Photosynthesis Research

, Volume 88, Issue 3, pp 343–350 | Cite as

Applying Pulse Amplitude Modulation (PAM) fluorometry to microalgae suspensions: stirring potentially impacts fluorescence

  • Jeffrey Cosgrove
  • Michael Borowitzka
Technical communication


The use of microalgae suspensions in PAM-fluorometers such as the Water-PAM (Walz GmbH, Germany) presents the problem of maintaining a homogeneous sample. The Water-PAM is marketed with an optional accessory for stirring the sample within the cuvette while in the emitter–detector (ED) unit. This stirring device can help to prevent cells from settling out of suspension over the time-course of chlorophyll-a fluorescence measurements. The ED unit was found to provide a vertically heterogeneous light environment and, therefore, cells within a single sample can exist in different quenched states. Enhancing cell movement by stirring was found to substantially influence measured fluorescence yield while performing induction curve and rapid light curve analyses. This is likely to result from relatively unquenched cells outside the main light-path moving into a higher light region and thus emitting disproportionately more fluorescence than quenched cells. Samples containing cells with high sinking rates or motile species may encounter similar (but reduced) problems. This effect can be mitigated by: (a) reducing analysis time to minimise the distance cells can sink/swim during the measurement procedure and avoiding the necessity of stirring; (b) limiting the proportion of sample outside the light path by minimising sample volume or; (c) by activating the stirrer only for short periods between saturation pulses and allowing enough time after stirring for quenching to stabilise before activation of the saturation pulse. Alternatively, modifications to the instrument providing a vertical dimension to the LED-array could resolve the issue by providing a more homogeneous light environment for the sample.


Chl-a fluorescence Electron transport rate Methodology Non-photochemical quenching PAM Quantum yield 


