A method for in-situ measurement of aquatic primary productivity based on fluorescence excitation spectrum three-wavelength fitting model

Abstract

The application discloses a kind of in-situ measurement methods of aquatic primary productivity based on fluorescence excitation spectrum three-wavelength fitting model, belong to environmental monitoring and aquatic ecosystem technical field.The application includes: in-situ collection target water body water sample and filter to remove large particle impurities;Water sample is moved into fluorescence spectrophotometer sample cell, and after dark chamber balancing, the fluorescence intensity of 438nm, 485nm, 624nm three characteristic wavelengths is measured;According to water sample type, the integral area of fluorescence excitation spectrum is calculated using corresponding first-order linear fitting equation;Through the correlation model of integral area and primary productivity, the primary productivity of target water body is calculated.The application does not need sample pretreatment, simple and fast, measurement period is ≤30min, realizes in-situ non-invasive monitoring;Cover a variety of photosynthetic pigment characteristic absorption peak, wide application range, can tolerate a variety of stress conditions;Measurement precision is high, sensitivity is strong.

Description

Technical Field

[0001] This invention belongs to the field of environmental monitoring and aquatic ecosystem technology, and particularly relates to an in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum. Background Technology

[0002] Aquatic primary productivity refers to the ability of aquatic photosynthetic plants or microorganisms to synthesize organic matter using solar energy within a specific time and space. It is a core indicator for measuring the health of aquatic ecosystems, assessing water quality levels, predicting algal blooms, and guiding aquaculture. Photosynthetic microalgae, as the main contributors to aquatic primary productivity, directly determine the level of primary productivity through their biomass and photosynthetic activity, while chlorophyll a (Chl a) content is a key parameter reflecting microalgal biomass.

[0003] Currently, the methods for measuring aquatic primary productivity mainly include the following categories: 1. Black and white bottle method: This method calculates primary productivity by measuring changes in dissolved oxygen in a closed system. It is a classic benchmark method, but it is cumbersome, time-consuming (requiring long-term incubation), the samples are easily contaminated, and it cannot achieve in-situ real-time monitoring. 2. Chlorophyll a extraction method: Chlorophyll a is extracted using organic solvents (such as methanol and acetone), and the content is determined using a spectrophotometer. Primary productivity is then estimated using empirical formulas. This method requires sample destruction, uses toxic organic solvents, has a complex extraction process, low sensitivity, and cannot be used for rapid on-site detection. 3. Chlorophyll fluorescence parameter method: such as the method based on the initial fluorescence Fo or the integral area of ​​the OJIP curve. The former is only applicable to healthy algal communities under non-stress conditions, while the latter uses a single excitation wavelength and cannot cover the characteristic absorption peaks of multiple photosynthetic pigments such as carotenoids and phycoerythrin in microalgae. Its accuracy is limited when applied to complex systems such as marine algae. 4. Remote sensing method: Chlorophyll a content is retrieved by satellite or airborne remote sensing to estimate primary productivity. Although it can achieve large-scale monitoring, it has low spatial resolution and is greatly affected by weather, and cannot meet the needs of in-situ accurate measurement. 5. Carbon isotope tracing method: It is complex to operate, costly, and time-consuming, making it difficult to promote its application in routine monitoring.

[0004] In summary, existing measurement methods generally suffer from drawbacks such as complex operation, long time consumption, high invasiveness, low sensitivity, limited applicability, or inability to monitor in situ. There is an urgent need to develop a rapid, accurate, non-invasive, and in-situ applicable aquatic primary productivity measurement technology. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum, thereby solving the problems in the background technology.

[0006] This invention provides the following technical solution: An in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum includes the following steps: S1: Water samples from the target aquatic environment are collected in situ and require no additional pretreatment after impurity filtration. S2: Fluorescence excitation spectrum detection of water samples is performed using a fluorescence spectrophotometer to obtain fluorescence intensity parameters at specific characteristic wavelengths; S3: Select the corresponding fitting equation based on the water sample type, and calculate the integral area of ​​the fluorescence excitation spectrum using the fluorescence intensity parameter; S4: The primary productivity of the target water body is calculated using the correlation model between integral area and primary productivity.

[0007] Preferably, the impurity filtration process in step S1 uses a 200-mesh filter, and the water sample is collected using an in-situ sampling device at a water depth of 1m, with a collection volume of 200-300mL.

[0008] Preferably, the detection parameters of the fluorescence spectrophotometer in step S2 are set as follows: emission wavelength fixed at 685 nm, excitation wavelength scanning range of 400 nm-650 nm, and scanning speed of 2000-3000 nm·min. -1 The spectral resolution is 0.1-0.3 nm; the specific characteristic wavelengths are 438 nm, 485 nm, and 624 nm, and the corresponding fluorescence intensity parameters are denoted as x1, x2, and x3.

