Photosynthetica 2005, 43(3):355-369 | DOI: 10.1007/s11099-005-0060-8

Chlorophyll fluorescence imaging of photosynthetic activity with the flash-lamp fluorescence imaging system

H. K. Lichtenthaler1,*, G. Langsdorf1, S. Lenk1, C. Buschmann1
1 Botanical Institute (Molecular Biology and Biochemistry of Plants), University of Karlsruhe, Karlsruhe, Germany

A flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) is described that allows to screen and image the photosynthetic activity of several thousand leaf points (pixels) of intact leaves in a non-destructive way within a few seconds. This includes also the registration of several thousand leaf point images of the four natural fluorescence bands of plants in the blue (440 nm) and green (520 nm) regions as well as the red (near 690 nm) and far-red (near 740 nm) Chl fluorescence. The latest components of this Karlsruhe FL-FIS are presented as well as its advantage as compared to the classical single leaf point measurements where only the fluorescence information of one leaf point is sensed per each measurement. Moreover, using the conventional He-Ne-laser induced two-wavelengths Chl fluorometer LITWaF, we demonstrated that the photosynthetic activity of leaves can be determined measuring the Chl fluorescence decrease ratio, RFd (defined as Chl fluorescence decrease Fd from maximum to steady state fluorescence Fs:Fd/Fs), that is determined by the Chl fluorescence induction kinetics (Kautsky effect). The height of the values of the Chl fluorescence decrease ratio RFd is linearly correlated to the net photosynthetic CO2 fixation rate PN as is indicated here for sun and shade leaves of various trees that considerably differ in their PN. Imaging the RFd-ratio of intact leaves permitted the detection of considerable gradients in photosynthetic capacity across the leaf area as well as the spatial heterogeneity and patchiness of photosynthetic quantum conversion within the control leaf and the stressed plants. The higher photosynthetic capacity of sun versus shade leaves was screened by Chl fluorescence imaging. Profile analysis of fluoresence signals (along a line across the leaf area) and histograms (the signal frequency distribution of the fluorescence information of all measured leaf pixels) of Chl fluorescence yield and Chl fluorescence ratios allow, with a high statistical significance, the quantification of the differences in photosynthetic activity between various areas of the leaf as well as between control leaves and water stressed leaves. The progressive uptake and transfer of the herbicide diuron via the petiole into the leaf of an intact plant and the concomitant loss of photosynthetic quantum conversion was followed with high precision by imaging the increase of the red Chl fluorescence F690. Differences in the availability and absorption of soil nitrogen of crop plants can be documented via this flash-lamp fluorescence imaging technique by imaging the blue/red ratio image F440/F690, whereas differences in Chl content are detected by collecting images of the fluorescence ratio red/far-red, F690/F740.

Additional key words: blue-green fluorescence; chlorophyll fluorescence decrease ratio; diuron absorption; flash light pulses; nitrogen supply; RFd-ratio; sun and shade leaves; water stress

Received: November 25, 2004; Accepted: April 26, 2005; Published: September 1, 2005  Show citation

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Lichtenthaler, H.K., Langsdorf, G., Lenk, S., & Buschmann, C. (2005). Chlorophyll fluorescence imaging of photosynthetic activity with the flash-lamp fluorescence imaging system. Photosynthetica, 43(3), 355-369. doi: 10.1007/s11099-005-0060-8
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    References

