In-situ fluorescence detection device and method for primary productivity of phytoplankton

Abstract

The invention discloses a phytoplankton primary productivity in-situ fluorescence detection device and method, and belongs to the technical field of aquatic ecology monitoring. The device adopts a modular integrated design and comprises a multi-band LED excitation light source, a laser induction module, a photomultiplier receiving system and an anti-vibration circuit structure, and synchronous detection of chlorophyll concentration and photosynthetic activity is realized through a short-optical-path optical design. The method is based on spectrum inversion and fluorescence kinetic analysis, a standard spectrum library is constructed, chlorophyll concentration distribution and photosynthetic parameters are calculated through least square fitting, and primary productivity data are output in combination with a photoresponse model. The device has the diameter of 67 mm, supports deployment of Argo and other mobile platforms, solves the problems that a traditional method is poor in timeliness and equipment is discrete, and has the advantages of being high in detection efficiency, high in anti-interference performance and wide in applicable water depth range.

Description

Technical Field

[0001] The present invention belongs to the technical field of phytoplankton primary productivity detection, and particularly relates to an in-situ fluorescence detection device and method for phytoplankton primary productivity. Background Art

[0002] The traditional methods for sampling and culturing phytoplankton primary productivity have a long cycle, cumbersome procedures, and poor timeliness, making it difficult to meet the current rapid monitoring requirements for phytoplankton primary productivity. As a tool for real-time and rapid analysis of the photosynthesis process of phytoplankton, the chlorophyll fluorescence method has shown broad application prospects in the field of primary productivity detection. However, the current primary productivity monitoring equipment on the market mainly faces the following technical problems: 1. Some commercial fluorometers (such as WATER-PAM) require manual operation and cannot be monitored in-situ for a long time; 2. Existing in-situ sensors (such as YSI chlorophyll sensors) only measure chlorophyll concentration and cannot directly calculate primary productivity; 3. There is a lack of an integrated device for simultaneously measuring chlorophyll concentration and primary productivity; 4. The equipment is large in volume, high in power consumption, low in sensitivity, and weak in pressure resistance, making it difficult to meet the in-situ monitoring requirements for phytoplankton primary productivity on mobile platforms such as Argo and underwater gliders. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides an in-situ fluorescence detection device and method for phytoplankton primary productivity.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] The first aspect of the present invention is to propose an in-situ fluorescence detection device for phytoplankton primary productivity, comprising:

[0006] A light window base as the main structure, with an excitation end window and a receiving end window concentrically assembled inside it. The excitation end window is made of quartz glass and is buffered from water pressure by a soft glue layer between it and the light window base;

[0007] A light source module, including multi-band LED lamp beads, white light LED lamp beads, and laser diodes distributed on the bevel of the light window base, which are respectively used for exciting fluorescence spectra, adjusting the light adaptation environment, and exciting fluorescence kinetics;

[0008] An optical receiving module, including a receiving end collimating mirror, a filter set, and a photomultiplier tube, for receiving and processing fluorescence signals;

[0009] A circuit module, which elastically fixes multi-layer detection circuit boards through support springs and connecting screws to achieve anti-vibration interference;

[0010] A sealed cabin body and a light shield, for realizing optical isolation and pressure protection for in-situ underwater detection.

[0011] In the above technical solution, the assembly gap between the excitation end window pane and the optical window base is +0.05 mm to +0.10 mm, a soft glue layer with a thickness of 0.05 mm to 0.1 mm is evenly coated on the contact surface between the two, and the thickness of the excitation end window pane is 0.1 mm to 0.15 mm shallower than that of the optical window base.

[0012] In the above technical solution, the light source module includes:

[0013] 3 groups of white light LED light sources, composed of natural white light LEDs and focusing mirrors;

[0014] 2 groups of laser diode light sources, including 454 nm blue and 624 nm red lasers;

[0015] 9 groups of multi-band LED light sources, covering a wavelength range of 380 - 750 nm.

[0016] In the above technical solution, the circuit module connects the optical window base and the circuit board fixing ring through an arc-shaped support frame, and the compression amount of the support spring is 7 - 11 mm to achieve dynamic buffering of the circuit board spacing.

[0017] In the above technical solution, the overall diameter of the device is ≤ 67 mm, and a threaded connection structure using a non-adhesive process is adopted. The cabin body and the watertight joint meet the underwater 1000 m pressure resistance requirement.

[0018] In the above technical solution, in the circuit module: the support spring is made of 304 stainless steel; the detection circuit boards are isolated by plastic cushion posts; the arc-shaped support frame connects the optical window base and the circuit board fixing ring.