[0009] Preferably, the fitting equation in step S3 is a first-order linear fitting equation, wherein: The fitting equation for the freshwater sample is: S = 1508.89 + 35.46x1 + 72.09x2 + 67.87x3; The fitting equation for the seawater sample is: S = 2388.57 + 82.86x1 + 11.57x2 + 74.34x3; The goodness of fit R of the fitting equation 2 ≥0.95, where S represents the integral area of ​​the fluorescence excitation spectrum.

[0010] Preferably, the correlation model in step S4 is a linear regression model, expressed as P = a × S × 10 -4 +b, where a and b are correction coefficients, P is primary productivity, and S is the integral area of ​​the fluorescence excitation spectrum.

[0011] Preferably, the association model is constructed in the following manner: a. Measure the chlorophyll a content (Chl a) of the water sample, and calculate the initial primary productivity (P) using the formula P=K×r×Chl a×DH, where K is an empirical constant of 1.97, r is the assimilation coefficient, and DH is the sunshine duration of the target water body area. b. Using the formula S=Σ(F Ex400nm-650nm Calculate the integral area S by multiplying F by 0.2, where F Ex400nm-650nm This indicates the emission fluorescence value in the excitation wavelength range of 400-650 nm; c. Using the integral area S as the independent variable and the initial primary productivity P as the dependent variable, a linear regression analysis is performed to obtain the aforementioned correlation model.

[0012] Preferably, the assimilation coefficient r is taken as follows: in a freshwater environment, r = 3.2 mg biomass h -1 mg -1 Chl a, r=5.62 mg biomass h in seawater -1 mg -1 Chl a.

[0013] Preferably, the method further includes model validation and correction steps: collecting a series of samples from a specific water body, measuring primary productivity using both this method and a benchmark measurement method, and obtaining at least three sets of paired data; performing linear regression analysis based on the paired data, correcting the coefficients in the correlation model, and ensuring that the goodness of fit R of the corrected model is... 2 ≥0.99.

[0014] Preferably, the method is applicable to aquatic environments such as freshwater lakes, reservoirs, aquaculture ponds, and nearshore areas, and can withstand high temperature, low temperature, high salinity, and high light stress conditions. It is also applicable to photosynthetic microalgae such as cyanobacteria, green algae, diatoms, and dinoflagellates.

[0015] Preferably, the deviation of the primary productivity measurement of the method is ≤7%, and the correlation coefficient r between the integral area and chlorophyll a content and cell density is >0.9 (p<0.01).

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides an in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum. The in-situ non-invasive measurement method requires no sample pretreatment (such as pigment extraction or culture) and can directly measure water samples without damaging the aquatic ecosystem. Combined with an in-situ fluorescence acquisition system, it can achieve real-time on-site monitoring. Fast and efficient: The entire measurement process (including sample equilibration, spectral scanning, and data calculation) takes ≤30 minutes, which is much faster than the traditional black and white bottle method (which requires 24 hours of incubation) and chlorophyll extraction method (which requires 24 hours of extraction). High precision and high sensitivity: The three-wavelength fitting model covers the characteristic absorption peaks of major photosynthetic pigments such as chlorophyll a / b, carotenoids, and phycoerythrin. The correlation coefficient between the integrated area and chlorophyll a content and cell density is r>0.9 (p<0.01). The deviation in primary productivity measurement is only 1%-11%, which is better than existing fluorescence methods. Wide range of applications: Suitable for various aquatic environments such as freshwater (lakes, reservoirs, aquaculture ponds) and seawater (nearshore, bays), it can withstand stress conditions such as high temperature, low temperature, high salinity, and high light, and is applicable to various photosynthetic microalgae such as cyanobacteria, green algae, diatoms, and dinoflagellates; Simple operation and low cost: No toxic organic solvents are required, the instrument is easy to operate, and it can be widely used in various scenarios such as environmental monitoring stations, aquaculture enterprises, and research institutions; It also has an algal bloom early warning function: by monitoring the mutation of integral area (indirectly reflecting cell density and chlorophyll a content), it can realize early warning of algal bloom outbreaks and provide technical support for water quality management. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 This is a flowchart of the process of the method of the present invention.

[0019] Figure 2 This is the fluorescence excitation spectrum curve of a typical aquatic sample.