    1. Babani, F., Lichtenthaler, H.K.: Light-induced and age-dependent development of chloroplasts in etiolated barley leaves as visualized by determination of photosynthetic pigments, CO2 assimilation rates and different kinds of chlorophyll fluorescence ratios. - J. Plant Physiol. 148: 555-566, 1996. Go to original source...
    2. Baker, N.R., Oxborough, K., Lawson, T., Morison, J.I.L.: High resolution imaging of photosynthetic activities of tissues, cells and chloroplasts in leaves. - J. exp. Bot. 52: 615-621, 2001. Go to original source...
    3. Barbagallo, R.P., Oxborough, K., Pallett, K.E., Baker, N.R.: Rapid, non-invasive screening for perturbation of metabolism and plant growth using chlorophyll fluorescence imaging. - Plant Physiol. 132: 485-493, 2003. Go to original source...
    4. Boardman, N.K.: Comparative photosynthesis of sun and shade plants. - Annu. Rev. Plant Physiol. 28: 355-377, 1977. Go to original source...
    5. Bolhar-Nordenkampf, H.R., Oquist, G.: Chlorophyll fluorescence as a tool in photosynthesis research. - In: Hall, D.O., Scurlock, J.M.O., Bolhar-Nordenkampf, H.R., Leegood, R.C., Long, S.P. (ed.): Photosynthesis and Production in a Changing Environment: A Field and Laboratory Manual. Pp. 193-206. Chapman & Hall, London-Glasgow-New York-Melbourne-Madras 1993. Go to original source...
    6. Bro, E., Meyer, S., Genty, B.: Heterogeneity of leaf CO2 assimilation during photosynthetic induction. - In: Mathis, P. (ed.): Photosynthesis: from Light to Biosphere. Vol. V. Pp. 607-610. Kluwer Academic Publ., Dordrecht-Boston-London 1995. Go to original source...
    7. Buschmann, C., Langsdorf, G., Lichtenthaler, H.K.: Imaging of the blue, green and red fluorescence emission of plants. - Photosynthetica 38: 483-491, 2000. Go to original source...
    8. Buschmann, C., Lichtenthaler, H.K.: Principles and characteristics of multi-colour fluorescence imaging of plants.-J. Plant Physiol. 152: 297-314, 1998. Go to original source...
    9. Chaerle, L., van der Straeten, D.: Seeing is believing: imaging techniques to monitor plant health.-Biochim. biophys. Acta 1519: 153-166, 2001. Go to original source...
    10. D'Ambrosio, N., Szabo, K., Lichtenthaler, H.K.: Increase of the chlorophyll fluorescence ration F690/F735 during the autumnal chlorophyll breakdown. - Radiat. environ. Biophys. 31: 51-62, 1992. Go to original source...
    11. Edner, H., Johansson, J., Svanberg, S., Lichtenthaler, H.K., Lang, M., Stober, F., Schindler, C., Bjorn, L.-O.: Remote multi-colour fluorescence imaging of selected broad-leaf plants. - EARSel Adv. remote Sensing 3: 2-14, 1995.
    12. Genty, B., Briantais, J.-M., Baker, N.R.: The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. - Biochim. biophys. Acta 990: 87-92, 1989. Go to original source...
    13. Gitelson, A.A., Buschmann, C., Lichtenthaler, H.K.: Leaf chlorophyll fluorescence corrected for reabsorption by means of absorption and reflectance measurements. - J. Plant Physiol. 152: 283-296, 1998. Go to original source...
    14. Govindjee: Sixty three years since Kautsky: Chlorophyll a fluorescence. - Aust. J. Plant Physiol. 22: 131-160, 1995. Go to original source...
    15. Govindjee: Chlorophyll a fluorescence, a bit of basics and history. - In: Papageorgiou, G.C., Govindjee (ed.): Chlorophyll Fluorescence: a Signature of Photosynthesis. Pp. 1-42. Springer, Dordrecht 2004. Go to original source...
    16. Hak, R., Lichtenthaler, H.K., Rinderle, U.: Decrease of the chlorophyll fluorescence ratio F690/F730 during greening and development of leaves. - Radiat. environ. Biophys. 28: 329-336, 1990. Go to original source...
    17. Hill, R., Schreiber, U., Gademann, R., Larkum, A.W.D., Kuhl, M., Ralph, P.J.: Spatial heterogeneity of photosynthesis and the effect of temperature-induced bleaching conditions in three species of corals. - Mar. Biol. 144: 633-640, 2004. Go to original source...
    18. Holub, O., Seufferheld, M.J., Gohlke, C., Govindjee, Clegg. R.M.: Fluorescence lifetime imaging (FLI) in real-time-a new technique in photosynthesis research. - Photosynthetica 38: 581-599, 2000. Go to original source...
    19. Kautsky, H., Hirsch, A.: Neue Versuche zur Kohlenstoffassimilation. - Naturwissenschaften 10: 1964, 1931.
