基于可变荧光统计法的藻类活体细胞密度宽范围检测实验

Experimental Investigation of Wide-Range Detection of Live Algal Cell Density Based on Variable Fluorescence Statistical Analysis

提出区间增益系数动态调整的可变荧光统计学分布检测方法,以可变荧光统计分析法为基础,随机抽样测量,统计可变荧光值的方差σ^(2)。分析不同细胞密度样本的可变荧光值方差σ^(2)与区间增益系数a之间的关系,利用区间增益系数动态调整方法准确测量宽细胞密度范围内的活体藻细胞密度。实验结果表明,该方法对羊角月牙藻细胞密度检测上限达到4640 cells·mL^(-1),较原始方法提高了近200倍;蛋白核小球藻、羊角月牙藻、斜生栅藻和杜氏盐藻检测结果与显微镜分析法的检测结果具有较高的一致性,相关系数R2均在0.999以上,相对误差绝对值均小于20.00%;直接测量楯形多甲藻与羊角月牙藻的混合藻种,检测结果与显微镜分析法的检测结果也具有较高的一致性,相对误差绝对值均小于20.00%。

Objective In planktonic community within the range of 10‒50μm,the predominant biological population is typically composed of phytoplankton,which are the primary producer in aquatic ecosystems.Phytoplankton plays a crucial role in aquatic ecological monitoring and serve as key indicators of algal bloom outbreaks in eutrophic water bodies.Simultaneously,it is a significant parameter to mitigate the invasion of alien species.The International Convention for the Control and Management of Ships’Ballast Water and Sediments specifies that the discharge standard for ship ballast water is a phytoplankton live cell density of<10 cells·mL^(-1).Therefore,rapid detection of phytoplankton live cell density is vital for early warning of algal blooms in eutrophic waters and compliance testing for ballast water.Traditional chlorophyll variable fluorescence methods do not require complex preprocessing,enabling rapid estimation of live algal cell numbers.However,cell density does not solely determine the fluorescence intensity of live algal cells,which is intricately linked to the type,size,and growth cycle of individual algal cells.Consequently,it is not feasible to rely solely on variable fluorescence intensities to precisely predict live algal cell numbers.The most recent research introduces a variable fluorescence statistical analysis method that calculates the central frequency B of the F_(v) value of live algal cells to estimate their density.This method overcomes the impact of individual differences in live algal cell characteristics and provides accurate measurements of live algal cell densities below 200 cells·mL^(-1).Nevertheless,the increase in live algal cell density leads to noticeable changes in the Gaussian distribution of the F_(v) dataset,the larger the density of live algal cell is,the steeper the Gaussian distribution curve is.The changes in central frequency B have not received sufficient attention;however,the variable fluorescence statistical analysis method exhibits significant deviations in the measurement results for live algal cell density above 200 cells·mL^(-1).Considering these challenges,this paper proposes a method of dynamically adjusting the interval gain coefficients based on the variable fluorescence statistical analysis method.Methods This study first conducts experiments with different algal species to explore the relationship between the central frequency B and interval gain coefficient a.Subsequently,the experimental results are validated through a self-constructed experimental system,and comparisons are performed with the original variable fluorescence statistical analysis method and the microscopic analysis method.This study aimed to verify whether the proposed method enhances the detection upper limit while ensuring the trace detection of live algal cells.Four algal species,Chlorella pyrenoidosa,Selenastrum capricornutum,Scenedesmus obliquus,and Dunaliella salina,were selected as experimental subjects.The system comprises sample processing,fluorescence excitation and collection,and signal acquisition and processing modules(Fig.2).The linkage between the sample pool and stirring device enables random and uniform sampling,while the coordinated operation of the three-way solenoid valve and peristaltic pump ensures precise injection and drainage of the samples.A monochromatic high-brightness laser diode(LD),combined with a vertical orthogonal optical path,efficiently and stably excites and collects the fluorescence of algal cells.Finally,the use of photomultiplier tubes and a high-speed AD(Analog to digital)acquisition circuit allows for high signal-to-noise ratio acquisition of weak algal cell fluorescence signals.Results and Discussions This study introduces a variable fluorescence statistical distribution detection method with dynamically adjusted interval gain coefficients based on the variable fluorescence statistical analysis method.Random sampling measurements and statistical analysis of the varianceσ^(2) of variable fluorescence values were conducted to analyze the relationship between the varianceσ^(2) of variable fluorescence values for samples with different cell densities and the interval gain coefficient a(Fig.4).Subsequently,this study establishes a dynamic adjustment method for interval gain coefficients that enables accurate counting of live algal cell density over a wide range of cell densities.The experimental results demonstrate that this method achieved a detection upper limit of 4640 cells·mL^(-1) for Selenastrum capricornutum,an increase of nearly 200 times that of the original method(Fig.5).The detection results for Chlorella pyrenoidosa,Selenastrum capricornutum,Scenedesmus obliquus,and Dunaliella salina show high consistency with microscopic examination results,with correlation coefficients R^(2) exceeding 0.999 and absolute values of relative errors below 20.00%(Fig.6).In conclusion,accuracy assessments of mixed algal densities were conducted for two algal species,Peridinium umbonatum and Selenastrum capricornutum,which exhibited substantial differences in single cell variable fluorescence(SCVF)across three distinct densities.The detection results show a high degree of consistency with microscopic examination results,with absolute relative errors consistently below 20.00%(Fig.7).Conclusions Existing live algal cell density detection methods currently face challenges in achieving precise counting for both high and low densities.Variable fluorescence statistical analysis can achieve accurate counting of low-density live algal cells;however,its detection range is limited,making it challenging to accurately detect live algal cell density in natural water bodies or during algal blooms.This study proposed a variable fluorescence statistical distribution detection method with dynamically adjusted interval gain coefficients based on this limitation.This method characterizes the relationship between the sample varianceσ^(2) of the F_(v) mean value and interval gain coefficient a to accurately count the live algal cell density over a wide range of cell densities.Results from tests on four algal species,Chlorella pyrenoidosa,Selenastrum capricornutum,Scenedesmus obliquus,and Dunaliella salina,show that the original variable fluorescence statistical analysis method maintains basic consistency with microscopic examination results only in the low-density domain,with significant deviations in high-density measurements,reaching a maximum absolute relative error of 81.59%.In contrast,the proposed variable fluorescence statistical distribution detection method demonstrated high consistency with microscopic examination results at high and low densities within 5×10^(3) cells·mL^(-1).The correlation coefficients R^(2) were all above 0.999,and the absolute values of relative errors were below 20.00%.The upper measurement limit of this method meets the requirements for algal density detection in natural water bodies and algal bloom warnings(2×10^(3) cells·mL^(-1)).Simultaneously,for samples with low cell densities,the proposed method ensures the measurement accuracy inherent in the original variable fluorescence statistical analysis.Relative errors in the measurement of algae with low cell densities consistently remained below 20.00%.The experimental results from mixed algae indicate that within the experimental system,the proposed method allows us to disregard the variable fluorescence masking phenomenon of high SCVF algae on low SCVF algae.Moreover,the proposed method enables direct measurement of live algal cell density in mixed cultures,and the density calculated using the proposed method maintains a high level of consistency with microscopic examination density,with relative errors consistently below 20.00%.The proposed method reduces the detection speed of the system and increases the algorithm complexity.It not only preserves the accuracy of traditional methods for low-density detection but also enhances the detection upper limit.Moreover,it can be applied for the direct measurement of mixed algal species,significantly improving the robustness and expanding the application potential of the system.