[0019] The second aspect of the present invention is to propose an in-situ fluorescence detection method for phytoplankton primary productivity, which is characterized by including the following steps:

[0020] a) Construct a standard spectral library S = [S a S c S t , where S a is a 9×N order spectral matrix of dominant algal species, S c is the yellow substance spectrum, and S t is the turbidity spectrum;

[0021] b) Measure the in-situ water sample excitation fluorescence spectrum F e , and solve R = (S T · S) -1 · (S T · F e ) through the iterative least squares method. After eliminating negative concentrations, obtain the chlorophyll distribution and the algal fluorescence spectrum F a ;

[0022] c) Under dark / light adaptation conditions, the fluorescence kinetic curve F of laser-induced fluorescence is used d , and based on the model F = F0 + F V (1 - e -EσT ), the photosynthetic activity parameters F V / F M and F’ V / F’ M are calculated by fitting;

[0023] d) Combining the normalized spectrum S w of the white light source, the photosynthetic activity parameters and F a , the gross primary productivity is calculated by GPP = E·Σ(s wi ·f ai ·F’ V / F’ M / F V / F M ).

[0024] In the above technical solution, the iterative process in step b) includes: if there are negative values in the concentration matrix R, locate the spectral column corresponding to the negative value and generate a new matrix S’; recalculate (S’ T ·S’) -1 and R’ = (S’ T ·S’) -1 ·(S’ T ·F e ); iterate until all values in R’ are non-negative, and assign a value of 0 to the concentration corresponding to the missing spectrum.

[0025] In the above technical solution, in step c): the fluorescence kinetic curve F d is determined by the gradient search method to obtain the best fitting value; the fitting variance is calculated using the weighted sum of squared residuals, and the weight is inversely proportional to the fluorescence intensity.

[0026] In the above technical solution, the light intensity E of the white light source is calibrated through the following steps: measuring the actual light intensity inside the light-shielding cover using an irradiance meter; establishing a linear calibration model between the LED drive current and the light intensity; multiplying the normalized spectrum S w by E to obtain the effective light radiation energy of each band.

[0027] Beneficial effects:

[0028] 1. The innovative design of the detection module and algorithm optimization integrate the functions of chlorophyll content detection and primary productivity measurement into the same instrument, changing the traditional mode that requires multiple independent devices for separate detection, and improving the detection efficiency at the same time.

[0029] 2. The design of the short optical path optical excitation and receiving lens can better ensure the excitation efficiency and receiving efficiency. At the same time, with the cooperation of the light shield, it can avoid the interference of scattered light and bring better measurement results.

[0030] 3. The miniaturized design can expand the application flexibility of the instrument (it can be deployed at fixed sites and for mobile monitoring, and can also be carried on mobile platforms such as Argo and underwater gliders) and reduce the complexity of manual operation. At the same time, the miniaturized design also brings lower power consumption.

[0031] 4. The nut is designed in cooperation with the spring elasticity. Through the elastic deformation characteristics of the spring, it can effectively buffer mechanical interferences such as external vibrations and impacts, avoid the displacement or loosening of the circuit board due to vibrations, and ensure its installation stability in a dynamic environment.

[0032] 5. Synchronously obtaining chlorophyll content and primary productivity data can directly construct a dynamic correlation model between the two, providing a more accurate basis for correlation analysis for water body ecological assessment, ocean / lake environmental monitoring, etc., and is more scientific and systematic than the data application of traditional batch detection.

[0033] 6. Using the least squares method to perform rapid fitting analysis on the data, quickly obtaining chlorophyll concentration and photosynthetic activity parameters, thereby improving the measurement accuracy of chlorophyll concentration and primary productivity. Brief Description of the Drawings

[0034] Figure 1 is the in-situ fluorescence detection device for phytoplankton primary productivity.

[0035] Figure 2 is the internal structure diagram of the in-situ fluorescence detection device for phytoplankton primary productivity.

[0036] Figure 3 is the isometric internal structure diagram of the in-situ fluorescence detection device for phytoplankton primary productivity.

[0037] Figure 4 is the optical detection structure diagram of the in-situ fluorescence detection device for phytoplankton primary productivity.

[0038] Figure 5 is the exploded view of the optical detection structure of the in-situ fluorescence detection device for phytoplankton primary productivity.

[0039] Figure 6 is the three-part LED angle distribution diagram of the in-situ fluorescence detection device for phytoplankton primary productivity.

[0040] Figure 7 is the schematic diagram of the in-situ fluorescence detection method for phytoplankton primary productivity.

[0041] Figure 8 is the detection result diagram of chlorophyll concentration.

[0042] Figure 9 It is a fluorescence spectrum diagram of algae.

[0043] Figure 10 It is a detection result diagram of photosynthetic activity.

