An apparatus for measuring the fluorescence kinetics of algal chlorophyll

Abstract

The present invention discloses an algae chlorophyll fluorescence kinetics measurement device, belonging to the technical field of water quality detection, which includes a sampling system, a light source system for exciting algae to generate fluorescence, a reference optical path for generating reference light, an optical detection system for converting fluorescence and reference light into electrical signals, and a signal processing unit for generating measurement data and reference data according to the electrical signals. The signal processing unit generates fluorescence kinetics curve data according to the measurement data E r (n) , and after performing discrete Fourier transform, low-pass filtering, and inverse Fourier transform on the reference data, generates stable reference data with the light source fluctuations removed f R (n) , and then calculates the optimized fluorescence kinetics curve data E R (n) = E r (n) / f R (n) . The present invention can solve the problem of the change in fluorescence excitation efficiency caused by the attenuation of the light source after long-term use, so that the optimized fluorescence kinetics curve is not affected by light source fluctuations and light source attenuation

Description

Technical Field

[0001] The present invention belongs to the technical field of water quality detection, and specifically relates to a fluorescence kinetics measurement device for detecting algal chlorophyll in water bodies. Background Art

[0002] Marine algae are the basis of the marine food chain. They mainly absorb carbon dioxide through photosynthesis and produce oxygen, being the main oxygen producers in the Earth's biosphere and an important way for the marine carbon sink. Therefore, studying the primary productivity of marine algae is crucial for understanding climate change and the functions of marine ecosystems.

[0003] By measuring the fluorescence kinetics curve of marine algae, information about the photosynthesis efficiency and light energy conversion rate of algae can be obtained, thereby estimating the primary productivity of marine phytoplankton and providing important data for studying the global biogeochemical cycle, marine ecosystems, and global climate change. At the same time, it can also be used for monitoring and warning water pollution. When pollution occurs, the fluorescence signal of algae will change, thus realizing the tracking of pollution sources.

[0004] At the present stage, when measuring the fluorescence kinetics curve of marine algae, the commonly used method is to irradiate a seawater sample with a light source of a specific wavelength to excite the algae in the seawater sample to produce fluorescence; use a photoelectric conversion device to receive the excited fluorescence and convert it into an electrical signal proportional to the fluorescence intensity, and then generate a fluorescence kinetics curve by fitting the collected electrical signal for later analysis of the photosynthesis and primary productivity of marine algae.

[0005] The main problems existing in the above fluorescence kinetics curve measurement method are: the light source often fluctuates during use, and there will be a problem of light source attenuation after long-term use. Both light source fluctuation and light source attenuation will cause changes in the fluorescence excitation efficiency, thereby resulting in deviations in the measurement results and affecting the accuracy of marine ecological analysis. Summary of the Invention

[0006] In order to solve the above problems existing in the prior art, the present invention proposes an algal chlorophyll fluorescence kinetics measurement device to reduce the influence of light source fluctuation or light source attenuation on the measurement results.

[0007] To solve the above technical problems, the present invention is implemented by adopting the following technical solutions:

[0008] An algal chlorophyll fluorescence kinetics measurement device, comprising:

[0009] A sample injection system for introducing the water body to be measured;

[0010] A light source system for emitting measurement light to excite the algae in the water body to be measured to produce fluorescence;

[0011] A reference optical path, which operates synchronously with the light source system and is used to generate reference light with the same wavelength but lower energy than the measurement light;

[0012] An optical detection system, which is used to collect the fluorescence respectively and convert it into a measurement electrical signal, and collect the reference light and convert it into a reference electrical signal;

[0013] A signal processing unit, which is used to perform analog-to-digital conversion on the measurement electrical signal, generate measurement data, and fit to generate fluorescence kinetic curve data E r (n); it is used to perform analog-to-digital conversion on the reference electrical signal, generate reference data, and perform the following processes:

[0014] Perform a discrete Fourier transform on the reference data to generate spectral data;

[0015] Filter the spectral data to filter out the frequencies of the noise signals;

[0016] Perform an inverse Fourier transform on the filtered spectral data to generate stable reference data f R (n) that removes the light source fluctuations;

[0017] Calculate the optimized fluorescence kinetic curve data E R (n) = E r (n) / f R (n).