[0020] Figure 3 The curves showing the correlation between the integral area of ​​the fluorescence excitation spectrum of Synechocystis PCC6803 and Chlorella H4 and cell density under different stress conditions are shown in the present invention.

[0021] Figure 4 The curves showing the correlation between the integral area of ​​the fluorescence excitation spectrum of Synechocystis PCC6803 and Chlorella H4 and the chlorophyll a content under different stress conditions of the present invention are shown.

[0022] Figure 5 This is the correlation curve between the integral area of ​​the fluorescence excitation spectrum of freshwater samples and primary productivity according to the present invention.

[0023] Figure 6 This is the correlation curve between the integral area of ​​the fluorescence excitation spectrum of seawater samples and primary productivity according to the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0025] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0026] Example 1: A method for in-situ measurement of aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum: The fluorescence intensity of three characteristic wavelengths (438 nm, 485 nm, and 624 nm) in the fluorescence excitation spectrum is used to calculate the integral area of ​​the fluorescence excitation spectrum through a first-order linear fitting model. Then, the correlation model between the integral area and primary productivity is combined to realize the in-situ measurement of aquatic primary productivity.

[0027] The specific steps are as follows: In-situ sample collection. Using NisKin water sampling bottles or similar in-situ sampling devices, collect 250mL water samples at a water depth of 1m at the sampling point in the target aquatic environment (freshwater lakes, reservoirs, aquaculture ponds, nearshore areas, etc.). If large particulate impurities are present in the water sample, filter them with a 200-mesh filter to remove the impurities; no other pretreatment is required.

[0028] Measurement of fluorescence excitation spectral characteristic parameters. The collected water sample was transferred into the sample cell (2 mL) of the fluorescence spectrophotometer, placed in a dark room for equilibration for 10 min, and then measured.

[0029] Instrument parameter settings: emission wavelength fixed at 685nm, excitation wavelength scanning range 400nm-650nm, scanning speed 2400nmmin -1 Spectral resolution 0.2 nm; Feature parameter extraction: The integral area S is calculated using the following formula: S = Σ(F Ex400nm-650nm ) × 0.2 (Formula 1), where F Ex400nm-650nm This represents the emission fluorescence value for an excitation wavelength range of 400-650 nm. Record the fluorescence intensity values ​​at excitation wavelengths of 438 nm, 485 nm, and 624 nm, denoted as x1 (438 nm), x2 (485 nm), and x3 (624 nm), respectively.

[0030] Establish a correlation model between integral area and primary productivity. The correlation model between integral area S and primary productivity P was constructed through the following steps: The chlorophyll a content (Chl a) of the water sample was measured, and the empirical formula P = K × r × Chl a × DH was used (where K is an empirical constant of 1.97, and r is the assimilation coefficient: 3.2 mg biomass h for freshwater). -1 mg -1 Chl a, seawater 5.62 mg biomass h -1 mg -1 Chl a, DH represents the duration of sunlight (for natural water bodies, primary productivity P is calculated based on local sunshine duration); linear regression analysis is performed with the integral area S as the independent variable and P as the dependent variable to obtain the correlation model: P = a × S × 10 -4 +b, where a and b are correction coefficients.

[0031] Aquatic primary productivity was calculated based on the integral area of ​​the fluorescence excitation spectrum using a three-wavelength fitting model.

[0032] Based on the water sample type (freshwater or seawater), the fluorescence intensity values ​​at three wavelengths (x1, x2, and x3) of the collected water samples were used to calculate the fluorescence excitation spectrum integral area S using the corresponding first-order linear fitting equation. The fitting equation for freshwater samples was S = 1508.89 + 35.46x1 + 72.09x2 + 67.87x3; the fitting equation for seawater samples was S = 2388.57 + 82.86x1 + 11.57x2 + 74.34x3. These fitting equations were obtained through linear regression analysis and first-order linear fitting of the fluorescence spectral data of the water samples, with a goodness of fit R0. 2 ≥0.95.

[0033] When assessing the primary productivity of a water body, the fluorescence intensity values ​​of x1, x2, and x3 of the collected water samples are substituted into the integral area S obtained by formula 2 or 3, and then substituted into formula 1 to calculate the primary productivity P of the target water body.

[0034] Example 2: Primary productivity measurement of freshwater body (a lake) Sample collection. In October 2021, five sampling points were set up in a lake (freshwater) in Kaifeng City. 250mL water samples were collected at a depth of 1m using NisKin water sampling bottles. Impurities were removed by filtering the samples through a 200-mesh filter and the samples were ready for use.

[0035] Instruments and reagents: Hitachi F-4700 fluorescence spectrophotometer; 2mL quartz sample cell; other equipment includes NisKin water sampling bottle, 200-mesh filter, centrifuge, etc.

