In this technique, the excitation light from the fluorometer passes through the untreated sample water and excites chlorophyll within the living cells of the algae present. Due to the nature of light, cells and other dissolved and particulate materials in the water will affect the excitation light before it reaches the chlorophyll molecules. Examples of interfering materials include other plant pigments and degradation products, dissolved organic matter, turbidity, and cell morphologies. Therefore, in vivo analysis is a semi-quantitative tool. In vivo numbers should correlate well with each other but rarely can they be used as actual chlorophyll a concentration measurements until correlated with extracted chlorophyll a data.
Temperature has an inverse relationship with fluorescence.
In a vertical profile, as temperature decreases, the fluorescence will increase independent of chlorophyll concentration. Light history will have significant affects on the fluorescence in algal cells. Cells will fluoresce more chlorophyll per cell when in darker environments than in well lit zones. One way of reducing the effects of light is to use the flow-through cap when sampling natural waters. If these factors are suspected to be significant it is worth conducting a quick study to look at the effects by comparing the fluorescence from filtered and non-filtered water samples from below the photic zone where chlorophyll concentrations would be at a minimum.