fluorescence correlation spectroscopy. Chem Phys 250:171–186Google Scholar
  14. 14.
    Jiménez-Banzo A, Nonell S, Hofkens J, Flors C (2008) Singlet oxygen photosensitization by EGFP and its chromophore HBDI. Biophys J 94:168–172Google Scholar
  15. 15.
    Bulina ME, Chudakov DM, Britanova OV, Yanushevich YG, Staroverov DB, Chepurnykh TV, Merzlyak EM, Shkrob MA, Lukyanov S, Lukyanov KA (2006) A genetically encoded photosensitizer. Nat Biotechnol 24:95–99Google Scholar
  16. 16.
    Hilborn RC (1982) Einstein coefficients, cross sections, f values, dipole moments and all that. Am J Phys 50:982–986Google Scholar
  17. 17.
    Kennis JTM, Larsen DS, van Stokkum IHM, Vengris M, van Thor JJ, van Grondelle R (2004) Uncovering the hidden ground state of green fluorescent protein. Proc Natl Acad Sci USA 101:17988–17993Google Scholar
  18. 18.
    Min W, Lu S, Chong S, Roy R, Holtom GR, Xie XS (2009) Imaging chromophores with undetectable fluorescence by stimulated emission microscopy. Nature 461:1105–1109Google Scholar
  19. 19.
    Pikas DJ, Kirkpatrick SM, Tewksbury E, Brott LL, Naik RR, Stone MO, Dennis WM (2002) Nonlinear saturation and lasing characteristics of green fluorescent protein. J Phys Chem B 106:4831–4837Google Scholar
  20. 20.
    Jung G, Ma Y, Prall BS, Fleming GR (2005) Ultrafast fluorescence depolarisation in the yellow fluorescent protein due to its dimerisation. Chem Phys Chem 6:1628–1632Google Scholar
  21. 21.
    Jung G, Wiehler J, Andreas Z (2005) The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222. Biophys J 88:1932–1947Google Scholar
  22. 22.
    Heikal AA, Hess ST, Webb WW (2001) Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specifity. Chem Phys 274:37–55Google Scholar
  23. 23.
    Strickler SJ, Berg RA (1962) Relationship between absorption intensity and fluorescence lifetime of molecules. J Chem Phys 37:814–822Google Scholar
  24. 24.
    Suhling K, Siegel J, Phillips D, French PMW, Lévêque-Fort S, Webb SED, Davis DM (2002) Imaging the environment of green fluorescent protein. Biophys J 83:3589–3595Google Scholar
  25. 25.
    Tregidgo C, Levitt JA, Suhling K (2008) Effect of refractive index on the fluorescence lifetime of green fluorescent protein. J Biomed Opt 13:031218Google Scholar
  26. 26.
    Van Manen H, Verkuijlen P, Wittendorp P, Subramaniam V, van den Berg TK, Roos D, Otto C (2008) Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy. Biophys J 94:L67–L69Google Scholar
  27. 27.
    Beuthan J, Minet O, Helfmann J, Herrig M, Müller G (1996) The spatial variation of the refractive index in biological cells. Phys Med Biol 41:369–382Google Scholar
  28. 28.
    Curl CL, Bellair CJ, Harris T, Allman BE, Harris PJ, Stewart AG, Roberts A, Nugent KA, Delbridge LMD (2005) Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy. Cytometry A 65:88–92Google Scholar
  29. 29.
    Yamauchi T, Iwai H, Miwa M, Yamashita Y (2008) Low-coherent quantitative phase microscope for nanometer-scale measurement of living cells morphology. Opt Express 16:12227–12238Google Scholar
  30. 30.
    Fu Y, Zhang J, Lakowicz JR (2008) Metal-enhanced fluorescence of single green fluorescent protein (GFP). Biochem Biophys Res Commun 376:712–717Google Scholar
  31. 31.
    Ormö M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395Google Scholar
  32. 32.
    Englman R, Jortner J (1970) The energy gap law for radiationless transitions in large molecules. Mol Phys 18:145–164Google Scholar
  33. 33.
    Freed KF (1978) Radiationless transitions in molecules. Acc Chem Res 11:74–80Google Scholar
  34. 34.
