, Volume 38, Issue 4, pp 581–599 | Cite as

Fluorescence Lifetime Imaging (FLI) in Real-Time - a New Technique in Photosynthesis Research

  • O. Holub
  • M.J. Seufferheld
  • C. Gohlke
  • Govindjee
  • R.M. Clegg


We describe an instrument that allows the rapid measurement of fluorescence lifetime-resolved images of leaves as well as sub-cellular structures of intact plants or single cells of algae. Lifetime and intensity fluorescence images can be acquired and displayed in real time (up to 55 lifetime-resolved images per s). Our imaging technique therefore allows rapid measurements that are necessary to determine the fluorescence lifetimes at the maximum (P level) fluorescence following initial illumination during the chlorophyll (Chl) a fluorescence transient (induction) in photosynthetic organisms. We demonstrate the application of this new instrument and methodology to measurements of: (1) Arabidopsis thaliana leaves showing the effect of dehydration on the fluorescence lifetime images; (2) Zea mays leaves showing differences in the fluorescence lifetimes due to differences in the bundle sheath cells (having a higher amount of low yield photosystem 1) and the mesophyll cells (having a higher amount of high yield photosystem 2); and (3) single cells of wild type Chlamydomonas reinhardtii and its non-photochemical quenching mutant NPQ2 (where the conversion of zeaxanthin to violaxanthin is blocked), with NPQ2 showing lowered lifetime of Chl a fluorescence. In addition to the lifetime differences referred to in (1) and (2), structural dependent heterogeneities in the fluorescence lifetimes were generally observed when imaging mesophyll cells in leaves.

Arabidopsis Chlamydomonas FLIM frequency domain homodyne microscopy modulation phase photosystems 1 and 2 stress time domain Zea 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Agati, G., Cerovic, Z.G., Moya, I.: The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum sativum: The role of the photosystem I contribution to the 735 nm fluorescence band.-Photochem. Photobiol. 72: 75-84, 2000.CrossRefPubMedGoogle Scholar
  2. Bazzaz, M.B., Govindjee: Photochemical properties of mesophyll and bundle sheath chloroplasts of maize.-Plant Physiol. 52: 257-262, 1973.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Berg, D., Maier, K., Otteken, D., Terjung, F.: Picosecond fluorescence decay studies on water-stressed pea leaves: energy transfer and quenching processes in Photosystem 2.-Photosynthetica 34: 97-106, 1997.CrossRefGoogle Scholar
  4. Briantais, J.-M., Dacosta, J., Goulas, Y., Ducruet, J.-M., Moya, I.: Heat stress induces in leaves an increase of the minimum level of chlorophyll fluorescence, F0: A time-resolved analysis.-Photosynth. Res. 48: 189-196, 1996.CrossRefPubMedGoogle Scholar
  5. Buschmann, C., Lichtenthaler, H.K.: Principles and characteristics of multi-colour fluorescence imaging of plants.-J. Plant Physiol. 152: 297-314, 1998.CrossRefGoogle Scholar
  6. Byrdin, M., Rimke, I., Schlodder, E., Stehlik, D., Roelofs, T.A.: Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: Are the kinetics of excited state decay trap-limited or transfer-limited?-Biophys. J. 79: 992-1007, 2000.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cerovic, Z.G., Goulas, Y., Gorbunov, M., Briantais, J.M., Camenen, L., Moya, I.: Fluorosensing of water stress in plants-diurnal changes of the mean lifetime and yield of chlorophyll fluorescence, measured simultaneously and at distance with a tau-LIDAR and a modified PAM-fluorimeter, in maize, sugar beet, and Kalanchoe.-Remote Sens. Environ. 58: 311-321, 1996.CrossRefGoogle Scholar
