Fluorometer readings are based on the relationship between in vivo fluorescence and the chlorophyll a concentration. Interferences cause changes to this relationship. Light history; temperature; turbidity, dissolved components and algal health can all have significant effects on the amount of fluorescence, independent of the chlorophyll a concentration. In some cases the degree of interference will depend on the instrument while in other cases the interference is independent of the instrumentation.

The impact of these factors can be controlled or corrected if the user is aware of their effects. The most effective means of generating accurate data is to take regular water samples for chlorophyll a extraction. The in vivo fluorescence value must be noted at the time of sampling. Once the exact chlorophyll a concentration is determined via extraction, the in vivo fluorescence and extracted data are compared and a correlation factor is developed. If samples are taken when there is significant changes in water quality and when the natural assemblage of phytoplankton changes due to changes in location and environment, a tight correlation will be obtained and accurate data will result. In practice it is unrealistic to obtain a water sample every time there is an environmental change but regular sampling, an awareness of the various factors and sound sampling and extraction practices will result in strong correlation between in vivo fluorescence and chlorophyll a concentrations.

1. Light History
Light history can have significant effects on the fluorescence in algal cells. At low light levels, algal cells can optimize the light uptake by pushing chloroplasts to the outer edge of the cell, or by producing more chlorophyll per cell. Both of these responses can result in fluorescence data that falsely represents the algal biomass. At the other end of the spectrum at high light intensities algal growth can be inhibited and fluorescence can under-estimate algal biomass. To avoid this, conduct profiles at night, use an opaque hose when sampling natural waters, or take samples for extraction. The transport time of the water in the hose will dark-adapt cells to an extent, significantly reducing fluorescence error caused by variations in the light history of the cells.

2. Temperature
All fluorophores are affected by temperature to varying extents. Temperature has an inverse relationship with fluorescence. For example, in a vertical profile, as the temperature decreases, the fluorescence will increase independent of chlorophyll concentration. The in vivo chlorophyll fluorescence changes at a rate of 1.4% per °C. A temperature drop of 10°C in a vertical profile would result in a 14% overestimation of chlorophyll due to temperature changes alone.

3. Water Quality
DOM, chlorophyll degradation products, (pheophytins), accessory pigments, (chlorophyll b and c), and turbidity can also affect fluorescence response. If these factors are suspected to be significant, a quick study should be conducted to look at the effects of comparing the fluorescence from filtered water and non-filtered water samples from below the photic zone where chlorophyll concentrations would be at a minimum.

4. Physiological State of the Algal Cells ('Health' of the Cell)

There are three outcomes of light energy that are absorbed by chlorophyll contained in algal cells; 1) it is channeled towards photosynthesis, 2)it is given off as heat, 3) it is re-emitted as fluorescence. Due to this relationship, as one process increases the others decrease. This is why 'healthy' algal cells will fluoresce less than a 'dying' cell.

Conclusion:
Despite the many environmental, physiological and instrument specific factors that result in variation in the relationship between in vivo fluorescence and actual chlorophyll a concentration, in natural assemblages of algae, containing a variety of species in a number of phases of growth, many of these factors tend to average out. In cases where measurement of mono-cultures of algae or algal blooms are occurring these factors may have a significant effect. Also, awareness of light effects, water quality and your instrumentation will give you the information you need to improve sampling practices and conduct the appropriate number of sampling and extractions that will result in accurate and informative in vivo fluorescence data sets.