Photosynthesis Research

, Volume 101, Issue 2–3, pp 119–133 | Cite as

Fluorescence measurement by a streak camera in a single-photon-counting mode



We describe here a recently developed fluorescence measurement system that uses a streak camera to detect fluorescence decay in a single photon-counting mode. This system allows for easy measurements of various samples and provides 2D images of fluorescence in the wavelength and time domains. The great advantage of the system is that the data can be handled with ease; furthermore, the data are amenable to detailed analysis. We describe the picosecond kinetics of fluorescence in spinach Photosystem (PS) II particles at 4–77 K as a typical experimental example. Through the global analysis of the data, we have identified a new fluorescence band (F689) in addition to the already established F680, F685, and F695 emission bands. The blue shift of the steady-state fluorescence spectrum upon cooling below 77 K can be interpreted as an increase of the shorter-wavelength fluorescence, especially F689, due to the slowdown of the excitation energy transfer process. The F685 and F695 bands seem to be thermally equilibrated at 77 K but not at 4 K. The simple and efficient photon accumulation feature of the system allows us to measure fluorescence from leaves, solutions, single colonies, and even single cells. The 2D fluorescence images obtained by this system are presented for isolated spinach PS II particles, intact leaves of Arabidopsis thaliana, the PS I super-complex of a marine centric diatom, Chaetoceros gracilis, isolated membranes of a purple photosynthetic bacterium, Acidiphilium rubrum, which contains Zn-BChl a, and a coral that contains a green fluorescent protein and an algal endosymbiont, Zooxanthella.


Streak camera Photosystem II Excitation energy transfer Time-resolved fluorescence spectroscopy Chlorophyll fluorescence Global analysis Temperature dependency 



Charge coupled device


Decay-associated spectrum


Excitation energy transfer

F685, F695 and F735

Fluorescence bands peaking at 685, 695, and 735 nm respectively


Light-harvesting chlorophyll a/b binding complex


Multichannel plate


Primary electron donor in PS II




Reaction center



This study was supported by a COE program for “The origin of the universe and matter” and by a grant-in-aid (No. 17370055) from the Japanese Ministry of Education, Science, Sports, and Culture to S·I. We are grateful to Mrs. Y. Nakamura and Drs. T. Tomi, Y. Shibata, H. Mino, A.M. Gilmore, Govindjee, A.W.D. Larkum, Z. Gombos, and Y. Ikeda who gave us a chance to measure variety of samples. We thank Govindjee for editing this manuscript.


