Identification Of Phytoplankton Cell Viability Based On Digital Holography And Its Application In The Study Of Marine Biological Pump

The sinking of particulate organic matter(POM)from the euphotic layer to the seafloor is an important component of the marine carbon cycle,leading to carbon migration and burial in the ocean.The formation of settleable POM is closely related to the phytoplankton viability.Quantifying the contribution of phytoplankton with different viability to settleable POM is of great significance for understanding the fate of upper organic matters and for evaluating efficiency of the biological pump in the ocean.The viability of phytoplankton is affected by various environmental factors.Before understanding the operation of marine ecosystem and evaluating the health status of marine ecosystem,it is fundamental to study the effects of environmental factors on phytoplankton viability at the individual level.However,the existing methods for identifying the viability of phytoplankton cells,such as staining,flow cytometry,chlorophyll fluorescence,and enzyme assays,mostly borrowed from the viability identification of animal cells.There are problems such as the need for staining,introduction of exogenous substances,lack of quantification,and inability to adapt to offshore applications,which greatly limit the development of research on marine phytoplankton.Therefore,focusing on the needs of phytoplankton cell viability identification in marine research and the limitations of existing identification methods,this study has carried out a series of studies and achieved the following results:(1)Based on differences in cell refractive index,staining results and growth curves,the viability states of phytoplankton were divided into three types:active,weak and dead.The modulation of light field by algal cells with different viability states was analyzed.It lays a theoretical foundation for the establishment of cell viability identification model based on digital holography and deep learning.(2)A new method for identification of phytoplankton cell viability based on digital holography and deep learning(Holo-deep method)was proposed.The hardware part of this method consists of a digital holography imaging system.Based on the principle of light interference,the interference pattern between the distorted object light illuminated on algal cells and the undistorted reference light was stored as a digital hologram.Then,the wavefront of the object light field was reconstructed by numerical diffraction calculation,which provided the amplitude and phase images of the algal cells.The algorithm part was based on the VGG16 convolution model of deep learning,which extracts high-dimensional features that can represent cell viability through multiple convolution operations in the holographic reconstructed images of phytoplankton cells,achieving three-classification of phytoplankton cell viability.The Holo-deep method was characterized by non-labeling,non-invasion,and not introducing exogenous materials.It can not only realize rapid viability identification of a large number of algal cells,but also carry out long-term tracking and observation of individual cells,which can not only reflect the dead and active cells,but also reflect the weak state between them.(3)The Holo-deep method was applied to the ecological study of marine phytoplankton,and the effects of environmental factors on phytoplankton viability were studied.Firstly,the different effects of different temperature processes on the viability of red tide algae were studied through the variable temperature experiment in the laboratory.It was proved that the heat damage caused by high temperature was the main reason for reducing the viability of algae cells and inhibiting the growth rate of population.This suggested that we should pay more attention to the microscopic characteristics of algal cells in the future monitoring of harmful algal blooms.Next,field observations of phytoplankton cell viability were conducted in the East China Sea to study the influence of environmental factors on the growth of phytoplankton population.The results showed that the viability of surface phytoplankton in the East China Sea in spring was affected by sea surface temperature and nutrient level.Among them,temperature mainly affected the weak cell ratio,but had no significant effect on the dead cell ratio.There was a threshold for the effect of nutrient level on algal cell viability.When nitrate concentration<7.0μmol·L-1 and phosphate concentration<0.6 μmol·L-1,the weak and dead cell ratios were positively correlated with the concentration of Chl a.But when nitrate concentration>7.0μmol·L-1 or phosphate concentration>0.6 μmol·L-1,the correlation was no longer established.(4)Holo-deep method was applied to the study of marine particulate organic carbon,and the quantitative relationship between different viability phytoplankton and settleable POM was explored.According to the size range of settleable POM,POC>75(i.e.,carbon content of POM with particle size larger than 75 μm)was used to characterize settleable POM.Firstly,through the laboratory culture of different species of algal cells,it was found that the cumulative number of dead cells observed daily was correlated with POC>75,indicating that settleable POM was mainly formed by the aggregation of detritus from the death and lysis of phytoplankton cells.Then,the field survey and incubation experiment were conducted in the East China Sea to explore the spatiotemporal relationship between phytoplankton death and POC>75 in natural seawater.The results showed that phytoplankton death was one of the main controlling factors of POC>75 formation,and there was the following relationship between the cumulative mass of POC>75 and the cumulative number of dead phytoplankton cells in the East China Sea as follows:Cumulative mass of POC>75(mg)=0.487×Cumulative number of dead cells(/104)+0.069.In this study,the quantitative relationship between phytoplankton cell viability and settleable POM was explained with the help of Holo-deep method,which provided a scientific basis for improving the carbon flux model in the East China Sea,and also deepened the understanding of marine biological carbon pumps.In summary,this paper not only carried out research on the identification methods of phytoplankton viability based on digital holography,but also explored the influence of environmental factors on phytoplankton viability and the contribution of phytoplankton death to settleable POM in the East China Sea.From the perspective of algal cell viability,this paper provides new insights into some issues in marine research,and provides a new approach for the field observation and research of marine phytoplankton.