Photosynthetica 2000, 38(4):483-491 | DOI: 10.1023/A:1012440903014

Imaging of the Blue, Green, and Red Fluorescence Emission of Plants: An Overview

C. Buschmann1, G. Langsdorf2, H.K. Lichtenthaler2
1 Botanical Institute, University of Karlsruhe, Karlsruhe, Germany
2 Botanical Institute, University of Karlsruhe, Karlsruhe, Germany

An overview is given on the fluorescence imaging of plants. Emphasis is laid upon multispectral fluorescence imaging in the maxima of the fluorescence emission bands of leaves, i.e., in the blue (440 nm), green (520 nm), red (690 nm), and far-red (740 nm) spectral regions. Details on the origin of these four fluorescence bands are presented including emitting substances and emitting sites within a leaf tissue. Blue-green fluorescence derives from ferulic acids covalently bound to cell walls, and the red and far-red fluorescence comes from chlorophyll (Chl) a in the chloroplasts of green mesophyll cells. The fluorescence intensities are influenced (1) by changes in the concentration of the emitting substances, (2) by the internal optics of leaves determining the penetration of excitation radiation and partial re-absorption of the emitted fluorescence, and (3) by the energy distribution between photosynthesis, heat production, and emission of Chl fluorescence. The set-up of the Karlsruhe multispectral fluorescence imaging system (FIS) is described from excitation with UV-pulses to the detection with an intensified CCD-camera. The possibilities of image processing (e.g., formation of fluorescence ratio images) are presented, and the ways of extraction of physiological and stress information from the ratio images are outlined. Examples for the interpretation of fluorescence images are given by demonstrating the information available for the detection of different developmental stages of plant material, of strain and stress of plants, and of herbicide treatment. This novel technique can be applied for near-distance screening or remote sensing.

Additional key words: photosynthetic activity; quality control; remote sensing; strain; stress detection

Prepublished online: August 1, 2000; Published: November 1, 2000  Show citation

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Buschmann, C., Langsdorf, G., & Lichtenthaler, H.K. (2000). Imaging of the Blue, Green, and Red Fluorescence Emission of Plants: An Overview. Photosynthetica38(4), 483-491. doi: 10.1023/A:1012440903014
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References

  1. Babani, F., Lichtenthaler, H.K.: Light-induced and age-dependent development of chloroplasts in etiolated barley leaves as visualised 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. Balota, M., Sowinska, M., Buschmann, C., Lichtenthaler, H.K., Heisel, F., Babani, F.: Fluorescence techniques as suitable methods to discriminate wheat genotypes under drought and high temperature condition.-SPIE 3707: 103-113, 1999. Go to original source...
  3. Björn, L.O., Forsberg, A.S.: Imaging by delayed light emission (phytoluminography) as a method for detecting damage to the photosynthetic system.-Physiol. Plant. 47: 215-222, 1979. Go to original source...
  4. Blaich, R., Bachmann, O., Baumberger, I.: Studies of photosynthesis inhibition by phytoluminography.-Z. Naturforsch. 37c: 452-457, 1982. Go to original source...
  5. Buschmann, C.: Induction kinetics of heat emission before and after photoinhibition in cotyledons of Raphanus sativus.-Photosynth. Res. 14: 229-240, 1987. Go to original source...
  6. 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...
  7. Buschmann, C., Schrey, H.: Fluorescence induction kinetics of green and etiolated leaves by recording the complete in-vivo emission spectra.-Photosynth. Res. 1: 233-241, 1981. Go to original source...
  8. Cerovic, Z.G., Samson, G., Morales, F., Tremblay, N., Moya, I.: Ultraviolet-induced fluorescence for plant monitoring: present state and prospects.-Agronomie: Agr. Environ. 19: 543-578, 1999. Go to original source...
  9. Chappelle, E.W., Wood, F.M., McMurtrey, J.E., Newcourt, W.W.: Laser-induced fluorescence of green plants. 3: LIF spectral signatures of five major plant types.-Appl. Optics 24: 74-80, 1985. Go to original source...
