Applying Pulse Amplitude Modulation (PAM) fluorometry to microalgae suspensions: stirring potentially impacts fluorescence

The use of microalgae suspensions in PAM-fluorometers such as the Water-PAM (Walz GmbH, Germany) presents the problem of maintaining a homogeneous sample. The Water-PAM is marketed with an optional accessory for stirring the sample within the cuvette while in the emitter-detector (ED) unit. This stirring device can help to prevent cells from settling out of suspension over the time-course of chlorophyll-a fluorescence measurements. The ED unit was found to provide a vertically heterogeneous light environment and, therefore, cells within a single sample can exist in different quenched states. Enhancing cell movement by stirring was found to substantially influence measured fluorescence yield while performing induction curve and rapid light curve analyses. This is likely to result from relatively unquenched cells outside the main light-path moving into a higher light region and thus emitting disproportionately more fluorescence than quenched cells. Samples containing cells with high sinking rates or motile species may encounter similar (but reduced) problems. This effect can be mitigated by: (a) reducing analysis time to minimise the distance cells can sink/swim during the measurement procedure and avoiding the necessity of stirring; (b) limiting the proportion of sample outside the light path by minimising sample volume or; (c) by activating the stirrer only for short periods between saturation pulses and allowing enough time after stirring for quenching to stabilise before activation of the saturation pulse. Alternatively, modifications to the instrument providing a vertical dimension to the LED-array could resolve the issue by providing a more homogeneous light environment for the sample.