Quantifying the uncertainties in multi-wavelength PAM fluorometry due to innate and irradiance-induced variability of fluorescence spectra


Multi-wavelength Chl a fluorometers are increasingly applied to assess phytoplankton photosynthetic capacity and composition, but their usefulness is limited by uncertainties in fluorescence excitation spectra (FES). We investigated this issue using the Phyto-PAM fluorometer to evaluate the effects of innate and irradiance-dependent variations in background (F) and variable (Fv) FES on analysis of three pigment groups (cyanobacteria, chlorophytes and chromophytes). The effects on group-specific estimates of minimum fluorescence (F0), a proxy for biomass, and Fv/Fm, the quantum yield of photochemistry, presented some challenges to the interpretation of group-specific results. F0 estimates usually had a 5–15% margin of error, even when measuring highly uneven mixtures, and applying imperfectly matched calibration FES or stressing samples with photosynthetically active and ultraviolet radiation; errors in Fv/Fm were commonly < 15%. Despite such relatively good accuracy, estimates for F0 and, especially, Fv/Fm are unreliable for groups at low relative abundance, and results can sometimes be reported for groups not actually present. We report margins of error for different levels of relative abundance to inform interpretation of measurements from natural communities and show that F and Fv spectra for some taxa can differ in ways that produce severe errors in F0 and Fv/Fm estimates if used uncritically.

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Availability of data

Data are available from authors on request.


Chl a :

Chlorophyll a

F 0 :

Dark-adapted minimum fluorescence

F :

Low-light adapted minimum fluorescence

F m :

Maximum fluorescence

F v :

Variable fluorescence

F v/F m :

Maximum quantum yield of photochemistry (dark adapted)


Fluorescence excitation spectrum/spectra


Light emitting diode




Non-metric multidimensional scaling


Non-photochemical quenching


PAR only treatment


PAR + UV-A treatment


PAR + UV-A + UV-B treatment


Pulse amplitude modulation


Photosynthetically active radiation






Photosystem II


Reference Spectrum/Spectra


Ultraviolet (radiation)


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The work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (R. Smith), with important additional support from Environment and Climate Change Canada (S. Watson). We thank two anonymous reviewers for their feedback that improved this manuscript.


Funding for this research was from a Natural Sciences and Engineering Research Council of Canada Discovery Grant (R. Smith), with important additional support from Environment and Climate Change Canada (S. Watson).

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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by L. Beecraft. The first draft of the manuscript was written by L. Beecraft, and all authors participated in critical revision of the manuscript. All authors read and approved the final manuscript for submission.

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Correspondence to Laura Beecraft.

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Beecraft, L., Watson, S.B. & Smith, R.E.H. Quantifying the uncertainties in multi-wavelength PAM fluorometry due to innate and irradiance-induced variability of fluorescence spectra. Aquat Ecol 55, 169–186 (2021). https://doi.org/10.1007/s10452-020-09821-6

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  • Phyto-PAM
  • Phytoplankton analysis
  • Multi-wavelength fluorometry
  • Variable fluorescence
  • Fluorescence excitation spectra
  • Methodological issues