[0044] Among them: 1 is the receiving end window pane, 2 is the excitation end window pane, 3 is the optical window base, 4 is the white light LED focusing mirror, 5 is the laser focusing mirror, 6 is the white light LED fixing seat, 7 is the laser focusing fixing ring, 8 is the laser LED fixing seat, 9 is the laser diode, 10 is the receiving end focusing mirror thread fixing, 11 is the receiving end focusing mirror, 12 is the receiving end focusing mirror fixing ring, 13 is the filter set, 14 is the multi-band LED lamp beads, 15 is the collimating mirror filter fixing ring, 16 is the receiving end collimating mirror, 17 is the receiving end collimating mirror isolation ring, 18 is the white light LED lamp beads, 19 is the light source connection circuit board, 20 is the photomultiplier tube fixing seat, 21 is the photomultiplier tube, 22 is the bow-shaped support frame, 23 is the circuit board connection screw, 24 is the circuit board bottom fixing ring, 25 is the support spring, 26 is the detection circuit board, 27 is the plastic cushion post, 28 is the nut, 29 is the plastic gasket, 30 is the top anti-vibration ring, 31 is the light shield, 32 is the cabin body, 33 is the top end cover, 34 is the watertight joint. Specific implementation mode

[0045] The present invention will be introduced in detail below in conjunction with the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the protection scope of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0046] Embodiment

[0047] Such as Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6As shown in the figure, this embodiment discloses an in-situ fluorescence detection device for primary productivity of phytoplankton, including: receiving end window pane 1, excitation end window pane 2, optical window base 3, white light LED focusing lens 4, laser focusing lens 5, white light LED fixing seat 6, laser focusing fixing ring 7, laser LED fixing seat 8, laser diode 9, receiving end focusing lens screw fixing 10, receiving end focusing lens 11, receiving end focusing lens fixing ring 12, filter set 13, multi-band LED lamp beads 14, collimating lens filter fixing ring 15, receiving end collimating lens 16, receiving end collimating lens isolation ring 17, white light LED lamp beads 18, light source connection circuit board 19, photomultiplier tube fixing seat 20, photomultiplier tube 21, bow-shaped support frame 22, circuit board connection screw 23, circuit board bottom fixing ring 24, support spring 25, detection circuit board 26, plastic cushion column 27, nut 28, plastic gasket 29, top anti-vibration ring 30, light-shielding cover 31, cabin body 32, top end cover 33, watertight joint 34.

[0048] The optical window base 3 is the installation main body. The excitation end window pane 2 and the optical window base 3 are concentrically assembled. The outer diameter of the optical window base 3 and the inner diameter of the excitation end window pane 2 ensure a gap of +0.05mm - +0.10mm, ensuring sufficient strength of the whole after being put in and fixed with glue. The material of the excitation end window pane 2 is quartz glass, and the material of the optical window base 3 is TC4. To avoid hard contact between the excitation end window pane 2 and the optical window base caused by water pressure at deep water depths and resulting in breakage, a layer of soft glue is evenly applied on the front plane of the excitation end window pane 2 and the bottom plane of the optical window base. The thickness of the glue layer is about 0.05mm - 0.1mm. Therefore, the thickness of the excitation end window pane 2 is 0.1mm - 0.15mm shallower than the depth of the optical window base 3.

[0049] The fixing glue is evenly applied on the outer cylindrical surface and the inner cylindrical surface of the excitation end window pane 2. Similarly, the fixing glue is evenly applied on the inner and outer cylindrical surfaces of the inner groove of the optical window base 3. After concentric installation, wait for the glue to cure.

[0050] The receiving end window pane 1 and the optical window base 3 are concentrically assembled. At the same time, the height of the receiving end window pane should be 0.5mm lower than the inner cylindrical step groove of the concentrically installed optical window base 3 to prevent the excitation light from scattering into the receiving end from the side. The fixing glue is evenly applied on the outer stepped cylindrical surface of the receiving end window pane 1. Similarly, the fixing glue is evenly applied on the inner and outer cylindrical surfaces of the inner groove of the optical window base 3. After concentric installation, wait for the glue to cure.

[0051] There are 9 multi - band LED bead mounting holes, 3 white - light LED mounting holes, and 2 laser diode mounting holes distributed at the upper end of the hypotenuse of the optical window base 3. (The light sources are divided into three parts according to their functions: 1. Laser diodes, which consist of 454 - nm blue and 624 - nm red laser diodes and a converging lens group, and are mainly used to stimulate and induce algae to generate a fluorescence kinetic rise curve; 2. White - light light sources, which consist of 3 natural white - light LEDs and a converging lens group, and are mainly used to generate a light - adapted environment during photosynthetic activity measurement, so as to analyze and obtain the photosynthetic activity under light - adapted conditions; 3. Multi - band LEDs, which consist of 9 LEDs in the range of 380 - 750 nm, and are mainly used to measure the excitation fluorescence spectrum of algae, analyze the community distribution, and the absorption of algal pigments).