    20. Krause, G.H., Weis, E.: Chlorophyll fluorescence and photosynthesis: the basics. - Annu. Rev. Plant Physiol. Plant mol. Biol. 42: 313-349, 1991. Go to original source...
    21. Lang, M., Lichtenthaler, H.K., Sowinska, M., Summ, P., Heisel, F.: Blue, green and red fluorescence signatures and images of tobacco leaves. - Bot. Acta 107: 230-236, 1994. Go to original source...
    22. Lang, M., Siffel, P., Braunova, Z., Lichtenthaler, H.K.: Investigations of the blue-green fluorescence emission of plant leaves. - Bot. Acta 105: 435-440, 1992. Go to original source...
    23. Langsdorf, G., Buschmann, C., Sowinska, M., Babani, F., Mokry, M., Timmermann, F., Lichtenthaler, H.K.: Multicolour fluorescence imaging of sugar beet leaves with different nitrogen status by flash lamp UV-excitation.-Photosynthetica 38: 539-551, 2000. Go to original source...
    24. Lichtenthaler, H.K.: Chlorophylls and carotenoids-pigments of photosynthetic biomembranes. - In: Colowick, S.P., Kaplan, N.O. (ed.): Methods in Enzymology. Vol. 148. Pp. 350-382. Academic Press, San Diego-New York-Berkeley-Boston-London-Sydney-Tokyo-Toronto 1987. Go to original source...
    25. Lichtenthaler, H.K. (ed.): Applications of Chlorophyll Fluorescence. - Kluwer Acad. Publ., Dordrecht-Boston-London 1988.
    26. Lichtenthaler, H.K.: The Kautsky effect: 60 years of chlorophyll fluorescence induction kinetics.-Photosynthetica 27: 45-55, 1992.
    27. Lichtenthaler, H.K.: Vegetation stress: an introduction to the stress concept in plants. - J. Plant Physiol. 148: 4-14, 1996. Go to original source...
    28. Lichtenthaler, H.K., Babani, F.: Detection of photosynthetic activity and water stress by imaging the red chlorophyll fluorescence. - Plant Physiol. Biochem. 38: 889-895, 2000. Go to original source...
    29. Lichtenthaler, H.K., Babani, F.: Light adaptation and senescence of the photosynthetic apparatus. Changes in pigment composition, chlorophyll fluorescence parameters and photosynthetic activity. - In: Papageorgiou, G.C., Govindjee (ed.): Chlorophyll Fluorescence: a Signature of Photosynthesis. Pp. 713-736. Springer, Dordrecht 2004. Go to original source...
    30. Lichtenthaler, H.K., Babani, F., Langsdorf, G., Buschmann, C.: Measurement of differences in red chlorophyll fluorescence and photosynthetic activity between sun and shade leaves by fluorescence imaging. - Photosynthetica 38: 521-529, 2000. Go to original source...
    31. Lichtenthaler, H.K., Burkart, S.: Photosynthesis and high light stress. - Bulg. J. Plant Physiol. 25: 3-16, 1999.
    32. Lichtenthaler, H.K., Buschmann, C., Doll, M., Fietz, H.-J., Bach, T., Kozel, U., Meier, D., Rahmsdorf, U.: Photosynthetic activity, chloroplast ultrastructure, and leaf characteristics of high-light and low-light plants and of sun and shade leaves. - Photosynth. Res. 2: 115-141, 1981. Go to original source...
    33. Lichtenthaler, H.K., Buschmann, C., Knapp, M.: How to correctly determine chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio RFd of leaves with the PAM fluorometer. - Photosynthetica 43: 379-393, 2005. Go to original source...
    34. Lichtenthaler, H.K., Kuhn, G., Prenzel, U., Meier, D.: Chlorophyll-protein levels and stacking degree of thylakoid stacking in radish chloroplasts from high-light, low-light and bentazon-treated plants. - Physiol. Plant. 56: 183-188, 1982. Go to original source...
    35. Lichtenthaler, H.K., Lang, M., Sowinska, M., Heisel, F., Miehe, J.A.: Detection of vegetation stress via a new high resolution fluorescence imaging system. - J. Plant Physiol. 148: 599-612, 1996. Go to original source...
    36. Lichtenthaler, H.K., Lang, M., Sowinska, M., Summ, P., Heisel, F., Miehe, J.A.: Uptake of th e herbicide diuron (DCMU) as visualized by the fluorescence imaging technique. - Bot. Acta 110: 158-163, 1997. Go to original source...
    37. Lichtenthaler, H.K., Miehe, J.A.: Fluorescence imaging as a diagnostic tool for plant stress. - Trends Plant Sci. 2: 316-320, 1997. Go to original source...
    38. Lichtenthaler, H.K., Rinderle U.: The role of chlorophyll fluorescence in the detection of stress conditions in plants. - CRC crit. Rev. anal. Chem. 19: S29-S85, 1988. Go to original source...