ED unit

emitter–detector unit


fluorescence yield


maximum fluorescence yield in the dark-adapted state


maximum fluorescence yield in the light-adapted state

Fm m

maximum F′m value


maximum photochemical yield in the dark-adapted state


light-emitting diode


non-photochemical quenching


photosystem II


pulse amplitude modulation


relative electron transport rate


maximum relative electron transport rate


rapid light curve


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  1. Beach K, Walters L, Vroom P, Smith C, Coyer J, Hunter C (2003) Variability in the ecophysiology of Halimeda spp. (Chlorophyta, Bryopsidales) on Conch Reef, Florida Keys, USA. J Phycol 39:633–643CrossRefGoogle Scholar
  2. Beardall J, Young EB, Roberts S (2001) Approaches for determining phytoplankton nutrient limitation. Aquat Sci 63:44–69CrossRefGoogle Scholar
  3. Beer S, Axelsson L (2004) Limitations in the use of PAM fluorometry for measuring photosynthetic rates of macroalgae at high irradiances. Eur J Phycol 39:1–7CrossRefGoogle Scholar
  4. Beer S, Ilan M, Eshel A, Weil A, Brickner I (1998a) Use of pulse amplitude modulated (PAM) fluorometry for in situ measurements of photosynthesis in two Red Sea faviid corals. Mar Biol 131:607–612CrossRefGoogle Scholar
  5. Beer S, Vilkenkin B, Weil A, Veste M, Susel L, Eshel A (1998b) Measuring photosynthetic rates in seagrasses by pulse amplitude modulated (PAM) fluorometry. Mar Ecol Prog Ser 174:293–300CrossRefGoogle Scholar
  6. Bienfang PK (1980) Phytoplankton sinking rates in oligotrophic waters off Hawaii, USA. Mar Biol 61:69–77CrossRefGoogle Scholar
  7. Campbell S, Miller C, Steven A, Stephens A (2003) Photosynthetic responses of two temperate seagrasses across a water quality gradient using chlorophyll fluorescence. J Exp Mar Biol Ecol 291:57–78CrossRefGoogle Scholar
  8. Clegg MR, Maberly SC, Jones RI (2003) Behavioural responses of freshwater phytoplanktonic flagellates to a temperature gradient. Eur J Phycol 38:195–203CrossRefGoogle Scholar
  9. Durako M, Kunzelman J, Kenworthy W, Hammerstrom K (2003) Depth-related variability in the photobiology of two populations of Halophila johnsonii and Halophila decipiens. Mar Biol 142:1219–1228Google Scholar
  10. Falkowski P, Wyman K, Ley A, Mauzerall D (1986) Relationship of steady state photosynthesis to fluorescence in eukaryotic algae. Biochim et Biophys Acta 849:183–192CrossRefGoogle Scholar
  11. Gilbert M, Domin A, Becker A, Wilhelm C (2000) Estimation of primary productivity by chlorophyll a in vivo fluorescence in freshwater phytoplankton. Photosynthetica 38:111–126CrossRefGoogle Scholar
  12. Guillard RRL, Ryther JH (1962) Studies on marine diatoms. 1 Cyclotella nana Husted and Detonula confervacea Gran. Can J Microbiol 8:229–239PubMedCrossRefGoogle Scholar
  13. Häder D, Lebert M, Figueroa F, Jiménez C, Viñegla B, Perez-Rodriguez E (1998) Photoinhibition in Mediterranean macroalgae by solar radiation measured on site by PAM fluorescence. Aquat Bot 61:225–236CrossRefGoogle Scholar
  14. Hill R, Ralph PJ (2005) Diel and seasonal changes in fluorescence rise kinetics of three scleractinian corals. Funct Plant Biol 32:549–559CrossRefGoogle Scholar
  15. Hill R, Schreiber U, Gademann R, Larkum AWD, Kühl M, Ralph PJ (2004) Spatial heterogeneity of photosynthesis and the effect of temperature-induced bleaching conditions in three species of corals. Mar Biol 144:633–640CrossRefGoogle Scholar
  16. Jakob T, Schreiber U, Kirchesch 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 83:343–361PubMedCrossRefGoogle Scholar
  17. Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations for determining chlorophylls a, b, c 1, and c 2 in higher plants, algae, and natural phytoplankton. Biochem Physiol Pflanz 167:191–194Google Scholar
  18. Kromkamp J, Peene J (1999) Estimation of phytoplankton photosynthesis and nutrient limitation in the Eastern Scheldt estuary using variable fluorescence. Aquat Ecol 33:101–104CrossRefGoogle Scholar
  19. Lazár D (1999) Chlorophyll a fluorescence induction. Biochim et Biophys Acta 1412:1–28CrossRefGoogle Scholar
  20. Lazár Da, Ilík P, Kruk J, Strzalka K, Naus J (2005) A theoretical study on effect of the initial redox state of cytochrome b 559 on maximal chlorophyll fluorescence level (FM): implications for photoinhibition of photosystem II. J Theor Biol 233:287–300CrossRefGoogle Scholar
  21. McMinn A, Hegseth E (2004) Quantum yield and photosynthetic parameters of marine microalgae from the southern Arctic Ocean, Svalbard. J Mar Biol Assoc UK 84:865–871CrossRefGoogle Scholar
  22. Platt T, Gallegos C, Harrison W (1980) Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J Mar Res 38:687–701Google Scholar
  23. Ralph PJ, Gademann R (2005) Rapid light curves: a powerful tool for the assessment of photosynthetic activity. Aquat Bot 82:222–237CrossRefGoogle Scholar
  24. Ralph PJ, Polk SM, Moore KA, Orth RJ, Smith Jr WO (2002) Operation of the xanthophyll cycle in the seagrass Zostera marina in response to variable irradiance. J Exp Mar Biol Ecol 271:189–207CrossRefGoogle Scholar
  25. Samson G, Prášil O, Yaakoubd B (1999) Photochemical and thermal phases of chlorophyll a fluorescence. Photosynthetica 37:163–182CrossRefGoogle Scholar
  26. Schäfer C, Björkman O (1989) Relationship between efficiency of photosynthetic energy conversion and chlorophyll fluorescence quenching in upland cotton (Gossypium hirsutum L.). Planta 178:367–376CrossRefGoogle Scholar
  27. Schreiber U, Gademann R, Ralph PJ, Larkum AWD (1997) Assessment of photosynthetic performance of Prochloron in Lissoclinum patella in hospite by chlorophyll fluorescence measurements. Plant Cell Physiol 38:945–951Google Scholar
  28. Schreiber U, Neubauer C, Schliwa U (1993) PAM fluorometer based on medium-frequency pulsed Xe-flash measuring light: a highly sensitive new tool in basic and applied photosynthesis research. Photosynth Res 36:65–72CrossRefGoogle Scholar
  29. Serôdio J, Cruz S, Vieira S, Brotas V (2005) Non-photochemical quenching of chlorophyll fluorescence and operation of the xanthophyll cycle in estuarine microphytobenthos. J Exp Mar Biol Ecol 326:157–169CrossRefGoogle Scholar
  30. Suggett D, Oxborough K, Baker N (2003) Fast repetition rate and pulse amplitude modulation chlorophyll a fluorescence measurements for assessment of photosynthetic electron transport in marine phytoplankton. Eur J Phycol 38:371–384CrossRefGoogle Scholar
  31. Villareal TA (2004) Single-cell pulse amplitude modulation fluorescence measurements of the giant diatom Ethmodiscus (Bacillariophyceae). J Phycol 40:1052–1061CrossRefGoogle Scholar
  32. White A, Critchley C (1999) Rapid light curves: a new fluorescence method to assess the state of the photosynthetic apparatus. Photosynth Res 59:63–72CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  1. 1.School of Biological Sciences and BiotechnologyMurdoch UniversityMurdochWestern Australia

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