Cosgrove Jeffrey, Borowitzka Michael
School of Biological Sciences and Biotechnology, Murdoch University, Murdoch, WA 6150, Western Australia.
Photosynth Res. 2006 Jun;88(3):343-50. doi: 10.1007/s11120-006-9063-y. Epub 2006 Jun 6.
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.
在诸如Water-PAM（德国瓦尔兹有限公司）等脉冲幅度调制荧光仪（PAM-fluorometers）中使用微藻悬浮液存在维持样品均匀性的问题。Water-PAM在销售时配备了一个可选附件，用于在发射-检测（ED）单元中搅拌比色皿内的样品。这种搅拌装置有助于防止细胞在叶绿素a荧光测量的时间过程中从悬浮液中沉降出来。人们发现ED单元提供了一个垂直不均匀的光环境，因此，单个样品中的细胞可能处于不同的猝灭状态。在进行诱导曲线和快速光曲线分析时，发现通过搅拌增强细胞运动对测量的荧光产量有显著影响。这可能是由于主光路外相对未猝灭的细胞移动到更高光照区域，从而比猝灭细胞发出不成比例更多的荧光。含有沉降速率高的细胞或游动物种的样品可能会遇到类似（但程度较轻）的问题。可以通过以下方法减轻这种影响：（a）减少分析时间，以最小化测量过程中细胞沉降/游动的距离，并避免搅拌的必要性；（b）通过最小化样品体积来限制光路外样品的比例；或者（c）仅在饱和脉冲之间的短时间内启动搅拌器，并在搅拌后留出足够时间使猝灭稳定，然后再启动饱和脉冲。或者，对仪器进行改进，为LED阵列提供垂直维度，可以通过为样品提供更均匀的光环境来解决这个问题。
