Advertisement

Marine Biology

, Volume 158, Issue 7, pp 1667–1675 | Cite as

Rapid assessment of different oxygenic phototrophs and single-cell photosynthesis with multicolour variable chlorophyll fluorescence imaging

  • Erik Trampe
  • Jörg Kolbowski
  • Ulrich Schreiber
  • Michael Kühl
Method

Abstract

We present a new system for microscopic multicolour variable chlorophyll fluorescence imaging of aquatic phototrophs. The system is compact and portable and enables microscopic imaging of photosynthetic performance of individual cells and chloroplasts using different combinations of blue, green, red or white light. Automated sequential exposure of microscopic samples to the three excitation colours enables subsequent deconvolution of the resulting fluorescence signals and colour marking of cells with different photopigmentation, i.e., cyanobacteria, green algae, red algae and diatoms. The photosynthetic activity in complex mixtures of phototrophs and natural samples can thus be assigned to different types of phototrophs, which can be quantified simultaneously. Here, we describe the composition and performance of the new imaging system and present applications with both natural phytoplankton and microalgal culture samples.

Keywords

Fluorescence Yield Photosynthetic Performance Actinic Light Rapid Light Curf Rapid Light Curf 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

This study was supported by grants from the Danish Natural Science Research Council (MK) and the Carlsberg Foundation (MK). We acknowledge the assistance of Egil Nielsen for mechanical constructions and the staff at the Heron Island Research Station for technical assistance and support.