[0052] The multi - band LED beads 14 are evenly distributed in the multi - band LED bead mounting holes at the upper end of the hypotenuse of the optical window base 3 through concentric assembly. The white - light LED focusing lens 4 is installed into the white - light LED mounting hole of the optical window base 3 through concentric assembly. The white - light LED fixing seat 6 is installed into the white - light LED mounting hole of the optical window base 3 through concentric assembly, and the fixation of the white - light LED focusing lens 4 and the white - light LED fixing seat 6 is completed by screw - rotation extrusion. The white - light LED bead 18 is installed into the inner through - hole of the white - light LED fixing seat 6 through concentric assembly. The bottom of the white - light LED fixing seat 6 is chamfered (to avoid blocking the white - light output light); The laser focusing lens 5 and the laser focusing fixing ring 7 are installed into the laser diode mounting hole of the optical window base 3 through concentric assembly. The laser focusing fixing ring 7 presses the end face of the laser focusing lens 5. The laser diode 9 is installed into the inner groove of the laser LED fixing seat 8 through concentric assembly and is fixed by using a set - screw through the threaded hole on the cylindrical surface of the laser LED fixing seat 8.

[0053] The laser LED fixing seat 8 is screwed into the laser diode mounting hole of the optical window base 3 through its own thread to squeeze the laser focusing fixing ring 7 to complete the overall fixation; The receiving - end focusing lens 11, the receiving - end focusing lens fixing ring 12, the filter set 13, the collimating - lens filter fixing ring 15, the receiving - end collimating lens 16, and the receiving - end collimating - lens isolation ring 17 are concentrically installed into the optical window base 3 in their respective installation sequences. The receiving - end threaded focusing - lens fixing 10 is screwed into the optical window base 3 through concentric assembly and thread to squeeze the receiving - end focusing lens 11 to complete the overall fixation; The height of the receiving - end threaded focusing - lens fixing 10 has a negative tolerance of - 0.05 mm - 0.1 mm (to ensure that there is no protrusion when installed into the optical window base 3).

[0054] The photomultiplier tube 21 is connected to the photomultiplier tube fixing base 20 by threads. The lamp source connection circuit board 19 passes through the square hole of the photomultiplier tube 21. The two circular holes of the lamp source connection circuit board 19 are concentrically corresponding to the two circular holes on the photomultiplier tube fixing base 20 and the optical window base 3. The positive and negative electrodes of the lamp beads at the inclined end of the optical window base 3 are soldered into the corresponding slots of the lamp source connection circuit board by electric soldering to complete the fixing of the lamp beads.

[0055] The circuit board connection screw 23 is screwed into the corresponding threaded hole of the circuit board bottom fixing ring 24 through concentric assembly threads and passes through. The nut 28 is concentrically assembled and tightened with the protruding circuit board connection screw 23. The support spring 25 passes through the bottom of the circuit board connection screw 23 concentrically and contacts the circuit board bottom fixing ring 24. The plastic gasket 29 passes through the circuit board connection screw 23 concentrically and contacts the support spring 25. The detection circuit board 26 passes through the circuit board connection screw 23 and contacts the plastic gasket 29. The plastic pad column 27 passes through the circuit board connection screw 23 and contacts the detection circuit board 26 (ensuring that the support spring 25 does not contact the detection circuit board 26). Similarly, the subsequent installation is assembled in the order described in the figure; it should be noted that: when the nut 28 at the upper end of the third circuit board is turned, the support spring 25 is compressed, ensuring that there is a certain redundant space for the components at the bottom of the bottom detection circuit board 26 and enabling the support spring 25 to have a certain compression pre-tightening force. The height of the support spring is 20 mm, and the length of the bottom detection circuit board 26 after compressing the lower support spring 25 is 13 mm.

[0056] The nut 28 at the upper end of the fourth detection circuit board 26 compresses the support spring 25 between the third and fourth circuit boards, making its compressed length 9 mm. The nut 28 above the sixth detection circuit board 26 is tightened to complete the fixing of the entire circuit board. The third support spring passes through the circuit board connection screw 23 and contacts the nut 28. The plastic gasket passes through the circuit board connection screw 23 and contacts the support spring 25. The top anti-vibration ring 30 passes through the circuit board connection screw 23 and contacts the plastic gasket 27. The nut 28 is screwed into the circuit board connection screw 23 by threads and compresses the support spring 25. The height of the third compression spring 25 after compression is 10 mm.

[0057] After the installation of the detection circuit part is completed, the bottom of the bow-shaped support frame 22 is fixed on the optical boat base 3 by screws passing through the lamp source connection circuit board 19 and the photomultiplier tube fixing base 20 concentrically. The top of the bow-shaped support frame 22 is fixed on the circuit board bottom fixing ring 24 by screws concentrically, and the overall internal structure is assembled.