    Henry BR, Siebrand W (1974) Radiationless transitions. In: Birks J (ed) Organic molecular photophysics, vol 1. Wiley, LondonGoogle Scholar
  35. 35.
    Bae JH, Rubini M, Jung G, Wiegand G, Seifert MHJ, Azim MK, Kim J, Zumbusch A, Holak TA, Moroder L, Huber R, Budisa N (2003) Expansion of the genetic code enables design of a novel “gold” class of green fluorescent proteins. J Mol Biol 328:1071–1081Google Scholar
  36. 36.
    Kummer AD, Kompa C, Lossau H, Pöllinger-Dammer F, Michel-Beyerle ME, Silva CM, Bylina EJ, Coleman WJ, Yang MM, Youvan DC (1998) Dramatic reduction in fluorescence quantum yield in mutants of green fluorescent protein due to fast internal conversion. Chem Phys 237:183–193Google Scholar
  37. 37.
    Martin ME, Negri F, Olivucci M (2004) Origin, nature, and fate of the fluorescent state of the green fluorescent protein chromophore at the CASPT2//CASSCF resolution. J Am Chem Soc 126:5452–5464Google Scholar
  38. 38.
    Maddalo S, Zimmer M (2006) The role of the protein matrix in green fluorescent protein fluorescence. Photochem Photobiol 82:367–372Google Scholar
  39. 39.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, BerlinGoogle Scholar
  40. 40.
    Hötzer B, Ivanov R, Brumbarova T, Altmeier S, Kappl R, Bauer P, Jung G (2010) Determination of copper(II) ion concentration by lifetime measurements of green fluorescent protein. unpublished resultsGoogle Scholar
  41. 41.
    Borst JW, Hink MA, van Hoek A, Visser AJWG (2005) Effects of refractive index and viscosity on fluorescence and anisotropy decays of enhanced cyan and yellow fluorescent proteins. J Fluoresc 15:153–160Google Scholar
  42. 42.
    Becker W (2005) Advanced time-correlated single photon counting technique. Springer series in chemical physics, vol 81. Springer, HeidelbergGoogle Scholar
  43. 43.
    Gadella TWJ (ed) (2009) FRET and FLIM techniques. Elsevier Science & Technology, AmsterdamGoogle Scholar
  44. 44.
    Gerritsen HC, Asselbergs MAH, Agronskaia AV, Van Sark WGJHM (2002) Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution. J Microsc 206:218–224Google Scholar
  45. 45.
    Suhling K, French PMW, Phillip D (2005) Time-resolved fluorescence microscopy. Photochem Photobiol Sci 4:13–22Google Scholar
  46. 46.
    Yasuda R (2006) Imaging spatiotemporal dynamics of neuronal signalling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy. Curr Opin Neurobiol 16:551–561Google Scholar
  47. 47.
    Pepperkok R, Squire A, Geley S, Bastiaens PIH (1999) Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Curr Biol 9:269–272Google Scholar
  48. 48.
    Kremers G, Goedhart J, van Munster EB, Gadella TWJ Jr (2006) Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius. Biochemistry 45:6570–6580Google Scholar
  49. 49.
    Kremers G, Goedhart J, van den Heuvel DJ, Gerritsen HC, Gadella TWJ Jr (2007) Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells. Biochemistry 46:3775–3783Google Scholar
  50. 50.
    Hess ST, Sheets ED, Wagenknecht-Wiesner A, Heikal AA (2003) Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells. Biophys J 85:2566–2580Google Scholar
  51. 51.
    Mauring K, Deich J, Rosell FI, McAnaney TB, Moerner WE, Boxer SG (2005) Enhancement of the fluorescence of the blue fluorescent proteins by high pressure or low temperature. J Phys Chem B 109:12976–12981Google Scholar
  52. 52.
    Jung G, Zumbusch A (2006) Improving autofluorescent proteins: comparative studies of the effective brightness of Green Fluorescent Protein (GFP) mutants. Microsc Res Tech 69:175–185Google Scholar
  53. 53.
    Bowen B, Woodbury N (2003) Single-molecule fluorescence lifetime and anisotropy measurements of the red fluorescent protein, DsRed, in solution. Photochem Photobiol 77:362–369Google Scholar
  54. 54.