  8. Ciscato, M.: Development of a Fluorescence Imaging System for the Quality Assessment of Fruits and Vegetables.-PhD. Thesis. Limburgs Universitair Centrum (LUC), Diepenbeek 2000.Google Scholar
  9. Clegg, R.M., Gadella, T.W.J., Jovin, T.M.: Lifetime-resolved fluorescence imaging.-Proc. SPIE 2137: 105-118, 1994.CrossRefGoogle Scholar
  10. Clegg, R.M., Schneider, P.C.: Fluorescence Lifetime-resolved Imaging Microscopy: A general description of the lifetime-resolved imaging measurements.-In: Slavik, J. (ed.): Fluorescence Microscopy and Fluorescent Probes. Pp. 15-33. Plenum Press, New York 1996.CrossRefGoogle Scholar
  11. Clegg, R.M., Schneider, P.C., Jovin, T.M.: Fluorescence Lifetime-resolved Imaging Microscopy.-In: Verga Scheggi, A.M., Martellucci, S., Chester, A.N., Pratesi, R. (ed.): Biomedical Optical Instrumentation and Laser-Assisted Biotechnology. Vol. 325. Pp. 143-156. Kluwer Academic Publ., Dordrecht-Boston-London 1996.CrossRefGoogle Scholar
  12. Crofts, A.R., Yerkes, C.T.: A molecular mechanism for qE-quenching. FEBS Lett. 352: 265-270, 1994.CrossRefPubMedGoogle Scholar
  13. Daley, P.F.: Chloropphyll fluorescence analysis and imaging in plant stress and disease.-Can. J. Plant Pathol. 17: 167-173, 1995.CrossRefGoogle Scholar
  14. Dau, H.: Molecular mechanisms and quantitative models of variable photosystem II fluorescence.-Photochem. Photobiol. 60: 1-23, 1994.CrossRefGoogle Scholar
  15. DeEll, J.R., Toivonen, P.M.A.: Chlorophyll fluorescence as a nondestructive indicator of broccoli quality during storage in modified-atmosphere packaging.-HortScience 35: 256-259, 2000.Google Scholar
  16. Demmig-Adams, B., Gilmore, A.M., Adams, W.W., III: In vivo function of carotenoids in higher plants.-FASEB J. 10: 403-412, 1996.PubMedGoogle Scholar
  17. Duschinsky, F.: Der zeitliche Intensitätsverlauf von intermittierend angeregter Resonanzstrahlung.-Z. Phys. 81: 7-22, 1933a.CrossRefGoogle Scholar
  18. Duschinsky, F.: Eine allgemeine Theorie der zur Messung sehr kurzer Leuchtdauern dienenden Versuchsanordnungen (Fluorometer); A general theory of the instruments for the measurement of very short after-glows (Fluorometer).-Z. Phys. 81: 23-42, 1933b.CrossRefGoogle Scholar
  19. Edwards, G.E., Furbank, R.T., Hatch, M.D., Osmond, C.B.: What does it take to be C4? Lessons from the evolution of C4 photosynthesis.-Plant Physiol. 125: 46-49, 2001.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Franck, F., Schoefs, B., Barthélemy, X., Myśliwa-Kurdziel, B., Strzałka, K., Popovic, R.: Protection of native chlorophyll(ide) forms and of photosystem II against photodamage during early stages of chloroplast differentiation.-Acta Physiol. Plant. 17: 123-132, 1995.Google Scholar
  21. Frank, H.A., Bautista, J.A., Josue, J.S., Young, A.J.: Mechanism of nonphotochemical quenching in green plants: Energies of the lowest excited singlet states of violaxanthin and zeaxanthin.-Biochemistry 39: 2831-2837, 2000.CrossRefPubMedGoogle Scholar
  22. French, T.: The Development of Fluorescence Lifetime Imaging and an Application in Immunology.-PhD. Thesis. University of Illinois at Urbana-Champaign, Urbana 1996.Google Scholar
  23. Gadella, T.W.J., Jr.: Fluorescence Lifetime Imaging Microscopy (FLIM): Instrumentation and applications.-In: Mason, W.T. (ed.): Fluorescent and Luminescent Probes for Biological Activity. Pp. 467-479. Academic Press, San Diego 1999.CrossRefGoogle Scholar