[0058] After the internal assembly is completed, a sealing ring is installed in the internal groove of the optical window base 3 for concentric assembly with the cabin body 32, and the 4 equally distributed through holes above the optical window base 3 correspond one by one to the 4 equally distributed threaded holes on the end face of the cabin body 32. The light shield 31 is concentrically assembled with the optical window base 3, and the 4 equally distributed through holes inside the light shield 3 correspond one by one to the 4 equally distributed through holes above the optical window base 3 and are fixed on the cabin body 32 by screws passing through the optical window base 3 and the light shield 31. After installing a sealing ring above the top end cover 33, it is concentrically assembled with the cabin body 32. At the same time, the 4 equally distributed through holes above the top end cover 33 correspond one by one to the 4 equally distributed threaded ports above the cabin body 32 and are fixed on the cabin body 32 by screws; the watertight joint 34 is concentrically assembled and screwed into the top end cover 33 through threaded connection.

[0059] In the instrument design, the included angle of the light source is one of the key parameters affecting the system performance. When the instrument design is limited by the miniaturization requirement, through experiments and theoretical analysis, it is determined that setting the included angle of the light source to 30° is a better choice. This angle is obtained after comprehensively considering multiple factors such as optical efficiency, spatial layout, and optical limitations brought by miniaturization, and can achieve a relatively good excitation effect in a limited space.

[0060] In the case where there is no size limit for the instrument, the included angle of the light source can be dynamically adjusted according to the change of the instrument shell size. Through research on aspects such as the optical propagation path, reflection and refraction characteristics, and the light uniformity of the target object under different shell sizes, it is found that flexibly adjusting the included angle of the light source can effectively optimize the lighting conditions, thereby achieving the best excitation effect and providing a strong guarantee for the efficient operation of the system.

[0061] Based on the above device, this embodiment also discloses a method for in-situ fluorescence detection of phytoplankton primary productivity, and the specific steps are as follows:

[0062] Step 1: Collect water samples with different spatial distributions in the research area, use the method of microscopic examination to confirm the main dominant algal species, select the same algal species cultured in the laboratory, and establish a standard spectral library S of the main dominant algal species a ;

[0063] Step 2: Take part of the water sample and place it in an autoclave for inactivation. Filter the water sample with a 0.75μm filter membrane, calibrate the yellow substance with the filtered sample to obtain the standard spectrum S of the yellow substance c , rinse the filter membrane with deionized water to obtain a suspension, calibrate the water turbidity with the suspension to obtain the standard spectrum S of the water turbidity t ;

[0064] Step 3: Combine S a , S c and S t to obtain the standard spectral library S, and transpose the standard spectral library S to matrix S TMultiply by S to obtain S T ·S, and then use Gauss-Jordan elimination to solve the inverse matrix (S T ·S) -1 ;

[0065] Step 4: Use the device to measure the excitation fluorescence spectrum F of the original water sample in the research area from 380 nm to 750 nm e , multiply S T by F e to obtain S T ·F e ;

[0066] Step 5: Multiply (S T ·S) -1 by S T ·F e to obtain the concentration result matrix R. If there are negative values in R, record the positions of the negative values, subtract the spectra at the corresponding positions in S to reproduce S, and repeat Steps 3, 4, and 5 until there are no negative values in R. Reassign the values of R according to the positions of the negative values to obtain the chlorophyll concentration. According to R and S a obtain the algal fluorescence spectrum F a ;

[0067] Step 6: Under dark adaptation conditions, use the device to measure the fluorescence kinetics curve F of the original water sample in the research area d , calculate the average fluorescence f d of the first 10 fluorescence values and the average fluorescence f 1st of the last 10 fluorescence values of F end respectively, and obtain the saturation fluorescence parameter E σ within the value range according to the fluorescence kinetics model, and form the intermediate matrix D;;

[0068] Step 7: Transpose the matrix D to obtain D T , multiply D by D to obtain the matrix D T ·D. Similarly, use Gauss-Jordan elimination to solve the inverse matrix (D T ·D) -1 , multiply D T by F d to obtain D T ·F d , multiply (D T ·D) -1 by D T ·F d to obtain the fluorescence result matrix Y. Search for the fluorescence result matrix Y with the minimum fitting variance within the value range of E σ to obtain the dark adaptation photosynthetic activity F V / F M ;

[0069] Step 8: Turn on the white light LED bead 18. Under the condition of light adaptation, repeat Steps 6 and 7 to obtain the light-adapted photosynthetic activity F’ V / F’ M;

[0070] Step 9: Input the light intensity E of the white light LED bead 18, the normalized light intensity spectrum S w , the algal fluorescence spectrum F a , the light-adapted photosynthetic activity F’ V / F’ M and the dark-adapted photosynthetic activity F V / F M into the primary productivity model to calculate and obtain the primary productivity GPP.