    Düser M, Zarrabi N, Bi Y, Zimmermann B, Dunn S, Börsch M (2006) 3D-localization of the a-subunit of FoF1-ATP synthase by time resolved single-molecule FRET. Proc SPIE 6092:60920Google Scholar
  55. 55.
    Hoffmann B, Zimmer T, Klöcker N, Kelbauskas L, König K, Benndorf K, Biskup C (2008) Prolonged irradiation of enhanced cyan fluorescent protein or Cerulean can invalidate Förster resonance energy transfer measurements. J Biomed Opt 13:031205Google Scholar
  56. 56.
    Jung G, Werner M, Schneider M (2008) Efficient photoconversion distorts the fluorescence lifetime of GFP in confocal microscopy: a model kinetic study on mutant Thr203Val. ChemPhysChem 9:1867–1874Google Scholar
  57. 57.
    Tramier M, Zahid M, Mevel J, Masse M, Coppey-Moisan M (2006) Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells. Microsc Res Tech 69:933–939Google Scholar
  58. 58.
    Lelimousin M, Noirclerc-Savoye M, Lazareno-Saez C, Paetzold B, Le Vot S, Chazal R, Macheboeuf P, Field MJ, Bourgeois D, Royant A (2009) Intrinsic dynamics in ECFP and Cerulean control fluorescence quantum yield. Biochemistry 48:10038–10046Google Scholar
  59. 59.
    Villoing A, Ridhoir M, Cinquin B, Erard M, Alvarez L, Vallverdu G, Pernot P, Grailhe R, Fe M, Pasquier H (2008) Complex fluorescence of the cyan fluorescent protein: comparisons with the H148D variant and consequences for quantitative cell imaging. Biochemistry 47:12483–12492Google Scholar
  60. 60.
    Scruggs AW, Flores CL, Wachter R, Woodbury NW (2005) Development and characterization of green fluorescent protein mutants with altered lifetimes. Biochemistry 44:13377–13384Google Scholar
  61. 61.
    Goedhart J, van Weeren L, Hink MA, Vischer NOE, Jalink K, Gadella TWJ Jr (2010) Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat Methods 7:137–139Google Scholar
  62. 62.
    Rizzo MA, Springer GH, Granada B, Piston DW (2004) An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22:445–449Google Scholar
  63. 63.
    Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6:178–182Google Scholar
  64. 64.
    Wachter RM, King BA, Heim R, Kallio K, Tsien RY, Boxer SG, Remington SJ (1997) Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein. Biochemistry 36:9759–9765Google Scholar
  65. 65.
    Malo GD, Pouwels LJ, Wang M, Weichsel A, Montfort WR, Rizzo MA, Piston DW, Wachter RM (2007) X-ray structure of Cerulean GFP: a tryptophan-based chromophore useful for fluorescence lifetime imaging. Biochemistry 46:9865–9873Google Scholar
  66. 66.
    Mena MA, Treynor TP, Mayo SL, Daugherty PS (2006) Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library. Nat Biotechnol 24:1569–1571Google Scholar
  67. 67.
    Kummer AD, Wiehler J, Schüttrigkeit TA, Berger BW, Steipe B, Michel-Beyerle ME (2002) Picosecond time-resolved fluorescence from blue-emitting chromophore variants Y66F and Y66H of the green fluorescent protein. Chem Bio Chem 3:659–663Google Scholar
  68. 68.
    Lossau HS, Kummer A, Heinecke R, Pöllinger-Dammer F, Kompa C, Beiser G, Johnsson T, Silva CM, Yang MM, Youvan DC, Michel-Beyerle ME (1996) Time-resolved spectroscopy of wild-type and mutant green fluorescent proteins reveals excited state deprotonation consistent with fluorophore-protein interactions. Chem Phys 213:1–16Google Scholar
  69. 69.
    Megley CM, Dickson LA, Maddalo SL, Chandler GJ, Zimmer M (2009) Photophysics and dihedral freedom of the chromophore in yellow, blue, and green fluorescent protein. J Phys Chem B 113:302–308Google Scholar
  70. 70.