[0036] Measurement steps Establish a correlation model between integral area and primary productivity.

[0037] The chlorophyll a content (Chl a) of water samples from five sampling points was measured, and the result was calculated using the empirical formula P = K × r × Chl a × DH (where K is an empirical constant of 1.97, and r is the assimilation coefficient: 3.2 mg biomass h for freshwater). -1 mg -1 Chl a, DH represents the duration of sunlight: the primary productivity P1-P5 of natural water bodies is calculated based on the local average sunshine duration of approximately 3.74 hours in Kaifeng. The filtered water sample was transferred into the sample cell and equilibrated in a dark room for 10 minutes. The emission wavelength was set to 685 nm, the excitation wavelength to 400-650 nm, and the scan rate to 2400 nm / min. -1 Record the integral areas of the fluorescence excitation spectra S1-S5; Using the integral areas S1-S5 as independent variables and P1-P5 as dependent variables, a linear regression analysis was performed, yielding the correlation model: P = 3.8012 × S × 10 -4 +0.6926.

[0038] Detection of fluorescence characteristic parameters of samples: A 250 mL water sample was collected at a depth of 1 m using a NisKin water sampling bottle. The filtered water sample was transferred to the sample cell and equilibrated in a dark room for 10 min. The emission wavelength was set to 685 nm, the excitation wavelength to 400-650 nm, and the scanning speed to 2400 nm / min. -1 The fluorescence intensities at 438 nm, 485 nm, and 624 nm were recorded: x1 = 49.33, x2 = 30.15, and x3 = 21.78.

[0039] Integral area calculation: The freshwater fitting equation is: S=1508.89+35.46×49.33+72.09×30.15+67.87×21.78≈6909.85.

[0040] Primary productivity calculation: Substituting into the correlation model, P = 3.8012 × S × 10 -4 +0.6926, therefore: P = 3.8012 × 6909.85 × 10 -4 +0.6926≈3.32×10 3 mgm -3 d -1 .

[0041] Result Validation The primary productivity of this sample, determined using the black-and-white bottle method, was 3.11 × 10⁻⁶. 3 mgm -3 d -1The measurement deviation of this method is (3.32-3.11) / 3.11×100%≈6.33%, which meets the accuracy requirements.

[0042] Example 3: Primary productivity measurement of seawater bodies (nearshore) Sample collection. In August 2024, five sampling points were set up in the coastal waters of Qingdao. 250 mL of seawater samples were collected at a depth of 1 m using NisKin water sampling bottles and filtered through a 200-mesh filter for later use.

[0043] Instruments and reagents: Hitachi F-4700 fluorescence spectrophotometer; 2mL quartz sample cell; other equipment includes NisKin water sampling bottle, 200-mesh filter, centrifuge, etc.

[0044] Measurement steps 3.1 Establish a correlation model between integral area and primary productivity.

[0045] The chlorophyll a content (Chl a) of water samples from five sampling points was measured, and the result was calculated using the empirical formula P = K × r × Chl a × DH (where K is an empirical constant of 1.97, and r is the assimilation coefficient: seawater 5.62 mg biomass h). -1 mg -1 Chl a, DH represents the duration of sunlight: primary productivity P1-P5 for natural water bodies is calculated based on the average daily sunshine duration of approximately 12 hours in the Qingdao coastal area. The filtered water sample was transferred into the sample cell and equilibrated in a dark room for 10 minutes. The emission wavelength was set to 685 nm, the excitation wavelength to 400-650 nm, and the scan rate to 2400 nm / min. -1 Record the integral areas of the fluorescence excitation spectra S1-S5; Using the integral areas S1-S5 as independent variables and P1-P5 as dependent variables, a linear regression analysis was performed, yielding the correlation model: P = 0.4209 × S × 10 -4 +0.2784; 3.2 Detection of Fluorescence Characteristic Parameters of Samples: A 250 mL water sample was collected at a depth of 1 m using a NisKin water sampling bottle. The filtered water sample was transferred to the sample cell and equilibrated in a dark room for 10 min. The emission wavelength was set to 685 nm, the excitation wavelength to 400-650 nm, and the scanning speed to 2400 nm / min. -1 The fluorescence intensities at 438nm, 485nm, and 624nm were recorded: x1=36.77, x2=25.58, and x3=22.47.

[0046] 3.3 Calculation of integral area: The seawater fitting equation is used: S=2388.57+82.86×36.77+11.57×25.58+74.34×22.47=7399.9926.