[0071] In this embodiment, the standard spectral library S of the main dominant algal species a is a 9×N order matrix, where 9 is the number of spectral points and N is the number of main dominant algal species;

[0072] In this embodiment, the standard spectrum S of the yellow substance c and the standard spectrum S of the water turbidity t are both 9×1 order matrices;

[0073] In this embodiment, the standard spectral library S = [S a S c S t ] is a 9×(N + 2) order matrix;

[0074] In this embodiment, the matrix S T · S is a (N + 2)×(N + 2) positive definite matrix, its determinant |S T · S| ≠ 0, and the inverse matrix (S T · S) -1 is a (N + 2)×(N + 2) positive definite matrix;

[0075] In this embodiment, the excitation fluorescence spectrum F e is a 9×1 order matrix. The multi-band LED bead 14 is used as the excitation light source. The excitation light is focused by the excitation end window 2 to form a light spot, which irradiates the water sample in the light-shielding cover 31 to induce the chlorophyll to generate multi-band fluorescence. The fluorescence is first converged by the receiving end window 1, then collimated by the receiving end collimating mirror 16, and then the stray light is filtered by the filter group 13 to obtain the emitted fluorescence at 680 nm. Finally, it is converted into an electrical signal by the photomultiplier tube 21 and output as the excitation fluorescence spectrum F e , and the matrix S T · F e is a (N + 2)×1 matrix;

[0076] In this embodiment, the concentration result matrix R = (S T · S) -1 · (S T · Fe ) = [r1..r i …r N r N+1 r N+2 T is an (N + 2)×1 matrix, where r i is the concentration of the i-th dominant algal species, r N+1 is the concentration of yellow substances, r N+2 is the water turbidity, and the algal fluorescence spectrum F a = [f a1 …f ai …f a9 is the superposition of the concentrations of each dominant algal species multiplied by the corresponding standard spectra, where f ai is the algal fluorescence at the i-th spectral point;

[0077] In this implementation, the fluorescence kinetics curve F d is a K×1 matrix. A laser diode 9 is used as the excitation light source. The excitation light is focused by the excitation end window 2 to form a light spot, which irradiates the water sample in the light-shielding cover 31 to induce the fluorescence kinetics rising process of chlorophyll. The fluorescence is first converged by the receiving end window 1, then collimated by the receiving end collimating mirror 16, and then the stray light is filtered by the filter group 13 to obtain the emitted fluorescence at 680 nm. Finally, it is converted into an electrical signal by the photomultiplier tube 21 and enters the signal detection circuit for processing and then outputs the fluorescence kinetics curve F d , and the fluorescence kinetics model is: F = F0 + F V (1 - e -EσT ), where F = [f1 f2…f K T is the variable fluorescence, F0 is the initial fluorescence, F V is the maximum variable fluorescence, T = [t1 t2…t K is the sampling time series, and the average value of the first 10 fluorescence f 1st = Σi = 10 i = 1f i / 10, and the average value of the last 10 fluorescence f end = Σi = K i = K - 9f i / 10. By inputting f 1st and f end into the fluorescence kinetics model, the value range of E σ can be obtained, and the intermediate matrix D = [D1…D i …D K T , where D i = [1 1 - e -Eσti ;

[0078] In this implementation, the matrix D T ·D is a 2×2 positive definite matrix, and the determinant |D​​​T ·D|≠0, the inverse matrix (D T ·D) -1 is a 2×2 matrix, and the matrix D T ·F d is a 2×1 matrix, and the fluorescence result matrix Y = (D T ·D) -1 ·(D T ·F d ) = [F0F V ) T is a 2×1 matrix;

[0079] In this implementation, the normalized spectral S of the light intensity of the white light LED lamp bead 18 w = [s w1 …s wi …s w9 is obtained by measuring with a spectrometer and normalizing the light intensity. s wi is the white light spectral response of the i-th spectral point. The primary productivity model is GPP = E·Σi = 9 i = 1s wi ·f ai ·F’ V / F’ M / F V / F M .

[0080] When performing chlorophyll concentration detection, the multi-band LED lamp beads 14 are sequentially lit. The excitation light forms a light spot after being focused by the excitation end window 2 and irradiates the water sample in the light-shielding cover 31, inducing the chlorophyll to generate multi-band fluorescence. The fluorescence is first converged by the receiving end window 1, then collimated by the receiving end collimating mirror 16, and then the stray light is filtered by the filter group 13 to obtain the emitted 680nm fluorescence. Finally, it is converted into an electrical signal by the photomultiplier tube 21 and enters the signal detection circuit for processing to output the excitation fluorescence spectrum. The least squares method is used to perform spectral inversion on the excitation fluorescence spectrum to obtain the chlorophyll concentration information.