  24. Gadella, T.W.J., Jr., Clegg, R.M., Jovin, T.M.: Fluorescence lifetime imaging microscopy: pixel-by-pixel analysis of phase-modulation data.-Bioimaging 2: 139-159, 1994.CrossRefGoogle Scholar
  25. Gadella, T.W.J., Jr., Jovin, T.M., Clegg, R.M.: Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale.-Biophys. Chem. 48: 221-239, 1993.CrossRefGoogle Scholar
  26. Genty, B., Meyer, S.: Quantitative mapping of leaf photosynthesis using chlorophyll fluorescence imaging.-Aust. J. Plant Physiol. 22: 277-284, 1995.CrossRefGoogle Scholar
  27. Gilmore, A.M., Govindjee: How higher plants respond to excess light: Energy dissipation in photosystem II.-In: Singhal, G.S., Renger, G., Sopory, S., Irrgang, K.D., Govindjee (ed.): Concepts in Photobiology. Pp. 513-548. Narosa Publ. House, Delhi-Madras-Bombay-Calcuta-London; Kluwer Academic Publ., Boston-Dordrecht-London 1999.CrossRefGoogle Scholar
  28. Gilmore, A.M., Hazlett, T.L., Debrunner, P.G., Govindjee: Comparative time-resolved photosystem II chlorophyll a fluorescence analyses reveal distinctive differences between photoinhibitory reaction center damage and xanthophyll cycle-dependent energy dissipation.-Photochem. Photobiol. 64: 552-563, 1996a.CrossRefPubMedGoogle Scholar
  29. Gilmore, A.M., Hazlett, T.L., Debrunner, P.G., Govindjee: Photosystem II chlorophyll a fluorescence lifetimes and intensity are independent of the antenna size differences between barley wild-type and chlorina mutants. Photochemical quenching and xanthophyll cycle-dependent nonphotochemical quenching of fluorescence.-Photosynth. Res. 48: 171-187, 1996b.CrossRefPubMedGoogle Scholar
  30. Gilmore, A.M., Hazlett, T.L., Govindjee: Xanthophyll cycle-dependent quenching of photosystem II chlorophyll a fluorescence: Formation of a quenching complex with a short fluorescence lifetime.-Proc. nat. Acad. Sci. USA 92: 2273-2277, 1995.CrossRefPubMedPubMedCentralGoogle Scholar
  31. Gilmore, A.M., Itoh, S., Govindjee: Global spectral-kinetic analysis of room temperature chlorophyll a fluorescence from light-harvesting antenna mutants of barley.-Phil. Trans. roy. Soc. London B 355: 1371-1384, 2000.CrossRefGoogle Scholar
  32. Gilmore, A.M., Shinkarev, V.P., Hazlett, T.L., Govindjee: Quantitative analysis of the effects of intrathylakoid pH and xanthophyll cycle pigments on chlorophyll a fluorescence lifetime distributions and intensity in thylakoids.-Biochemistry 37: 13582-13593, 1998.CrossRefPubMedGoogle Scholar
  33. Govindjee: Sixty-three years since Kautsky: Chlorophyll a fluorescence.-Aust. J. Plant Physiol. 22: 131-160, 1995.CrossRefGoogle Scholar
  34. Govindjee, Amesz, J., Fork, D.C. (ed.): Light Emission by Plants and Bacteria.-Academic Press, Orlando-San Diego-New York-Austin-Boston-London-Sydney-Tokyo-Toronto 1986.Google Scholar
  35. Harris, E.H.: The Chlamydomonas Sourcebook.-Academic Press, San Diego-New York-Berkeley-Boston-London-Sydney-Tokyo-Toronto 1989.Google Scholar
  36. Hartel, H., Lokstein, H., Grimm, B., Rank, B.: Kinetic studies on the xanthophyll cycle in barley leaves. Influence of antenna size and relations to nonphotochemical chlorophyll fluorescence quenching.-Plant Physiol. 110: 471-482, 1996.CrossRefPubMedPubMedCentralGoogle Scholar
  37. Holzwarth, A.R.: Excited-state kinetics in chlorophyll systems and its relationship to the functional organization of the photosystems.-In: Scheer, H. (ed.): Chlorophylls. Pp. 1125-1151. CRC Press, Boca Raton-Ann Arbor-Boston-London 1991.Google Scholar