  1. Amunts A, Drory O, Nelson N (2007) The structure of a plant photosystem I supercomplex at 3.4 Ǻ resolution. Nature 447:58–63PubMedCrossRefGoogle Scholar
  2. Andrizhiyevskaya EG, Frolov D, van Grondelle R, Dekker JP (2004) On the role of the CP47 core antenna in the energy transfer and trapping dynamics of photosystem II. Phys Chem Chem Phys 6:4810–4819CrossRefGoogle Scholar
  3. Andrizhiyevskaya EG, Chojnicka A, Bautista JA, Diner BA, van Grondelle R, Dekker P (2005) Origin of the F685 and F695 fluorescence in photosystem II. Photosynth Res 84:173–180PubMedCrossRefGoogle Scholar
  4. Berthold DA, Babcock GT, Yocum CF (1981) A highly resolved, oxygen-evolving photosystem II preparation from spinach thylakoid membranes: EPR and electron-transport properties. FEBS Lett 134:231–234CrossRefGoogle Scholar
  5. Boekema EJ, van Roon H, Calkoen F, Bassi R, Dekker JP (1999) Multiple types of association of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes. Biochemistry 38:2233–2239PubMedCrossRefGoogle Scholar
  6. Brody SS, Brody M (1963) Aggregated chlorophyll in vivo. Natl Acad Sci-Natl Res Council Publ 1145:455–478Google Scholar
  7. Broess K, Trinkunas G, van der Weij-de Wit CD, Dekker JP, van Hoek A, van Amerongen H (2006) Excitation energy transfer and charge separation in photosystem II membranes revisited. Biophys J 91:3776–3786PubMedCrossRefGoogle Scholar
  8. Cho F, Govindjee (1970a) Low temperature (4–77 K) spectroscopy of Chlorella: temperature dependence of energy transfer efficiency. Biochim Biophys Acta 216:139–150PubMedCrossRefGoogle Scholar
  9. Cho F, Govindjee (1970b) Low temperature (4–77 K) spectroscopy of Anacystis: temperature dependence of energy transfer efficiency. Biochim Biophys Acta 216:151–161PubMedCrossRefGoogle Scholar
  10. Cho F, Spencer J, Govindjee (1966) Emission spectra of Chlorella at very low temperatures (−269 to −196°C). Biochim Biophys Acta 126:174–176PubMedCrossRefGoogle Scholar
  11. Croce R, Dorra D, Holzwarth AR, Jennings RC (2000) Fluorescence decay and spectral evolution in intact photosystem I of higher plants. Biochemistry 39:6341–6348PubMedCrossRefGoogle Scholar
  12. De Weerd FL, Palacios MA, Andrizhiyevskaya EG, Dekker JP, Van Grondelle R (2002a) Identifying the lowest electronic states of the chlorophylls in the CP47 core antenna protein of photosystem II. Biochemistry 41:15224–15233PubMedCrossRefGoogle Scholar
  13. De Weerd FL, van Stokkum IHM, van Amerongen H, Dekker JP, Van Grondelle R (2002b) Pathways for energy transfer in the core light-harvesting complexes CP43 and CP47 of photosystem II. Biophys J 82:1586–1597PubMedCrossRefGoogle Scholar
  14. Dekker JP, van Grondelle R (2001) Primary charge separation in photosystem II. Photosynth Res 63:195–208CrossRefGoogle Scholar
  15. Dekker JP, Jassoldt A, Pettersson A, van Roon H, Groot ML, van Grondelle R (1995) On the nature of the F695 and F685 emission of photosystem II. In: Mathis P (ed) Photosynthesis: from light to biosphere, vol 1. Kluwer, Dordrecht, pp 53–56Google Scholar
  16. Domonkos I, Malec P, Sallai A, Kovács L, Itoh K, Shen G, Ughy B, Bogos B, Sakurai I, Kis M, Strzalka K, Wada H, Itoh S, Farkas T, Gombos Z (2004) Phosphatidylglycerol is essential for oligomerization of photosystem I reaction center. Plant Physiol 134:1471–1478PubMedCrossRefGoogle Scholar
  17. Fleming GR, Morris JM, Robinson GW (1977) Picosecond fluorescence spectroscopy with a streak camera. Austr J Chem 30:2338–2352Google Scholar
  18. Fukushima Y, Okajima K, Shibata Y, Ikeuchi M, Itoh S (2005) Primary intermediate in the photocycle of a blue-light sensory BLUF FAD-protein, Tll0078, of Thermosynechococcus elongatus BP-1. Biochemistry 44:5149–5158PubMedCrossRefGoogle Scholar
  19. Gasanov R, Abilov ZK, Gazanchyan RM, Kurbonova UM, Khanna R, Govindjee (1979) Excitation-energy transfer in photosystem-I and photosystem-II from grana and in photosystem-I from stroma lamellae, and identification of emission bands with pigment-protein complexes at 77 K. Z Pflanzenphysiol 95:149–169Google Scholar
  20. Gilmore AM (2004) Excess light stress: Probing excitation dissipation mechanisms through global analysis of time- and wavelength-resolved chlorophyll a fluorescence. In: Papageorgiou GC, Govindjee (eds) Chlorophyll a fluorescence: a signature of photosynthesis. Dordrecht, SpringerGoogle Scholar
