Monitoring the responses of Haematococcus pluvialis under different culture conditions by using FLIM

As one of the organisms with the highest astaxanthin content in nature, <i>Haematococcus pluvialis</i> (<i>H. pluvialis</i>) has great value in pharmaceutical and cosmetic industries in recent years. Both light and salinity are important factors involved in the growth status of <i>H. pluvialis</i>. However, monitoring the real-time response of <i>H. pluvialis</i> to different culture conditions requires new approaches. Currently, fluorescence lifetime imaging microscopy (FLIM) has been used in biological fields since it can provide quantitative data of biological samples in a non-invasive way. Here, we have applied FLIM method into studying the responses of <i>H. pluvialis</i> under different culture conditions. The <i>H. pluvialis</i> were cultured under different levels of light or salinity, whose auto-fluorescent images were obtained by using FLIM and then analyzed by the SPCImage software to provide their fluorescence lifetime values. The results showed that in both experimental settings, the fluorescence lifetime values of the microalgae group under normal culture condition kept relatively constant. The lifetime values of the group in darkness declined continuously till 48 hours, while lifetime values of the group in higher light intensities increased in first 24 hours followed by a decrease in the next 24 hours. Meanwhile, the lifetime values of the group in a culture medium with threefold salinity level kept decrease till 72 hours. By contrast, in the group under sixfold salinity stress, the lifetime values presented a rapid rise in the first day and dropped back in 3 days. These changes of the fluorescence lifetime values of <i>H. pluvialis</i> may be explained by the variations of the internal microenvironment and the physiological status of the microalgae. In conclusion, this work has shown that FLIM can be applied into detecting autofluorescence of microalgae in a non-invasive way, providing the fluorescence lifetime values of individual microalga for quantitative analysis of their responses to environmental alternations.