Measurement of the fluorescence of photosynthetic pigments

Method principle

Photoautotrophic as well as other organisms need energy for the production of organic compounds. Photoautotrophs employ the energy of light, which they absorb, however absorbed light is not entirely employed for photochemical reactions. Part of the energy is dissipated as heat and another one is emitted as fluorescence. Fluorescence is an optical phenomenon in which molecular absorption of light photons triggers the emission of photons with longer (less energetic) wavelength. Measurement of fluorescence may be used for the quantification of phytoplankton in samples from an aquatic environment.

Photosystem-II of all photoautotrophic organisms is created by chlorophyll-a and other accessorial pigments which are specific for each group of phytoplankton. Based on the presence of pigments, photoautotrophic organisms may be categorized to following groups: "blue" (cyanobacteria), "green" (green algae), "brown" (bacillariophytes - diatoms, dinophytes), "red" (rhodophytes) and "mixed" (cryptophytes). Some of photosynthetic pigments are fluorescent, when they are excited by light. In case of an appropriate configuration of measuring instruments, pigment fluorescence may serve as an endpoint for the determination of total chlorophyll-a concentration and provides an information about the presence of various groups of phytoplankton in measured sample. Main advantage of fluorescence measurement is easiness and non-destructiveness of the method. Measurement of fluorescence is suitable for the determination of phytoplankton biomass not only in a laboratory conditions, but also in situ.





Various groups of phytoplankton may be determined by submersible probe FluoroProbe (bbe Moldaenke GmBH, Kiel, Germany). The instrument enables excitation of photosynthetic pigments at six wavelengths (i.e. 370 nm, 470 nm, 525 nm, 570 nm, 590 nm and 610 nm respectively). Fluorescence emission is measured at fixed wavelength of 680 nm. Based on characteristics of their excitation spectra distinguishing of following phytoplankton groups is possible: Chlorophyceae (green algae), Cyanophyceae (cyanobacteria), Bacillariophyceae (diatoms) together with Dinophyceae and Cryptophyceae. Amount of phytoplankton biomass is expressed as chlorophyll-a concentration (in µg per ml of sample). Detection limit as was stated by manufacturer is 1 µg L-1 and according to our experience, upper limit for the detection, especially when cyanobacteria dominate in the measured sample, is approximately 50 µg L-1 (when higher concentration of chlorophyll-a in the sample is expected, it is necessary to dilute the sample appropriately). FluoroProbe may be used in the laboratory (with the cuvette adapter) as well as in situ in entire profile of a dam (up to the depth of 100 meters depending on the length of measuring-cable) or a stream. A use of a special adapter enables the determination of chlorophyll-a concentration in benthic mats. Recently, methodology for the determination of Microcystis sp. cyanobacteria in sediments using FluoroProbe has been processed at the CCT.

Algae Online Monitor (AOM)

Another instrument working based on a principle of chlorophyll-a fluorescence measurement used in our labs is Algae Online Monitor (AOM). AOM has been constructed by (Photon Systems Instruments, Brno, Czech Republic). Fluorescence of photosynthetic pigments is excited by two LED diodes (emitting at 455 nm and 590 nm), thus the instrument enables distinguishing of two different groups of phytoplankton – both cyanobacteria as well as green algae. The instrument has been adjusted for continual in situ measurement. Its limit of detection is about 300 to 400 cells of Microcystis aeruginosa per ml of measured sample, corresponding to the chlorophyll-a concentration of 0.02 µg L-1 approximately. AOM will be tested in natural conditions during the season 2009-2010.

Algae Online Monitor - data

Algae Online Monitor











CCT publications to above mentioned issue:

  • Gregor, J., Marsalek, B. (2004): Freshwater phytoplankton quantification by chlorophyll a: a comparative study of in vitro, in vivo and in situ methods. Water Research 38(3): 517-522.
  • Gregor, J., Marsalek, B. (2005): A simple in vivo fluorescence method for the selective detection and quantification of freshwater cyanobacteria and eukaryotic algae. Acta Hydrochimica et Hydrobiologica 33(2): 142-148.
  • Gregor, J., Marsalek, B., Sipkova, H. (2007): Detection and estimation of potentially toxic cyanobacteria in raw water at the drinking water treatment plant by in vivo fluorescence method. Water Research 41(1): 228-234.
  • Gregor, J., Jancula, D., Marsalek, B. (2008): Growth assays with mixed cultures of cyanobacteria and algae assessed by in vivo fluorescence: One step closer to real ecosystems? Chemosphere 70(10): 1873-1878.