Chlorophyll α specific absorption and fluorescence excitation spectra for light-limited phytoplankton

Methods are described for the measurement of spectral absorption coefficients, fluorescence excitation, and fluorescence yields for pigmented particles retained on filters. The corrections required for absorption coefficients include determining increased optical pathlength while corrections for fluorescence include determining system spectral variability, mean light level and reabsorption. The empirical technique is consistent with and validated by theoretical relationships for light transmission and fluorescence of absorbing particulate material embedded in a medium with intense scattering.These methods were applied to a study of photoadaptation in several phytoplankton species and revealed variations in the blue for chlorophyll α specific absorption [αph*(λ)] and fluorescence excitation [F*(λ)] of greater than 3− and 10-fold, respectively. Variations in the spectral shapes and the magnitude of αph*(λ) and F*(λ) with photoadaptation are determined largely by the effect of pigment absorption in discrete particles, sometimes referred to as the sieve or package effect. A model is presented expressing F*(λ) in terms of α*(λ) which predicts large variability in F*(λ) due to cell size and cellular pigmentation and which may help reconcile the previously reported, but unexplained variations in F*(λ). Spectral variations in the fluorescence yield appear to be caused by variations in the fraction of light absorbed by photosystem II which fluoresces as compared to photosystem I or photoprotective pigments which do not fluoresce. The techniques presented provide a rapid, reproducible, and simple approach for routine analysis, particularly for field applications where particle densities are too low for direct analysis of absorption spectra.