References

  1. Baker NR (2008) Chlorophyll fluorescence: a probe of photosynthesis in vivo. Ann Rev Plant Biol 59:89–113CrossRefGoogle Scholar
  2. Behrendt L, Trampe E, Larkum AWD, Qvortrup K, Norman A, Chen M, Ralph PJ, Sørensen SJ, Kühl M (2011) Endolithic chlorophyll d-containing phototrophs. ISME J (advance online publication 16 Dec 2010) doi: 10.1038/ismej.2010.195 CrossRefGoogle Scholar
  3. Berman-Frank I, Lungren P, Chen Y-B, Kuepper H, Kolber Z, Bergman B, Falkowski P (2001) Segregation of nitrogen fixation and oxygenic photosynthesis in the marine cyanobacterium Trichodesmium. Science 294:1534–1537CrossRefGoogle Scholar
  4. Beutler M, Wiltshire KH, Meyer B, Moldaenke C, Lüring C, Meyerhöfer M (2002) A fluorometric method for the differentiation of algael populations in vivo and in situ. Photosynth Res 72:39–53CrossRefGoogle Scholar
  5. Boulding EG, Platt TR (1986) Variation in photosynthetic rates among individual cells of a marine dinoflagellate. Mar Ecol Progr Ser 29:199–203CrossRefGoogle Scholar
  6. Dijkman NA, Kromkamp JC (2006) Photosynthetic characteristics of the phytoplankton in the Scheldt estuary: community and single-cell fluorescence measurements. Eu J Phycol 41:425–434CrossRefGoogle Scholar
  7. Gorbunov MY, Kolber ZS, Falkowski PG (1999) Measuring photosynthetic parameters in individual algal cells by fast repetition rate fluorometry. Photosynth Res 62:141–153CrossRefGoogle Scholar
  8. Jakob T, Schreiber U, Kirschesch V, Langner U, Wilhelm C (2005) Estimation of chlorophyll content and daily primary production of the major algal groups by means of multiwavelength-excitation PAM chlorophyll fluorometry: performance and methodological limits. Photosynth Res 83:343–361CrossRefGoogle Scholar
  9. Kolber Z, Prasil O, Falkowski PG (1998) Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols. Biochem Biophys Acta 1367:88–106PubMedGoogle Scholar
  10. Kolbowski J, Schreiber U (1995) Computer-controlled phytoplankton analyzer based on 4-wavelengths PAM chlorophyll fluorometer. In: Mathis P (ed) Photosynthesis: from light to biosphere, vol V. Kluwer Academic Publishers, Dordrecht, pp 825–828CrossRefGoogle Scholar
  11. Kromkamp JC, Forster RM (2003) The use of variable fluorescence measurements in aquatic ecosystems: differences between multiple and single turnover measuring protocols and suggested terminology. Europ J Phycol 38:103–112CrossRefGoogle Scholar
  12. Kühl M, Polerecky L (2008) Functional and structural imaging of phototrophic microbial commmunities and symbioses. Aq Microb Ecol 53:99–118CrossRefGoogle Scholar
  13. Kühl M, Glud RN, Borum J, Roberts R, Rysgaard S (2001) Photosynthetic performance of surface associated algae below sea ice as measured with a pulse amplitude modulated (PAM) fluorometer and O2 microsensors. Mar Ecol Progr Ser 223:1–14CrossRefGoogle Scholar
  14. Mitchell BG, Kiefer DA (1988) Variability in pigment specific particulate fluorescence and absorption spectra in the northeeastern Pacific Ocean. Deep-Sea Res 35:665–689CrossRefGoogle Scholar
  15. Morán XAG, Estrada M (2001) Short-term variability of photosynthetic parameters and particulate and dissolved primary production in the Alboran Sea (SW Mediterranean). Mar Ecol Progr Ser 212:53–67CrossRefGoogle Scholar
  16. Olson RJ, Sosik HM, Chekalyuk AM (1999) Photosynthetic characteristics of marine phytoplankton from pump-during-probe fluorometry of individual cells at sea. Cytometry 37:1–13CrossRefGoogle Scholar
  17. Oxborough K (2004) Imaging of chlorophyll a fluorescence: theoretical and practical aspects of an emerging technique for the monitoring of photosynthetic performance. J Exp Bot 55:1195–1205CrossRefGoogle Scholar
  18. Oxborough K, Hanlon ARM, Underwood GJC, Baker NR (2000) In vivo estimation of the photosystem II photochemical efficiency of individual microphytobenthic cells using high-resolution imaging of chlorophyll a fluorescence. Limnol Oceanogr 45:1420–1425CrossRefGoogle Scholar
  19. Papageorgiou GC, Govindjee (2004) Chlorophyll fluorescence: a signature of photosynthesis. Kluwer Academic Publishers, DordrechtGoogle Scholar
  20. Ralph PJ, Gademann R (2005) Rapid light curves: a powerful tool to assess photosynthetic activity. Aquat Bot 82:222–237CrossRefGoogle Scholar
  21. Ralph PJ, Larkum AWD, Kühl M (2005) Temporal patterns in zooxanthellae expulsion during bleaching conditions. J Exp Mar Biol Ecol 316:17–28CrossRefGoogle Scholar
  22. Schreiber U (2004) Pulse-amplitude-modulation (PAM) fluorometry and saturation pulse method: an overview. In: Papageorgiou GCG (ed) Chlorophyll fluorescence: a signature of photosynthesis. Kluwer, Dordrecht, pp 279–319CrossRefGoogle Scholar
  23. Šetlíková E, Šetlík I, Küpper H, Kasalický V, Prášil O (2005) The photosynthesis of individual algal cells during the cell cycle of Scenedesmus quadricauda studied by chlorophyll fluorescence kinetic microscopy. Photosynth Res 84:113–120CrossRefGoogle Scholar
  24. Snel JFH, Dassen HHA (2000) Measurement of light and pH dependence of single-cell photosynthesis by fluorescence microscopy. J Fluoresc 10:269–273CrossRefGoogle Scholar
  25. Suggett DJ, Oxborough K, Baker NR, MacIntyre HL, Kana TM, Geider RJ (2003) Fast repetition rate and pulse amplitude modulation chlorophyll a fluorescence measurements for assessment of photosynthesis electron transport in marine phytoplankton. Europ J Phycol 38:371–384CrossRefGoogle Scholar
  26. Thar R, Kühl M, Holst G (2001) A fiber-optic fluorometer for microscale mapping of photosynthetic pigments in microbial communities. Appl Environ Microbiol 67:2823–2828CrossRefGoogle Scholar
  27. Villareal TA (2004) Single-cell pulse amplitude modulation fluorescence measurements of the giant diatom Ethmodiscus (Bacillariophyceae). J Phycol 40:1052–1061CrossRefGoogle Scholar
  28. White AJ, Critchley C (1999) Rapid light curves: a new fluorescence method to assess the state of the photosynthetic apparatus. Phot Res 59:63–72CrossRefGoogle Scholar
  29. Xupeng HU, Rongguo SU, Zhang F, Wang X, Wang H, Zheng Z (2010) Multiple excitation wavelength fluorescence emission spectra technique for discrimination of phytoplankton. J Ocean Univ China (Ocean Coastal Sea Res) 9:16–24Google Scholar
  30. Yentsch CS, Phinney DA (1985) Spectral fluorescence: a taxonomic tool for studying the structure of phytoplankton populations. J Plankton Res 7:617–632CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Erik Trampe
    • 1
  • Jörg Kolbowski
    • 2
  • Ulrich Schreiber
    • 2
  • Michael Kühl
    • 1
    • 3
  1. 1.Marine Biological Section, Department of BiologyUniversity of CopenhagenHelsingørDenmark
  2. 2.Julius-von-Sachs Institut für Biowissenschaften, Lehrstuhl Botanik IUniversity of WürzburgWürzburgGermany
  3. 3.Plant Functional Biology and Climate Change ClusterUniversity of Technology SydneyUltimo SydneyAustralia

Personalised recommendations