  21. Gilmore AM, Itoh S, Govindjee (2000) Global spectral-kinetic analysis of room temperature chlorophyll a fluorescence from light-harvesting antenna mutants of barley. Philos Trans R Soc Lond B 355:1371–1384CrossRefGoogle Scholar
  22. Gilmore AM, Matsubara S, Ball MC, Barker DH, Itoh S (2003a) Excitation energy flow at 77 K in the photosynthetic apparatus of overwintering evergreens. Plant Cell Environ 26:1021–1034CrossRefGoogle Scholar
  23. Gilmore AM, Larkum AWD, Salih A, Itoh S, Shibata Y, Bena C, Yamasaki H, Papina M, van Woesik R (2003b) Simultaneous time resolution of the emission spectra of fluorescent proteins and Zooxanthella Chlorophyll in reef-building corals. Photochem Photobiol 77:515–523PubMedCrossRefGoogle Scholar
  24. Gobets B, Van Grondelle R (2001) Energy transfer and trapping in photosystem I. Biochim Biophys Acta 1507:80–99PubMedCrossRefGoogle Scholar
  25. Gobets B, Stokkum IHM, Rögner M, Kruip J, Schlodder E, Karapetyan N, Dekker JP, van Grondelle R (2001) Time-resolved fluorescence emission measurements of photosystem I particles of various cyanobacteria: a unified compartmental model. Biophys J 81:407–424PubMedCrossRefGoogle Scholar
  26. Govindjee, Yang L (1966) Structure of the red fluorescence band in chloroplasts. J Gen Physiol 49:763–780PubMedCrossRefGoogle Scholar
  27. Govindjee, Amesz J, Fork DC (eds) (1986) Light emission by plants and bacteria. Academic Press, OrlandoGoogle Scholar
  28. Groot ML, Peterman EJG, Van Kan PJM, Van Stokkum IHM, Dekker JP, Van Grondelle R (1994) Temperature-dependent triplet and fluorescence quantum yields of the photosystem II reaction center described in a thermodynamic model. Biophys J 67:318–330PubMedCrossRefGoogle Scholar
  29. Groot ML, Peterman EJG, van Stokkum IHM, Dekker JP, Van Grondelle R (1995) Triplet and fluorescing states of the CP47 antenna complex of photosystem II studied as a function of temperature. Biophys J 68:281–290PubMedCrossRefGoogle Scholar
  30. Groot ML, Frese RN, de Weerd FL, Bromek K, Pettersson Å, Peterman EJG, van Stokkum IHM, van Grondelle R, Dekker JP (1999) Spectroscopic properties of the CP43 core antenna protein of photosystem II. Biophys J 77:3328–3340PubMedCrossRefGoogle Scholar
  31. Guskov A, Kern J, Gabdulkhakov A, Broser M, Zouni A, Saenger W (2009) Cyanobacterial photosystem II at 2.9-Å resolution and the role of quinones, lipids, channels and chloride. Nature Struct Mol Biol 16:334–342CrossRefGoogle Scholar
  32. Horton P, Ruban AV, Rees D, Pascal AA, Noctor G, Young AJ (1991) Control of the light-harvesting function of chloroplast membranes by aggregation of the LHCII chlorophyll-protein complex. FEBS Lett 292:1–4PubMedCrossRefGoogle Scholar
  33. Ihalainen JA, Van Stokkum IHM, Gibasiewicz K, Germano M, Van Grondelle R, Dekker JP (2005) Kinetics of excitation trapping in intact photosystem I of Chlamydomonas reinhardtii and Arabidopsis thaliana. Biochim Biophys Acta 1706:267–275PubMedCrossRefGoogle Scholar
  34. Ikeda Y, Komura M, Watanabe M, Minami C, Koike H, Itoh S, Kashino Y, Satoh K (2008) Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis. Biochim Biophys Acta 1777:351–361PubMedCrossRefGoogle Scholar
  35. Ito T, Hiramatsu M, Hosoda M, Tsuchiya Y (1991) Picosecond time-resolved absorption spectrometer using a streak camera. Rev Sci Instrum 62:1415–1419CrossRefGoogle Scholar
  36. Itoh S, Sugiura K (2004) Photosystem I fluorescence. In: Papageorgiou GC, Govindjee (eds) Chlorophyll a fluorescence: a signature of photosynthesis. Dordrecht, SpringerGoogle Scholar
  37. Jordan P, Fromme P, Witt HT, Klukas O, Saenger W, Krauβ N (2001) Three-dimensional structure of cyanobacterial photosystem I at 2.5 Ǻ resolution. Nature 411:909–917PubMedCrossRefGoogle Scholar
  38. Komura M, Shibata Y, Itoh S (2006) A new fluorescence band F689 in photosystem II revealed by picosecond analysis at 4–77 K: function of two terminal energy sinks F689 and F695 in PS II. Biochim Biophys Acta 1757:1657–1668PubMedCrossRefGoogle Scholar
  39. Krausz E, Hughes JL, Smith PJ, Pace RJ, Årsköld SP (2005) Assignment of the low-temperature fluorescence in oxygen-evolving photosystem II. Photosynth Res 84:193–199PubMedCrossRefGoogle Scholar
