Saturation fluorimetry of complex organic compounds with a high local concentration of fluorophores (by the example of phytoplankton)

Saturation of fluorescence of complex organic compounds with a high local concentration of fluorescing molecules (fluorophores), when singlet–singlet annihilation makes a noticeable contribution to saturation, is considered. The fluorescence saturation curve is obtained analytically for the case of a rectangular temporal and spatial distribution of photons in a laser pulse. It is shown that the fluorescence saturation curve depends on the parameter Φ₀, which is proportional to the concentration of fluorescing molecules, and on the parameters A, B, and α describing the influence of singlet–singlet annihilation, bleaching of an optically thin layer, and nonstationarity of excitation, respectively. The fluorescence saturation curves are studied experimentally for compounds with a high local concentration of fluorescing molecules such as molecules of a monoculture of diatomic alga Thalassiosira weissflogii. The experimental fluorescence saturation curves are well described by the obtained analytic expression. The values of the parameter Φ₀, proportional to the concentration of chlorophyll a, and the parameter A (for the first time) are obtained from the alga fluorescence saturation curves.