  10. Daley, P.F., Raschke, K., Ball, J.T., Berry, J.A.: Topography of photosynthetic activity of leaves obtained from video imaging of chlorophyll fluorescence.-Plant Physiol. 90: 1233-1238, 1989. Go to original source...
  11. D'Ambrosio, N., Szabo, K., Lichtenthaler, H.K.: Increase of the chlorophyll fluorescence ratio F690/F735 during the autumnal chlorophyll breakdown.-Radiat. environ. Biophys. 31: 51-62, 1992. Go to original source...
  12. Demmig-Adams, B., Adams, W.W., III: Photoprotection and other responses of plants to high light stress.-Annu. Rev. Plant Physiol. Plant mol. Biol. 43: 599-626, 1992. Go to original source...
  13. Edner, H., Johansson, J., Svanberg, S., Lichtenthaler, H.K., Lang, M., Stober, F., Schindler, C., Björn, L.-O.: Remote multi-colour fluorescence imaging of selected broad-leaf plants.-EARSel Adv. remote Sens. 3: 2-14, 1995.
  14. Edner, H., Johansson, J., Svanberg, S., Wallinder, E.: Fluorescence lidar multicolour imaging of vegetation.-Appl. Optics 33: 2471-2478, 1994. Go to original source...
  15. Ellenson, J.L., Amundson, R.G.: Delayed light imaging for the early detection of plant stress.-Science 215: 1104-1106, 1982. Go to original source...
  16. Fry, S.C.: Phenolic compounds of the primary cell wall and their possible role in the hormonal regulation of growth.-Planta 146: 343-351, 1979. Go to original source...
  17. Fry, S.C.: Phenolic components of the primary cell wall.-Biochem. J. 203: 493-504, 1982. Go to original source...
  18. Genty, B., Meyer, S.: Quantitative mapping of leaf photosynthesis using chlorophyll fluorescence imaging.-Aust. J. Plant Physiol. 22: 277-284, 1994. Go to original source...
  19. Gilroy, S.: Fluorescence microscopy of living plant cells.-Annu. Rev. Plant Physiol. Plant mol. Biol. 48: 165-190, 1997. Go to original source...
  20. Gitelson, A.A., Buschmann, C., Lichtenthaler, H.K.: Leaf chlorophyll fluorescence corrected for re-absorption by means of absorption and reflectance measurements.-J. Plant Physiol. 152: 283-296, 1998. Go to original source...
  21. Goodwin, R.H.: Fluorescent substances in plants.-Annu. Rev. Plant Physiol. 4: 283-304, 1953. Go to original source...
  22. Hák, 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...
  23. Harris, P.J., Hartley, R.D.: Detection of bound ferulic acid in cell walls of the gramineae by ultraviolet fluorescence microscopy.-Nature 259: 508-510, 1976. Go to original source...
  24. Hartley, R.D.: Carbohydrate esters of ferulic acid as components of cell walls of Lolium multiflorum.-Phytochemistry 12: 661-665, 1973. Go to original source...
  25. Heisel, F., Sowinska, M., Khalili, E., Eckert, C., Miehé, J.A., Lichtenthaler, H.K.: Laser-induced fluorescence imaging for monitoring nitrogen fertilizing treatments of wheat.-SPIE 3059: 10-21, 1997. Go to original source...
  26. Heisel, F., Sowinska, M., Miehé, J.A., Lang, M., Lichtenthaler, H.K.: Detection of nutrient deficiencies of maize by laser induced fluorescence imaging.-J. Plant Physiol. 148: 622-631, 1996. Go to original source...
  27. Holzwarth, A.R., Wendler, J., Haehnel, W.: Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in Chlorella vulgaris. Resolution of photosystem I fluorescence.-Biochim. biophys. Acta 807: 155-167, 1987. Go to original source...
  28. Johnson, G.A., Mantha, S.V., Day, T.A.: A spectrofluorometric survey of UV-induced blue-green fluorescence in foliage of 35 species.-J. Plant Physiol. 156: 242-252, 2000. Go to original source...