  40. Krey A, Govindjee (1964) Fluorescence change in Porphyridium exposed to green light of different intensity: a new emission band at 693 nm and its significance to photosynthesis. Proc Natl Acad Sci USA 52:1568–1572PubMedCrossRefGoogle Scholar
  41. Kwa SLS, Volker S, Tilly NT, van Grondelle R, Dekker JP (1994) Polarized site-selection spectroscopy of chlorophyll a in detergent. Photochem Photobiol 59:219–228CrossRefGoogle Scholar
  42. Loll B, Kern J, Saenger W, Zouni A, Biesiadka J (2005) Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II. Nature 438:1040–1044PubMedCrossRefGoogle Scholar
  43. Masters V, Smith PJ, Krausz E, Pace R (2001) Stark shifts and exciton coupling in PSII ‘Supercores’. J Lumin 94–95:267–270CrossRefGoogle Scholar
  44. Melkozernov AN (2001) Excitation energy transfer in photosystem I from oxygenic organisms. Photosyn Res 70:129–153PubMedCrossRefGoogle Scholar
  45. Miloslavina Y, Szczepaniak M, Müller MG, Sander J, Nowaczyk M, Rögner M, Holzwarth AR (2006) Charge separation kinetics in intact photosystem II core particles is trap-limited. A picosecond fluorescence study. Biochemistry 45:2436–2442PubMedCrossRefGoogle Scholar
  46. Mimuro M, Tamai N, Yamazaki T, Yamazaki I (1987) Excitation energy transfer in spinach chloroplasts. FEBS Lett 213:119–122CrossRefGoogle Scholar
  47. Mino H, Itoh S (2005) EPR properties of a g = 2 broad signal trapped in S1 state in the Ca2+-depleted photosystem II. Biochim Biophys Acta 1708:42–49PubMedCrossRefGoogle Scholar
  48. Murata N, Nishimura M, Takamiya A (1966) Fluorescence of chlorophyll in photosynthetic system. III. Emission and action spectra of fluorescence-three emission bands of chlorophyll a and the energy transfer between two pigment systems. Biochim Biophys Acta 126:234–243PubMedCrossRefGoogle Scholar
  49. Ono T, Inoue Y (1986) Effects of removal and reconstitution of the extrinsic 33, 24 and 16 kDa proteins on flash oxygen yield in photosystem II. Biochim Biophys Acta 850:380–389CrossRefGoogle Scholar
  50. Papageorgiou GC, Govindjee (eds) (2004) Chlorophyll a fluorescence: a signature of photosynthesis. Springer, DordrechtGoogle Scholar
  51. Slavov C, Ballottari M, Morosinotto T, Bassi R, Holzwarth R (2008) Trap-limited charge separation kinetics in higher plant photosystem I complexes. Biophys J 94:3601–3612PubMedCrossRefGoogle Scholar
  52. Tomi T, Shibata Y, Ikeda Y, Taniguchi S, Haik C, Mataga N, Shimada K, Itoh S (2007) Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy. Biochim Biophys Acta 1767:22–30PubMedCrossRefGoogle Scholar
  53. Tsukatani Y, Miyamoto R, Itoh S, Oh-Oka H (2004) Function of a PscD subunit in a homodimeric reaction center complex of the photosynthetic green sulfur bacterium Chlorobium tepidum studied by insertional gene inactivation. Regulation of energy transfer and ferredoxin-mediated NADP+ reduction on the cytoplasmic side. J Biol Chem 279:51122–51130PubMedCrossRefGoogle Scholar
  54. Van Dorssen RJ, Plijter JJ, Dekker JP, Den Ouden A, Amesz J, Van Gorkom HJ (1987a) Spectroscopic properties of chloroplast grana membranes and of the core of photosystem II. Biochim Biophys Acta 890:134–143CrossRefGoogle Scholar
  55. Van Dorssen RJ, Breton J, Plijter JJ, Satoh K, Van Gorkom HJ, Amesz J (1987b) Spectroscopic properties of the reaction center and of the 47-kDa chlorophyll protein of photosystem II. Biochim Biophys Acta 893:267–274CrossRefGoogle Scholar
  56. Van Stokkum IHM, van Oort B, van Mourik F, Gobets B, van Amerongen H (2008) (Sub)-picosecond spectral evolution of fluorescence studied with a Synchroscan streak-camera system and target analysis. In: Aartsma TJ, Matysik J (eds) Biophysical techniques in photosynthesis (Advances in Photosynthesis and Respiration), vol 26. Springer, Dordrecht, pp 223–240CrossRefGoogle Scholar
  57. Vlakova R (2000) Chlorophyll a self-assembly in polar solvent-water mixtures. Photochem Photobiol 71:71–83CrossRefGoogle Scholar
  58. Wakao N, Yokoi N, Isoyama N, Hiraishi A, Shimada K, Kobayashi M, Kise H, Iwaki M, Itoh S, Takaichi S, Sakurai Y (1996) Discovery of natural photosynthesis using Zn-containing bacteriochlorophyll in an aerobic bacterium Acidiphilium rubrum. Plant Cell Physiol 37:889–893Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  1. 1.Division of Material Science (Physics), Graduate School of ScienceNagoya UniversityNagoyaJapan

Personalised recommendations