[0081] When performing dark adaptation photosynthetic activity detection, first turn off the white light LED lamp bead 18 to ensure that the inside of the light-shielding cover 31 is in a dark adaptation environment. Then turn on the laser diode 9 as the excitation light source. The excitation light forms a light spot after being focused by the excitation end window 2 and irradiates the water sample in the light-shielding cover 31, inducing the chlorophyll to generate the rising process of fluorescence kinetics. The fluorescence is first converged by the receiving end window 1, then collimated by the receiving end collimating mirror 16, and then the stray light is filtered by the filter group 13 to obtain the emitted 680nm fluorescence. Finally, it is converted into an electrical signal by the photomultiplier tube 21 and enters the signal detection circuit for processing to output the fluorescence kinetics curve. The least squares method is used to perform fitting inversion on the fluorescence kinetics curve to obtain the dark adaptation photosynthetic activity.

[0082] When performing light - adapted photosynthetic activity detection, first turn on the white - light LED lamp bead 18 to ensure that the inside of the light - shielding cover 31 is in a light - adapted environment. Then turn on the laser diode 9 as the excitation light source. The excitation light forms a light spot after being focused by the excitation - end window pane 2 and irradiates the water sample inside the light - shielding cover 31, inducing the fluorescence kinetic rise process of chlorophyll. The fluorescence is first converged by the receiving - end window pane 1, then collimated by the receiving - end collimating mirror 16, and then the stray light is filtered out by the filter set 13 to obtain the emitted fluorescence at 680 nm. Finally, it is converted into an electrical signal by the photomultiplier tube 21 and enters the signal detection circuit for processing, and then outputs the fluorescence kinetic curve. The least - squares method is used to fit and invert the fluorescence kinetic curve to obtain the light - adapted photosynthetic activity.

[0083] When performing primary productivity detection, chlorophyll concentration detection, dark - adapted photosynthetic activity detection, and light - adapted photosynthetic activity detection are carried out in sequence to obtain key fluorescence parameters, and the primary productivity parameters are obtained by inputting them into the primary productivity model.

[0084] When performing the rapid light - response curve detection, based on the primary productivity detection, the light intensity of the white - light LED lamp bead 18 is gradually adjusted from low to high, and the primary productivity parameters under different light intensities are obtained. Fitting the primary productivity under different light intensities can obtain the rapid light - response curve.

[0085] As Figure 7 shown, a method for in - situ fluorescence detection of phytoplankton primary productivity is as follows: Step 1: Collect water samples with different spatial distributions in the research area, use microscopic examination to confirm the main dominant algal species, select the same algal species cultured in the laboratory, and establish the standard spectral library S of the main dominant algal species a ;

[0086] Step 2: Take part of the water sample and place it in an autoclave for inactivation. Filter the water sample with a 0.75 - μm filter membrane, calibrate the yellow substance with the filtered sample to obtain the standard spectrum S of the yellow substance c , rinse the filter membrane with deionized water to obtain a suspension, calibrate the water turbidity with the suspension to obtain the standard spectrum S of the water turbidity t ;

[0087] Step 3: Combine S a , S c and S t to obtain the standard spectral library S;

[0088] Step 4: Use the device to measure the excitation fluorescence spectrum F at 380 nm - 750 nm of the original water sample in the research area e , use the least - squares method for spectral inversion to obtain the concentration result matrix R and the algal fluorescence spectrum F a ;

[0089] Step 5. Under dark adaptation conditions, use the device to measure the fluorescence kinetic curve F of the original water sample in the research area, and use the least squares method for fitting inversion to obtain the dark adaptation photosynthetic activity F d / F V ; M ;

[0090] Step 6. Turn on the white light LED bead 18. Under light adaptation conditions, use the device to measure the fluorescence kinetic curve F’ d of the original water sample in the research area, and use the least squares method for fitting inversion to obtain the light adaptation photosynthetic activity F’ V / F’ M;

[0091] Step 7. Input the light intensity E of the white light LED bead 18, the normalized light intensity spectrum S w , the algal fluorescence spectrum F a , the light adaptation photosynthetic activity F’ V / F’ M and the dark adaptation photosynthetic activity F V / F M into the primary productivity model to calculate and obtain the primary productivity GPP.

[0092] Specifically, the in-situ fluorescence detection effect of phytoplankton primary productivity is as follows:

[0093] Select Dongpu Reservoir as the research area, collect water samples from different areas, establish an algal standard spectral library by microscopic examination, take some water samples and inactivate them in an autoclave, filter the water samples with a 0.75μm filter membrane, calibrate the yellow substances with the filtered samples to obtain the yellow substance standard spectrum, rinse the filter membrane with deionized water to obtain the suspension, calibrate the water turbidity with the suspension to obtain the water turbidity standard spectrum, and combine the algal, yellow substance and turbidity standard spectra to generate the standard spectral library. Use the device for primary productivity detection. The chlorophyll concentration detection results are as Figure 8 shown. The total chlorophyll concentration is 12.63μg / L, of which green algae account for 12.5% and diatoms account for 87.5%. Combining with the algal standard spectral library, the algal fluorescence spectrum can be obtained as Figure 9 shown. The photosynthetic activity detection results are as Figure 10 shown. The dark adaptation photosynthetic activity is 0.44, and the light adaptation photosynthetic activity is 0.38. Input the detected parameters into the primary productivity model, and the primary productivity can be analyzed to be 47.92nmol(e) / m 3 / s.