  29. Kocsányi, L., Haitz, M., Lichtenthaler, H.K.: Measurement of the laser-induced chlorophyll fluorescence kinetics using a fast acoustooptic device.-In: Lichtenthaler, H.K. (ed.): Applications of Chlorophyll Fluorescence. Pp. 99-107. Kluwer Academic Publ., Dordrecht-Boston-London 1988. Go to original source...
  30. Krause, G.H., Somersalo, S., Zumbusch, E., Weyers, B., Laasch, H.: On the mechanism of photoinhibition in chloroplasts. Relationship between changes in fluorescence and activity of photosystem II.-J. Plant Physiol. 136: 472-479, 1990. Go to original source...
  31. 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...
  32. Lang, M., Lichtenthaler, H.K., Sowinska, M., Heisel, F., Miehé, J.A.: Fluorescence imaging of water and temperature stress in plant leaves.-J. Plant Physiol. 148: 613-621, 1996. Go to original source...
  33. 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...
  34. Lang, M., Stober, F., Lichtenthaler, H.K.: Fluorescence emission spectra of plant leaves and plant constituents.-Radiat. environ. Biophys. 30: 333-347, 1991. Go to original source...
  35. Lichtenthaler, H.K.: The Kautsky effect: 60 years of chlorophyll fluorescence induction kinetics.-Photosynthetica 27: 45-55, 1992.
  36. Lichtenthaler, H.K., Babani, F.: Detection of photosynthetic activity and water stress by imaging the red chlorophyll fluorescence.-Plant Physiol. Biochem. 38: (in press), 2000. Go to original source...
  37. 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: 523-531, 2000. Go to original source...
  38. Lichtenthaler, H.K., Burkart, S., Schindler, C., Stober, F.: Changes in photosynthetic pigments and in vivo chlorophyll fluorescence parameters under photoinhibitory growth conditions.-Photosynthetica 27: 343-353, 1992b.
  39. Lichtenthaler, H.K., Buschmann, C., Rinderle, U., Schmuck, G.: Application of chlorophyll fluorescence in ecophysiology.-Radiat. environ. Biophys. 25: 297-308, 1986. Go to original source...
  40. Lichtenthaler, H.K., Hak, R., Rinderle, U.: The chlorophyll fluorescence ratio F690/F730 in leaves of different chlorophyll content.-Photosynth. Res. 25: 295-298, 1990. Go to original source...
  41. Lichtenthaler, H.K., Lang, M., Sowinska, M., Heisel, F., Miehé, 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...
  42. Lichtenthaler, H.K., Lang, M., Sowinska, M., Summ, P., Heisel, F., Miehé, J.A.: Uptake of the herbicide diuron (DCMU) as visualized by the fluorescence imaging technique.-Bot. Acta 110: 158-163, 1997. Go to original source...
  43. Lichtenthaler, H.K., Miehé, J.A.: Fluorescence imaging as a diagnostic tool for plant strees.-Trends Plant Sci. 2: 316-320, 1997. Go to original source...
  44. 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...
  45. 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...
  46. Lichtenthaler, H.K., Stober, F., Lang, M.: The nature of the different laser-induced fluorescence signatures of plants.-EARSeL Adv. remote Sens. 1: 20-32, 1992a.
  47. Morales, F., Cerovic, Z.G., Moya, I.: Time-resolved blue-green fluorescence of sugar beet (Beta vulgaris L.) leaves. Spectroscopic evidence for the presence of ferulic acid as the main fluorophore of the epidermis.-Biochim. biophys. Acta 1273: 251-262, 1996. Go to original source...
  48. Morales, F., Cerovic, Z.G., Moya, I.: Time-resolved blue-green fluorescence of sugar beet leaves. Temperature-induced changes and consequences for the potential use of blue-green fluorescence as a signature for remote sensing of plants.-Aust. J. Plant Physiol. 25: 325-334, 1998. Go to original source...
  49. Nilsson, H.-E.: Remote sensing and image analysis in plant pathology.-Annu. Rev. Phytopathol. 33: 489-527, 1995. Go to original source...