    Barondeau DP, Kassmann CJ, Tainer JA, Getzoff ED (2002) Structural chemistry of a green fluorescent protein Zn biosensor. J Am Chem Soc 124:3522–3524Google Scholar
  71. 71.
    Ai HW, Shaner NC, Cheng Z, Tsien RY, Campbell R (2007) Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry 46:5904–5910Google Scholar
  72. 72.
    Tramier M, Gautier I, Piolot T, Ravalet S, Kemnitz K, Coppey J, Durieux C, Mignotte V, Coppey-Moisan M (2002) Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells. Biophys J 83:3570–3577Google Scholar
  73. 73.
    Demachy I, Ridard J, Laguitton-Pasquier H, Durnerin E, Vallverdu G, Archirel P, Lévy B (2005) Cyan fluorescent protein: molecular dynamics, simulations, and electronic absorption spectrum. J Phys Chem B 109:24121–24133Google Scholar
  74. 74.
    Chattoraj M, King BA, Bublitz GA, Boxer SG (1996) Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. Proc Natl Acad Sci USA 93:862–8367Google Scholar
  75. 75.
    Olson S, McKenzie RH (2010) A dark excited state of fluorescent chromophores, considered as Brooker dyes. Chem Phys Lett 492:150–156Google Scholar
  76. 76.
    Pal PP, Bae JH, Azim MK, Hess P, Friedrich R, Huber R, Moroder L, Budisa N (2005) Structural and spectral response of Aequorea victoria green fluorescent proteins to chromophore fluorination. Biochemistry 44:3663–3672Google Scholar
  77. 77.
    Wiehler J, Jung G, Seebacher C, Zumbusch A, Steipe B (2003) Mutagenic stabilization of the photocycle intermediate of green fluorescent protein (GFP). ChemBioChem 4:1164–1171Google Scholar
  78. 78.
    Cotlet M, Hofkens J, Maus M, Gensch T, Van der Auweraer M, Michiels J, Dirix G, Van Guyse M, Vanderleyden J, Visser AJWG, De Schryver FC (2001) Excited-state dynamics in the enhanced green fluorescent protein mutant probed by picosecond time-resolved single photon counting spectroscopy. J Phys Chem B 105:4999–5006Google Scholar
  79. 79.
    Striker G, Subramaniam V, Seidel CAM, Volkmer A (1999) Photochromicity and fluorescence lifetimes of green fluorescent protein. J Phys Chem B 103:8612–8617Google Scholar
  80. 80.
    Stavrov SS, Solntsev KM, Tolbert LM, Huppert D (2006) Probing the decay coordinate of the green fluorescent protein: arrest of cis-trans isomerization by the protein significantly narrows the fluorescence spectra. J Am Chem Soc 128:1540–1546Google Scholar
  81. 81.
    Bagchi B, Fleming GR, Oxtoby DW (1983) Theory of electronic relaxation in solution in the absence of an activation barrier. J Chem Phys 78:7375–7385Google Scholar
  82. 82.
    Kummer AD, Wiehler J, Rehaber H, Kompa C, Steipe B, Michel-Beyerle ME (2000) Effects of threonine 203 replacements on excited-state dynamics and fluorescence properties of the green fluorescent protein (GFP). J Phys Chem B 104:4791–4798Google Scholar
  83. 83.
    Schwille P, Kummer S, Heikal AA, Moerner WE, Webb Watt W (2000) Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins. Proc Natl Acad Sci USA 97:151–156Google Scholar
  84. 84.
    Weber W, Helms V, McCammon JA, Langhoff PW (1999) Shedding light on the dark and weakly fluorescent states of green fluorescent proteins. Proc Natl Acad Sci USA 96:6177–6182Google Scholar
  85. 85.
    Baffour-Awuah NYA, Zimmer M (2004) Hula-twisting in green fluorescent protein. Chem Phys 303:7–11Google Scholar
  86. 86.
    Andresen M, Wahl MC, Stiel AC, Gräter F, Schäfer LV, Trowitzsch S, Weber G, Eggeling C, Grubmüller H, Hell SW, Jakobs S (2005) Structure and mechanism of the reversible photoswitch of a fluorescent protein. Proc Natl Acad Sci USA 102:13070–13074Google Scholar
  87. 87.
