Photosynthesis Research

, Volume 114, Issue 1, pp 59–68 | Cite as

An LED-based fluorometer for chlorophyll quantification in the laboratory and in the field

  • Jacob J. Lamb
  • Julian J. Eaton-Rye
  • Martin F. Hohmann-Marriott
Technical Communication

Abstract

The chlorophyll content is an important experimental parameter in agronomy and plant biology research. In this report, we explore the feasibility of determining total concentration of extracts containing chlorophyll a and chlorophyll b by chlorophyll fluorescence. We found that an excitation at 457 nm results in the same integrated fluorescence emission for a molecule of chlorophyll a and a molecule of chlorophyll b. The fluorescence yield induced by 457 nm is therefore proportional to total molar chlorophyll concentration. Based on this observation, we designed an instrument to determine total chlorophyll concentrations. A single light emitting diode (LED) is used to excite chlorophyll extracts. After passing through a long-pass filter, the fluorescence emission is assessed by a photodiode. We demonstrate that this instrument facilitates the determination of total chlorophyll concentrations. We further extended the functionality of the instrument by including LEDs emitting at 435 and 470 nm wavelengths, thereby preferentially exciting chlorophyll a and chlorophyll b. This instrument can be used to determine chlorophyll a and chlorophyll b concentrations in a variety of organisms containing different ratios of chlorophylls. Monte-Carlo simulations are in agreement with experimental data such that a precise determination of chlorophyll concentrations in carotenoid-containing biological samples containing a concentration of less than 5 nmol/mL total chlorophyll can be achieved.