  50. Ning, L., Edwards, G.E., Strobel, G.A., Daley, L.S., Callis, J.B.: Imaging fluorometer to detect pathological and physiological change in plants.-Appl. Spectrosc. 49: 1381-1389, 1995. Go to original source...
  51. Ning, L., Petersen, B.E., Edwards, G.E., Daley, L.S., Callis, J.B.: Recovery of digital information stored in living plant leaf photosynthetic apparatus as fluorescence signals.-Appl. Spectrosc. 51: 1-9, 1997. Go to original source...
  52. Omasa, K.: Image instrumentation methods of plant analysis.-In: Linskens, H.-F., Jackson, J.F. (ed.): Modern Methods of Plant Analysis. Vol. 11. Pp. 203-243. Springer-Verlag, Berlin 1990. Go to original source...
  53. Omasa, K., Shimazaki, K.-I., Aiga, I., Larcher, W., Onoe, M.: Image analysis of chlorophyll fluorescence transients for diagnosing the photosynthetic system of attached leaves.-Plant Physiol. 84: 748-752, 1987. Go to original source...
  54. Osmond, C.B., Berry, J.A., Balachandran, S., Büchen-Osmond, C., Daley, P.F., Hodgson, R.A.J.: Potential consequences of virus infection for shade-sun acclimation in leaves.-Bot. Acta 103: 226-229, 1990. Go to original source...
  55. Osmond, C.B., Daley, P.F., Badger, M.R., Lüttge, U.: Chlorophyll fluorescence quenching during photosynthetic induction in leaves of Abutilon striatum Dicks. infected with abutilon mosaic virus, observed with a field-portable imaging system.-Bot. Acta 111: 390-397, 1998. Go to original source...
  56. Osmond, C.B., Kramer, D., Lüttge, U.: Reversible, water stress-induced non-uniform chlorophyll fluorescence quenching in wilting leaves of Potentilla reptans may not be due to patchy stomatal response.-Plant Biol. 1: 618-624, 1999a. Go to original source...
  57. Osmond, C.B., Schwartz, O., Gunning, B.: Photoinhibitory printing on leaves, visualised by chlorophyll fluorescence imaging and confocal microscopy, is due to diminished fluorescence from grana.-Aust. J. Plant Physiol. 26: 717-724, 1999b. Go to original source...
  58. Oxborough, K., Baker, N.R.: Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components - calculation of qP and Fv'/Fm' without measuring Fo'.-Photosynth. Res. 54: 135-142, 1997a. Go to original source...
  59. 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, 1997b. Go to original source...
  60. Pfündel, E.: Estimating the contribution of Photosystem I to total leaf chlorophyll fluorescence.-Photosynth. Res. 56: 185-195, 1998. Go to original source...
  61. Rost, F.W.D.: Fluorescence Microscopy.-Cambridge University Press, Cambridge 1995.
  62. Saito, Y., Hatake, K., Nomura, E., Kawahara, T.D., Nomura, A., Sugimoto, N., Itabe, T.: Range-resolved image detection of laser-induced fluorescence of natural trees for vegetation distribution monitoring.-Jap. J. appl. Phys. 36: 7024-7027, 1997a. Go to original source...
  63. Saito, Y., Kanoh, M., Hatake, K., Kawahara, T.D., Nomura, A.: Investigation of laser-induced fluorescence of several natural leaves for application to lidar vegetation monitoring.-Appl. Opt. 37: 431-437, 1998. Go to original source...
  64. Saito, Y., Saito, R., Kawahara, T.D., Nomura, A., Takeda, S.: Development and performance characteristics of laser-induced fluorescence imaging lidar for forestry applications.-Forest Ecol. Managem. 128: 129-137, 2000. Go to original source...
  65. Saito, Y., Saito, R., Nomura, A., Kawahara, T.D., Nomura, A., Takaragaki, S., Ida, K., Takeda, S.: Performance check of vegetation fluorescence imaging lidar through in vivo and remote estimation of chlorophyll concentration inside plant leaves.-Opt. Rev. 6: 155-159, 1999. Go to original source...