  38. Horváth, G., Droppa, M., Mustárdy, L., Faludi-Dániel, Á.: Functional characteristics of intact chloroplasts isolated from mesophyll protoplasts and bundle sheath cells of maize.-Planta 141: 239-244, 1978.CrossRefPubMedGoogle Scholar
  39. Jahns, P., Depka, B., Trebst, A.: Xanthophyll cycle mutants from Chlamydomonas reinhardtii indicate a role for zeaxanthin in the D1 protein turnover.-Plant Physiol. Biochem. 38: 371-376, 2000.CrossRefGoogle Scholar
  40. Jalink, H., van der Schoor, R., Frandas, A., van Pijlen, J.G., Bino, R.J.: Chlorophyll fluorescence of Brassica oleracea seeds as a non-destructive marker for seed maturity and seed performance.-Seed Sci. Res. 8: 437-443, 1998.CrossRefGoogle Scholar
  41. Jameson, D.M.: The Seminal Contributions of Gregorio Weber to Modern Fluorescence Spectroscopy. Methods and Applications of Fluorescence Spectroscopy.-Springer-Verlag, Heidelberg 2001.Google Scholar
  42. Jameson, D.M., Gratton, E., Hall, R.D.: The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry.-Appl. Spectrosc. Rev. 20: 55-106, 1984.CrossRefGoogle Scholar
  43. Karukstis, K.K.: Chlorophyll fluorescence as a physiological probe of the photosynthetic apparatus.-In: Scheer, H. (ed.): Chlorophylls. Pp. 769-795. CRC Press, Boca Raton-Ann Arbor-Boston-London 1991.Google Scholar
  44. Kautsky, H., Hirsch, A.: Neue Versuche zur Kohlensäure-assimilation.-Naturwissenschaften 19: 964, 1931.CrossRefGoogle Scholar
  45. König, K., Boehme, S., Leclerc, N., Ahuja, R.: Time-gated autofluorescence microscopy of motile green microalga in an optical trap.-Cell. mol. Biol. 44: 763-770, 1998.PubMedGoogle Scholar
  46. Kramer, D.M., Crofts, A.R.: Control and measurement of photosynthetic electron transport in vivo.-In: Baker, N.R. (ed.): Photosynthesis and the Environment. Pp. 25-66. Kluwer Academic Publ., Dordrecht-Boston-London 1996.Google Scholar
  47. Lavorel, J., Breton, J., Lutz, M.: Methodological principles of measurement of light emitted by photosynthetic systems.-In: Govindjee, Amesz, J., Fork, D.C. (ed.): Light Emission by Plants and Bacteria. Pp. 57-98. Academic Press, Orlando-San Diego-New York-Austin-Boston-London-Sydney-Tokyo-Toronto 1986.CrossRefGoogle Scholar
  48. Lazár, D.: Chlorophyll a fluorescence induction.-Biochim. biophysica Acta 1412: 1-28, 1999.CrossRefGoogle Scholar
  49. Li, X.P., Björkman, O., Shih, C., Grossman, A.R., Rosenquist, M., Jansson, S., Niyogi, K.K.: A pigment-binding protein essential for regulation of photosynthetic light harvesting.-Nature 403: 391-395, 2000.CrossRefPubMedGoogle Scholar
  50. Malkin, S., Kok, B.: Fluorescence induction studies in isolated chloroplast. I. Number of components involved in the reaction and quantum yields.-Biochim. biophys. Acta 126: 413-432. 1966.CrossRefPubMedGoogle Scholar
  51. Mazza, C.A., Boccalandro, H.E., Giordano, C.V., Battista, D., Scopel, A.L., Ballare, C.L.: Functional significance and induction by solar radiation of ultraviolet-absorbing sunscreens in field-grown soybean crops.-Plant Physiol. 122: 117-125, 2000.CrossRefPubMedPubMedCentralGoogle Scholar
  52. Morales, F., Belkhodja, R., Goulas, Y., Abadia, J., Moya, I.: Remote and near-contact chlorophyll fluorescence during photosynthetic induction in iron-deficient sugar beet leaves.-Remote Sens. Environ. 69: 170-178, 1999.CrossRefGoogle Scholar
  53. Moya, I., Sebban, P., Haehnel, W.: Lifetime of excited states and quantum yield of chlorophyll a fluorescence in vivo.-In: Govindjee, Amesz, J., Fork, D.C. (ed.): Light Emission by Plants and Bacteria. Pp. 161-190. Academic Press, Orlando-San Diego-New York-Austin-Boston-London-Sydney-Tokyo-Toronto 1986.CrossRefGoogle Scholar