Keywords

Fluorometer Chlorophyll Light emitting diode Portable 

Supplementary material

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References

  1. Arar EJ, Collins GB (1997) In vitro determination of chlorophyll a and pheophytin a in marine and freshwater algae by fluorescence. EPA method 445.0. US Environmental Protection Agency, CincinnatiGoogle Scholar
  2. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24:1–15PubMedCrossRefGoogle Scholar
  3. Boardman NK, Thorne SW (1971) Sensitive fluorescence method for the determination of chlorophyll a−chlorophyll b ratios. Biochim Biophys Acta 253:222–231.Google Scholar
  4. Camejo D, Rodriguez P, Morales MA, Dell’Amico JM, Torrecillas A, Alarcon JJ (2005) High temperature effects on photosynthetic activity of two tomato cultivars with different heat susceptibility. J Plant Physiol 162:281–289PubMedCrossRefGoogle Scholar
  5. Cantrell KM Jr, Ingle JD (2003) The SLIM spectrometer. Anal Chem 75:27–35PubMedCrossRefGoogle Scholar
  6. Cavender-Bares J, Bazzaz FA (2004) From leaves to ecosystems: using chlorophyll fluorescence to assess photosynthesis and plant function in ecological studies. In: Papageorgiou GC, Govindjee (eds) Chlorophyll a fluorescence: a signature of photosynthesis. Advances in photosynthesis and respiration, vol 19. Springer, Dordrecht, pp 737–755Google Scholar
  7. Chew AG, Bryant DA (2007) Chlorophyll biosynthesis in bacteria: the origins of structural and functional diversity. Annu Rev Microbiol 61:113–129PubMedCrossRefGoogle Scholar
  8. Dunn J, Turnbull JD, Robinson SA (2004) Comparison of solvent regimes for the extraction of photosynthetic pigments from leaves of higher plants. Funct Plant Biol 31:195–202CrossRefGoogle Scholar
  9. Forster LS, Livingston R (1952) The absolute quantum yields of the fluorescence of chlorophyll solutions. J Chem Phys 20:1315–1320CrossRefGoogle Scholar
  10. Goodwin RH (1947) Fluorometric method for estimating small amounts of chlorophyll a. Anal Chem 19:789–794Google Scholar
  11. Jeffrey SW (1974) Profiles of photosynthetic pigments in the ocean using thin-layer chromatography. Marine Biol 26:101–110CrossRefGoogle Scholar
  12. Kiang NY, Siefert J, Govindjee, Blankenship RE, Meadows VS (2007) Spectral signatures of photosynthesis I: review of earth organisms. Astrobiol 7:222–251CrossRefGoogle Scholar
  13. Kouril R, Ilik P, Naus J, Schoefs B (1999) On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio. Photosynth Res 62:107–116CrossRefGoogle Scholar
  14. Lichtenthaler HK, Wellburn AR (1983) Determination of total carotenoids and chlorophyll a and b of leaf extracts in different solvents. Biochem Soc Trans 603:591–593Google Scholar
  15. Lichtenthaler HK, Buschmann C, Doll M, Fietz H-J, Bach T, Kozel U, Meier D, Rahmsdorf U (1981) Photosynthetic activity, chloroplast ultrastructure, and leaf characteristics of high-light and low-light plants and of sun and shade leaves. Photosynth Res 2:115–141CrossRefGoogle Scholar
  16. Lim CK (2003) High-performance liquid chromatography and mass spectrometry of porphyrins, chlorophylls and bilins. Methods in chromatography, vol 2. World Scientific Publishing Co, SingaporeGoogle Scholar
  17. Maróti P (2008) Kinetics and yields of bacteriochlorophyll fluorescence: redox and conformational changes in reaction center of Rhodobacter sphaeroids. Eur Biophys J 37:1175–1184PubMedCrossRefGoogle Scholar
  18. Nichols BW (1963) Separation of the lipids of photosynthetic tissues: improvements in analysis by thin-layer chromatography. Bioch Biophys Acta 70:417–422CrossRefGoogle Scholar
  19. Porra RJ (2002) The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res 73:149–156PubMedCrossRefGoogle Scholar
  20. Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975:384–394CrossRefGoogle Scholar
  21. Scarpari LM, Meinhardt LW, Maizzafera P, Pomella AWV, Schiavinato MA, Cascardo JCM, Pereira GAG (2005) Biochemical changes during the development of witches’ broom: the most important disease of cocoa in Brazil caused by Crinipellis perniciosa. J Exp Bot 56:865–877PubMedCrossRefGoogle Scholar
  22. Stomp M, Huisman J, Stal LJ, Matthijs HC (2007) Colorful niches of phototrophic microorganisms shaped by vibrations of the water molecule. ISME J 1:271–282PubMedGoogle Scholar
  23. Strain HH, Thomas MR, Katz JJ (1963) Spectral absorption properties of ordinary and fully deuterated chlorophylls a and b. Biochim Biophys Acta 75:306–311PubMedCrossRefGoogle Scholar
  24. Talbot MFJ, Sauer K (1997) Spectrofluorimetric method for the determination of large chlorophyll a/b ratios. Photosynth Res 53:73–79CrossRefGoogle Scholar
  25. Timperio AM, D’Amici GM, Barta C, Loreto F, Zolla L (2007) Proteomics, pigment composition, and organization of thylakoid membranes in iron-deficient spinach leaves. J Exp Bot 58:3695–3710PubMedCrossRefGoogle Scholar
  26. Van Heukelem L, Thomas CS (2001) Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments. J Chromatogr A 910:31–49PubMedCrossRefGoogle Scholar
  27. Vernon LP, Seely GR (1966) The chlorophylls. Academic Press, NYGoogle Scholar
  28. Weber G, Teale FWJ (1957) Determination of the absolute quantum yield of fluorescent solutions. Trans Faraday Soc 53:646–655CrossRefGoogle Scholar
  29. Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307–313Google Scholar
  30. Yentsch CS, Menzel DW (1963) A method for the determination of phytoplankton chlorophyll and phaeophytin by fluorescence. Deep-Sea Res 10:221–231Google Scholar
  31. Zechmeister L (1962) Cis-trans isomeric carotenoids vitamins A and arylpolyenes. Springer, ViennaCrossRefGoogle Scholar
  32. Zechmeister L, Polgar A (1943) Cis-trans isomerization and spectral characteristics of carotenoids and some related compounds. J Am Chem Soc 65:1522–1528CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Jacob J. Lamb
    • 1
  • Julian J. Eaton-Rye
    • 1
  • Martin F. Hohmann-Marriott
    • 1
    • 2
  1. 1.Department of BiochemistryUniversity of OtagoDunedinNew Zealand
  2. 2.Department of BiotechnologyNorwegian University of Science and TechnologyTrondheimNorway

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