    Nifosi R, Tozzini V (2006) Cis-trans photoisomerization of the chromophore in the green fluorescent protein variant E2GFP: a molecular dynamics study. Chem Phys 323:358–368Google Scholar
  88. 88.
    Mizuno H, Mal TK, Wälchi M, Kikuchi A, Fukano T, Ando R, Jeyakanthan TJ, Shiro Y, Ikura M, Miyawaki A (2008) Light-dependent regulation of structural flexibility in a photochromic fluorescent protein. Proc Natl Acad Sci USA 105:9227–9232Google Scholar
  89. 89.
    Schleifenbaum F, Blum C, Elgass K, Subramaniam V, Meixner AJ (2008) New insights into the photophysics of DsRed by multiparameter spectroscopy on single proteins. J Phys Chem B 112:7669–7674Google Scholar
  90. 90.
    Bateman RJ, Chance RR, Hornig JF (1974) Fluorescence reabsorption in anthracene single crystals: lifetime variations with emission wavelength and temperature. Chem Phys 4:402–408Google Scholar
  91. 91.
    Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90:1103–1163Google Scholar
  92. 92.
    Müller-Taubenberger A, Anderson KI (2007) Recent advances using green and red fluorescent protein variants. Appl Microbiol Biotechnol 77:1–12Google Scholar
  93. 93.
    Heikal AA, Hess ST, Baird GS, Tsien RY, Webb WW (2000) Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc Natl Acad Sci USA 97:11996–12001Google Scholar
  94. 94.
    Schüttrigkeit TA, Zachariae U, von Feilitzsch T, Wiehler J, von Hummel J, Steipe B, Michel-Beyerle ME (2001) Picosecond time-resolved FRET in the fluorescent protein from Discosoma Red (wt-DsRed). ChemPhysChem 2:325–328Google Scholar
  95. 95.
    Nienhaus GU, Wiedenmann J (2009) Structure, dynamics and optical properties of fluorescent proteins: perspectives for marker development. Chem Phys Chem 2009:1369–1379Google Scholar
  96. 96.
    Schmid JA, Neumeier H (2005) Evolutions in science triggered by green fluorescent protein (GFP). Chem Bio Chem 6:1149–1156Google Scholar
  97. 97.
    Seefeldt B, Kasper R, Seidel T, Tinnefeld P, Dietz K, Heilemann M, Sauer M (2008) Fluorescent proteins for single-molecule fluorescence applications. J Biophoton 1:74–82Google Scholar
  98. 98.
    Shaner N, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572Google Scholar
  99. 99.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909Google Scholar
  100. 100.
    Cox G, Matz M, Salih A (2007) Fluorescence lifetime imaging of coral fluorescent proteins. Microsc Res Tech 70:243–251Google Scholar
  101. 101.
    Hasegawa J, Ise T, Fujimoto KJ, Kikuchi A, Fukumura E, Miyawaki A, Shiro Y (2010) Excited states of fluorescent proteins, mKO and DsRed: chromophore-protein electrostatic interaction behind the color variations. J Phys Chem B 114:2971–2979Google Scholar
  102. 102.
    Yan W, Zhang L, Xie D, Zeng J (2007) Electronic excitations of green fluorescent proteins: modeling solvatochromatic shifts of red fluorescent protein chromophore model compound in aqueous solutions. J Phys Chem B 111:14055–14063Google Scholar
  103. 103.
    Liu RSH, Yang L, Liu J (2007) Mechanisms of photoisomerization of polyenes in confined media: from organic glasses to protein binding cavities. Photochem Photobiol 83:2–10Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  • Gregor Jung
    • 1
  • Andreas Brockhinke
    • 2
  • Thomas Gensch
    • 3
  • Benjamin Hötzer
    • 1
  • Stefanie Schwedler
    • 2
  • Seena Koyadan Veettil
    • 1
  1. 1.Biophysical ChemistrySaarland UniversitySaarbrückenGermany
  2. 2.Physical Chemistry 1University of BielefeldBielefeldGermany
  3. 3.Institute of Structural Biology and Biophysics 1 (Cellular Signaling, ISB-1)Forschungszentrum JülichJülichGermany

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