[0094] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. An in-situ fluorescence detection device for primary productivity of phytoplankton, characterized in that: include: The light window base (3) serves as a main structure, and is provided with an excitation end window sheet (2) and a receiving end window sheet (1) which are concentrically assembled. The excitation end window sheet (2) is made of quartz glass, and a soft rubber layer is used between the light window base (3) to buffer water pressure. The light source module comprises a multi-band LED lamp bead (14), a white light LED lamp bead (18) and a laser diode (9) distributed on the oblique edge of the light window base (3), and is used for stimulating fluorescence spectrum, light adaptation environment regulation and fluorescence dynamics stimulation respectively; An optical receiving module, comprising a receiving end collimator (16), a filter set (13) and a photomultiplier tube (21), and used for receiving and processing fluorescent signals; The circuit module elastically fixes the multi-layer detection circuit board (26) through the support spring (25) and the circuit board connecting screw (23) to achieve anti-vibration interference; The sealed cabin (32) and the light shield (31) realize optical isolation and pressure protection for underwater in-situ detection.

2. The device according to claim 1, characterized in that The assembly gap between the excitation end window sheet (2) and the light window base (3) is +0.05mm-+0.10mm, the contact surfaces of the two are evenly coated with a 0.05mm-0.1mm thick soft glue layer, and the thickness of the excitation end window sheet is 0.1mm-0.15mm shallower than that of the light window base.

3. The device according to claim 1, characterized in that The light source module comprises: 3 sets of white light LED light sources, consisting of white light LED lamp beads (18) and white light LED focusing lenses (4); 2 sets of laser diode light sources, including 454nm blue and 624nm red lasers; 9 sets of multi-band LED light sources, covering the wavelength range of 380-750nm.

4. The device according to claim 1, characterized in that The circuit module is connected to the light window base (3) and the fixing ring (24) at the bottom of the circuit board via the arched support frame (22); the compression amount of the support spring (25) is 7-11 mm, thereby achieving dynamic buffering of the circuit board spacing.

5. The device according to claim 1, characterized in that The overall diameter of the device is ≤67 mm, and a threaded connection structure without adhesive technology is adopted. The cabin (32) and the watertight joint (34) meet the pressure resistance requirements of 1000 m underwater.

6. The device according to claim 1, characterized in that In the circuit module: the support spring (25) is made of 304 stainless steel; the detection circuit boards (26) are isolated by plastic pads (27); and the arched support frame (22) connects the light window base (3) and the circuit board fixing ring (24).

7. A method for in situ fluorescence detection of phytoplankton primary productivity, characterized in that: The following steps are involved: a) Construct a standard spectral library S = [S a S c S t ], where S a is the 9×N-order spectrum matrix of dominant algae species, S c is the spectrum of yellow substance, S t is the turbidity spectrum; b) Measure the fluorescence spectrum of the in-situ water sample F e , solve R=(S T ·S) -1 ·(S T ·F e ), and then eliminate the negative concentration to obtain the chlorophyll distribution and algae fluorescence spectrum F a ; c) Under dark / light adaptation conditions, laser-induced fluorescence kinetic curve F d , based on the model F=F0+F V (1-e -EσT ) Fitting calculation of photosynthetic activity parameter F V / F M and F' V / F' M ; d) Combined with the normalized spectrum S of the white light source w , photosynthetic activity parameters and F a , through GPP = E · Σ (s wi ·f ai ·F' V / F' M / F V / F M )Calculate primary productivity.

8. The method according to claim 7, characterized in that The iterative process in step b) includes: if there are negative values ​​in the concentration matrix R, locate the spectrum column corresponding to the negative value and generate a new matrix S'; recalculate (S' T ·S') -1 and R'=(S' T ·S') -1 ·(S' T ·F e );Iterate until all R' values ​​are non-negative, and the concentration corresponding to the missing spectrum is assigned to 0.

9. The method according to claim 7, characterized in that: In step c): fluorescence kinetic curve F d The best fit value was determined by gradient search method; the fitting variance was calculated using weighted residual sum of squares, with the weights inversely proportional to the fluorescence intensity.

10. The method according to claim 7, characterized in that The light intensity E of the white light source is calibrated by the following steps: using a radiometer to measure the actual light intensity in the light shield (31); establishing a linear correction model between LED driving current and light intensity, and converting the normalized spectrum S w Multiplying by E gives the effective light radiation energy in each band.

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