  66. Saito, Y., Takahashi, K., Nomura, E., Mineuchi, K., Kawahara, T.D., Nomura, A., Kobayashi, S., Ishii, H.: Visualization of laser-induced fluorescence of plants influenced by environmental stress with a microfluorescence imaging system and a fluorescence imaging lidar system.-SPIE 3059: 190-198, 1997b. Go to original source...
  67. Schindler, C., Lichtenthaler, H.K.: Is there a correlation between light-induced zeaxanthin accumulation and quenching of variable chlorophyll a fluorescence?-Plant Physiol. Biochem. 32: 813-823, 1994.
  68. Schweiger, J., Lang, M., Lichtenthaler, H.K.: Differences in fluorescence excitation spectra of leaves between stressed and non-stressed plants.-J. Plant Physiol. 148: 536-547, 1996. Go to original source...
  69. ©esták, Z.: Chlorophyll fluorescence kinetic depends on age of leaves and plants.-In: Argyroudi-Akoyunoglou, J.H., Senger, H. (ed.): The Chloroplast: From Molecular Biology to Biotechnology. Pp. 291-296. Kluwer Academic Publ., Dordrecht-Boston-London 1999. Go to original source...
  70. ©esták, Z., ©iffel, P.: Leaf-age related differences in chlorophyll fluorescence.-Photosynthetica 33: 347-369, 1997.
  71. Siebke, K., Weis, E.: Assimilation images of leaves of Glechoma hederacea: Analysis of non-synchronous stomata related oscillations.-Planta 196: 155-165, 1995a. Go to original source...
  72. Siebke, K., Weis, E.: Imaging of chlorophyll-a-fluorescence in leaves: Topography of photosynthetic oscillations in leaves of Glechoma hederacea.-Photosynth. Res. 45: 225-237, 1995b. Go to original source...
  73. ©iffel, P., ©esták, Z.: Low temperature fluorescence spectra of chloroplasts: methodical aspects and possible applications.-In: Lichtenthaler, H.K. (ed.): Applications of Chlorophyll Fluorescence. Pp. 55-61. Kluwer Academic Publ., Dordrecht-Boston-London 1988. Go to original source...
  74. Slavik, J. (ed.): Fluorescence Microscopy and Fluorescent Probes.-Plenum, New York 1996. Go to original source...
  75. Sowinska, M., Cunin, B., Deruyver, A., Heisel, F., Miehé, 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...
  76. Stober, F., Lang, M., Lichtenthaler, H.K.: Studies on the blue, green and red fluorescence signatures of green, etiolated and white leaves.-Remote Sens. Environ. 47: 65-71, 1994. Go to original source...
  77. Stober, F., Lichtenthaler, H.K.: Changes of the laser-induced blue, green and red fluorescence signatures during greening of etiolated leaves of wheat.-J. Plant Physiol. 140: 673-680, 1992. Go to original source...
  78. Stober, F., Lichtenthaler, H.K.: Characterization of the laser-induced blue, green and red fluorescence signatures of leaves of wheat and soybean grown under different irradiance.-Physiol. Plant. 88: 696-704, 1993a. Stober, F., Lichtenthaler, H.K.: Studies on the localization and spectral characteristics of the fluorescence emission of differ-ently pigmented wheat leaves.-Bot. Acta 106: 365-370, 1993b. Go to original source...
  79. Strasser, R.J., Butler, W.L.: Fluorescence emission spectra of Photosystem I, Photosystem II and the light-harvesting chlorophyll a/b complex of higher plants.-Biochim. biophys. Acta 462: 307-313, 1977. Go to original source...
  80. Sundbom, E., Björn, L.O.: Phytoluminography: imaging plants by delayed light emission.-Physiol. Plant. 40: 39-41, 1977. Go to original source...
  81. Valcke, R., Ciscato, M., Heisel, F., Miehé, J.-A., Sowinska, M.: Analysis of heavy metal stressed plants by fluorescence imaging.-SPIE 3707: 82-90, 1999. Go to original source...
  82. Wang, X.F., Herman, B.: Fluorescence Imaging Spectroscopy and Microscopy.-John Wiley &; Sons, New York 1996.