    39. Lichtenthaler, H.K., Schweiger, J.: Cell wall bound ferulic acid, the major substance of the blue-green fluorescence emission of plants. - J. Plant Physiol. 152: 272-282, 1998. Go to original source...
    40. Meier, D., Lichtenthaler, H.K.: Ultrastructural development of chloroplasts in radish seedlings grown at high and low light conditions and in the presence of the herbicide bentazon. - Protoplasma 107: 195-207, 1981. Go to original source...
    41. Nedbal, L., Soukupova, J., Kaftan, D., Whitmarsh, J., Trtilek, M.: Kinetic imaging of chlorophyll fluorescence using modulated light. - Photosynth. Res. 66: 3-12, 2000. Go to original source...
    42. Nedbal, L., Whitmarsh, J.: Chlorophyll fluorescence imaging of leaves and fruits. - In: Papageorgiou, G., Govindjee (ed.): Chlorophyll Fluorescence, a Signature of Photosynthesis. Pp. 389-407. Springer, Dordrecht 2004. Go to original source...
    43. Osmond, C.B., Kramer, D., Luttge, U.: Reversible, water stress-induced non-uniform chlorophyll fluorescence quenching in wilting leaves of Potentilla reptans may not be due to patchy stomatal responses. - Plant Biol. 1: 618-624, 1999. Go to original source...
    44. Oxborough, K.: Imaging of chlorophyll a fluorescence: theoretical and practical aspects of an emerging technique for the monitoring of photosynthetic performance. - J. exp. Bot. 55: 1195-1205, 2004. Go to original source...
    45. Papageorgiou, G.C., Govindjee (ed.): Chlorophyll a Fluorescence, a Signature of Photosynthesis.-Springer, Dordrecht 2004. Go to original source...
    46. Santrucek, J., Siffel, P., Lang, M., Lichtenthaler, H.K., Schindler, C., Synkova, H., Konecna, V., Szabo, K.: Photosynthetic activity and chlorophyll fluorescence parameters in aurea and green forms of Nicotiana tabacum. - Photosynthetica 27: 529-543, 1992.
    47. Schindler, C., Lichtenthaler, H.K.: Photosynthetic CO2 assimilation, chlorophyll fluorescence and zeaxanthin accumulation in field grown maple trees in the course of a sunny and a cloudy day. - J. Plant Physiol. 148: 399-412, 1996. Go to original source...
    48. Schreiber, U., Schliwa, U., Bilger, W.: Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorimeter. - Photosynth. Res. 10: 51-62, 1986. Go to original source...
    49. Siffel, P., Santrucek, J., Lang, M., Braunova, Z., Simkova, M., Synkova, H., Lichtenthaler, H.K.: Age dependence of photosynthetic activity, chlorophyll fluorescence parameters and chloroplast ultrastructure in aurea and green forms of Nicotiana tabacum Su/su mutant. - Photosynthetica 29: 81-94, 1993.
    50. Sowinska, M., Cunin, B., Deruyver, A., Heisel, F., Miehe, J.-A., Langsdorf, G., Lichtenthaler, H.K: Near-field measurements of vegetation by laser-induced fluorescence imaging. - SPIE 3868: 120-131, 1999. Go to original source...
    51. Stober, F., Lang, M., Lichtenthaler, H.K.: Blue, green and red fluorescence emission signatures of green, etiolated, and white leaves. - Remote Sens. Environ. 47: 65-71, 1994. Go to original source...
    52. Stober, F., Lichtenthaler, H.K.: Studies on the constancy of the blue and green fluorescence yield during the chlorophyll fluorescence induction kinetics (Kautsky effect). - Radiat. environ. Biophys. 32: 357-365, 1993. Go to original source...
    53. Strasser, R.J., Srivastava, A., Tsimilli-Michael, M.: The fluorescence transient as a tool to characterize and screen photosynthetic samples. - In: Yunus, M., Pathre, U., Mohanty, P., (ed.): Probing Photosynthesis: Mechanism, Regulation and Adaptation. Pp. 445-483. Taylor & Francis, London 1998.
    54. Szabo, K., Lichtenthaler, H.K., Kocsanyi, L., Richter, P.: A CCD-OMA device for the measurement of complete chlorophyll fluorescence emission spectra of leaves during the fluorescence induction kinetics. - Radiat. environ. Biophys. 31: 153-160, 1992. Go to original source...
    55. Tuba, Z., Lichtenthaler, H.K., Csintalan, Z., Nagy, Z., Szente, K.: Reconstitution of chlorophylls and photosynthetic CO2 assimilation upon rehydration of the desiccated poikilo-chlorophyllous plant Xerophyta scabrida (Pax) Th. Dur. et Schinz. - Planta 192: 414-420, 1994. Go to original source...

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