[0047] 3.4 Primary Productivity Calculation: Substituting into the seawater correlation model, P = 0.4209 × S × 10 -4 +0.2784, therefore: P = 0.4209 × 7399.9926 × 10 -4 +0.2784≈0.59×10 3 mgm -3 d -1 .

[0048] Result Validation The measured value using the black and white bottle method is 0.55 × 10³¹⁰. 3 mgm -3 d -1 The measurement deviation of this method is (0.59-0.55) / 0.55×100%≈6.78%, which meets the accuracy requirements.

[0049] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can be modified and varied in various ways. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for in-situ measurement of aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum, characterized in that, Includes the following steps: S1: Water samples from the target aquatic environment are collected in situ and require no additional pretreatment after impurity filtration. S2: Fluorescence excitation spectrum detection of water samples is performed using a fluorescence spectrophotometer to obtain fluorescence intensity parameters at specific characteristic wavelengths; S3: Select the corresponding fitting equation based on the water sample type, and calculate the integral area of ​​the fluorescence excitation spectrum through the fluorescence intensity parameter; S4: The primary productivity of the target water body is calculated using the correlation model between integral area and primary productivity.

2. The in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum according to claim 1, characterized in that, The impurity filtration process described in step S1 uses a 200-mesh filter screen, and the water sample is collected using an in-situ sampling device at a water depth of 1m, with a collection volume of 200-300mL.

3. The in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum according to claim 1, characterized in that, The detection parameters of the fluorescence spectrophotometer in step S2 are set as follows: emission wavelength is fixed at 685 nm, excitation wavelength scanning range is 400 nm-650 nm, and scanning speed is 2000-3000 nm·min. -1 The spectral resolution is 0.1-0.3 nm; the specific characteristic wavelengths are 438 nm, 485 nm, and 624 nm, and the corresponding fluorescence intensity parameters are denoted as x1, x2, and x3.

4. The in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum according to claim 1, characterized in that, The fitting equation described in step S3 is a first-order linear fitting equation, where: The fitting equation for the freshwater sample is: S = 1508.89 + 35.46x1 + 72.09x2 + 67.87x3; The fitting equation for the seawater sample is: S = 2388.57 + 82.86x1 + 11.57x2 + 74.34x3; The goodness of fit R of the fitting equation 2 ≥0.95, where S represents the integral area of ​​the fluorescence excitation spectrum.

5. The in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum according to claim 1, characterized in that, The correlation model mentioned in step S4 is a linear regression model, expressed as P = a × S × 10 -4 +b, where a and b are correction coefficients, P is primary productivity, and S is the integral area of ​​the fluorescence excitation spectrum.

6. The in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum according to claim 5, characterized in that, The association model is constructed in the following way: a. Measure the chlorophyll a content (Chl a) of the water sample, and calculate the initial primary productivity (P) using the formula P=K×r×Chl a×DH, where K is an empirical constant of 1.97, r is the assimilation coefficient, and DH is the sunshine duration of the target water body area. b. Using the formula S=Σ(F Ex400nm-650nm Calculate the integral area S by multiplying F by 0.2, where F Ex400nm-650nm This indicates the emission fluorescence value in the excitation wavelength range of 400-650 nm; c. Using the integral area S as the independent variable and the initial primary productivity P as the dependent variable, a linear regression analysis is performed to obtain the aforementioned correlation model.

7. The in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum according to claim 6, characterized in that, The assimilation coefficient r is taken as follows: in freshwater environment, r = 3.2 mg biomass h -1 mg -1 Chl a, r=5.62mg biomass h in seawater environment -1 mg -1 Chl a.

8. The in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum according to claim 1, characterized in that, It also includes model validation and correction steps: collecting a series of samples from a specific water body, measuring primary productivity using this method and the benchmark measurement method respectively, and obtaining at least 3 sets of paired data; Linear regression analysis was performed based on paired data to correct the coefficients in the correlation model, thereby improving the goodness of fit R0 of the corrected model. 2 ≥0.

99.

9. The in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum according to claim 1, characterized in that, The method is applicable to aquatic environments such as freshwater lakes, reservoirs, aquaculture ponds, and nearshore areas. It can withstand high temperature, low temperature, high salinity, and high light stress conditions and is suitable for photosynthetic microalgae such as cyanobacteria, green algae, diatoms, and dinoflagellates.

10. The in-situ measurement method for aquatic primary productivity based on a three-wavelength fitting model of fluorescence excitation spectrum according to claim 1, characterized in that, The method has a primary productivity measurement deviation of ≤11%, and the correlation coefficient r between the integral area and chlorophyll a content and cell density is >0.9.

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