  54. Murata, N., Nishimura, M., Takamiya, A.: Fluorescence of chlorophyll in photosynthetic systems. II. Induction of fluorescence in isolated spinach chloroplasts.-Biochim. biophys. Acta 120: 23-33, 1966.CrossRefPubMedGoogle Scholar
  55. Niyogi, K.K.: Photoprotection revisited: Genetic and molecular approaches.-Annu. Rev. Plant Physiol. Plant mol. Biol. 50: 333-359, 1999.CrossRefPubMedGoogle Scholar
  56. Niyogi, K.K., Björkman, O., Grossman, A.R.: Chlamydomonas xanthophyll cycle mutants identified by video imaging of chlorophyll fluorescence quenching.-Plant Cell 9: 1369-1380, 1997.CrossRefPubMedPubMedCentralGoogle Scholar
  57. Oxborough, K., Baker, N.R.: An instrument capable of imaging chlorophyll a fluorescence from intact leaves at very low irradiance and at cellular and subcellular levels of organization.-Plant Cell Environ. 20: 1473-1483, 1997.CrossRefGoogle Scholar
  58. Papageorgiou, G.C.: The photosynthesis of cyanobacteria (blue bacteria) from the perspective of signal analysis of chlorophyll a fluorescence.-J. sci. ind. Res. 55: 596-617, 1996.Google Scholar
  59. Samson, G., Prášil, O., Yaakoubd, B.: Photochemical and thermal phases of chlorophyll a fluorescence.-Photosynthetica 37: 163-182, 1999.CrossRefGoogle Scholar
  60. Sanders, R., Van Zandvoort, M.A.M.J., Draaijer, A., Levine, Y.K., Gerritsen, H.C.: Confocal fluorescence lifetime imaging of chlorophyll molecules in polymer matrices.-Photochem. Photobiol. 64: 817-820, 1996.CrossRefGoogle Scholar
  61. Schneider, P.C., Clegg, R.M.: Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications.-Rev. sci. Instrum. 68: 4107-4119, 1997.CrossRefGoogle Scholar
  62. Scholes, J.D., Rolfe, S.A.: Photosynthesis in localised regions of oat leaves infected with crown rust (Puccinia coronata). Quantitative imaging of chlorophyll fluorescence.-Planta 199: 573-582, 1996.CrossRefGoogle Scholar
  63. Spencer, R.D., Weber, G.: Measurement of subnanosecond fluorescence lifetimes with a cross-correlation phase fluorometer.-Ann. New York Acad. Sci. 158: 361-376, 1969.CrossRefGoogle Scholar
  64. Stirbet, A., Govindjee, Strasser, B.J., Strasser, R.J.: Chlorophyll a fluorescence induction in higher plants: Modelling and numerical simulation.-J. theor. Biol. 193: 131-151, 1998.CrossRefGoogle Scholar
  65. Verveer, P.J., Squire, A., Bastiaens, P.I.H.: Global analysis of fluorescence lifetime imaging microscopy data.-Biophys. J. 78: 2127-2137, 2000.CrossRefPubMedPubMedCentralGoogle Scholar
  66. Wang, X.F., Periasamy, A., Herman, B., Coleman, D.M.: Fluorescence lifetime imaging microscopy (FLIM): instrumentation and applications.-Crit. Rev. anal. Chem. 23: 369-395, 1992.CrossRefGoogle Scholar
  67. Weber, G.: Resolution of the fluorescence lifetimes in a heterogeneous system by phase and modulation measurements.-J. phys. Chem. 85: 949-953, 1981.CrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • O. Holub
    • 1
  • M.J. Seufferheld
    • 2
  • C. Gohlke
    • 1
  • Govindjee
    • 2
  • R.M. Clegg
    • 3
  1. 1.Department of Physics, Laboratory for Fluorescence DynamicsUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  2. 2.Department of Plant Biology, 265 Morrill HallUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  3. 3.Department of Physics, Laboratory for Fluorescence DynamicsUniversity of Illinois at Urbana-ChampaignUrbanaUSA

Personalised recommendations