[0018] In some embodiments of the present application, the formula for performing a discrete Fourier transform on the reference data can be configured as:

[0019]

[0020] Among them, F r (k) is the spectral data generated after the discrete Fourier transform; f r (n) is the reference data; k is the frequency of the frequency-domain signal; n is the sampling point in the time domain; is the complex exponential function; N is the sequence length; j is the imaginary unit;

[0021] The calculation formula for filtering the spectral data is:

[0022] F R (k) = α * F r (k) + (1 - α)F R (k - 1);

[0023] Among them, F R (k) is the spectral data after filtering out the noise; α is the filtering coefficient;

[0024] The formula for performing inverse Fourier transform on the filtered spectral data is as follows:

[0025]

[0026] In some embodiments of the present application, in order to remove noise and improve the signal-to-noise ratio, the signal processing unit may be configured to process the measurement data generated after analog-to-digital conversion as follows:

[0027] Smooth the measurement data using the smooth function;

[0028] Generate a fluorescence kinetics curve from the smoothed data through a fitting regression model;

[0029] Judge and eliminate the residual value points according to the fluorescence kinetics curve to obtain the processed fluorescence kinetics curve data E r (n).

[0030] In some embodiments of the present application, in order to prevent the amplitude of the optimized fluorescence kinetics curve from attenuating excessively and affecting the accurate judgment of the overall waveform, the energy of the reference light emitted by the reference optical path may be configured to be less than 10% of the energy of the measurement light emitted by the light source system.

[0031] In some embodiments of the present application, a light source fixing member is further provided in the algal chlorophyll fluorescence kinetics measurement device. It is configured to have a hollow cavity, and the sampling system is installed in the cavity. A light source installation hole penetrating the cavity is formed on the light source fixing member. The light source system may be configured to include several light sources, and the light sources are installed in the light source installation holes, and the light-emitting surfaces of the light sources face the water body to be measured in the cavity, so as to ensure that as much light source energy as possible can act on the water body to be measured in the sampling system; at the same time, the inner wall of the cavity is subjected to vacuum aluminum electroplating treatment to form a reflecting surface with a light reflection efficiency of more than 90%, so as to enhance the strong excitation of transient fluorescence, thereby closing the photosynthetic reaction center of the algae and blocking the photosynthetic electron transport chain to obtain a more accurate fluorescence kinetics curve.

[0032] In some embodiments of the present application, a cuvette made of a transparent material may be provided in the sample injection system for accommodating the water body to be measured; the cuvette is installed in the cavity of the light source fixing member; in order to detect various algae in the water body to be measured, a plurality of LED light sources for exciting the fluorescence of dinoflagellates and green algae, a plurality of LED light sources for exciting the fluorescence of cryptophytes, and a plurality of LED light sources for exciting the fluorescence of cyanobacteria may be provided in the light source system; a plurality of LED light sources with the same use may be installed in the light source mounting holes on the same layer of the light source fixing member, and the water body to be measured on the same layer in the cuvette is irradiated from different directions to improve the excitation efficiency of the fluorescence signal. At the same time, the reference optical path may be designed to be configured with a reference light source identical to each wavelength of the LED light source in the light source system, the reference light source is installed in the light source mounting hole on the light source fixing member, and a beam splitter is provided in the light source mounting hole where the reference light source is installed to split the light emitted by the reference light source to generate the reference light.

[0033] In some embodiments of the present application, two LED light sources with a wavelength of 440 nm and two LED light sources with a wavelength of 470 nm may be configured to excite the fluorescence of dinoflagellates and green algae; two LED light sources with a wavelength of 520 nm may be configured to excite the fluorescence of cryptophytes; two LED light sources with a wavelength of 645 nm may be configured to excite the fluorescence of cyanobacteria; four reference light sources are provided in the reference optical path, namely an LED light source with a wavelength of 440 nm, an LED light source with a wavelength of 470 nm, an LED light source with a wavelength of 520 nm, and an LED light source with a wavelength of 645 nm; among them, three LED light sources with a wavelength of 440 nm and three LED light sources with a wavelength of 470 nm may be respectively installed in different light source mounting holes on the same layer of the light source fixing member, and the LED light sources of the two wavelengths are arranged alternately and evenly around the cuvette for one week; three LED light sources with a wavelength of 520 nm and three LED light sources with a wavelength of 645 nm are respectively installed in different light source mounting holes on another layer of the light source fixing member, and the LED light sources of the two wavelengths are arranged alternately and evenly around the cuvette for one week. In this way, when two LED light sources for emitting measurement light with a certain wavelength irradiate the water body to be measured in the cuvette radially at the same time, light disturbance caused by the two LED light sources facing each other can be prevented.

[0034] In some embodiments of the present application, in order to realize the automatic sampling and discharging of the water body to be measured, a flow pump may be provided in the measuring device, which is arranged outside the light source fixing member, and the pipeline connecting the flow pump is connected to the bottom of the cuvette to pump the water body to be measured into the cuvette or completely pump it out of the cuvette.

[0035] In some embodiments of the present application, a floating ring can also be provided in the measuring device and installed in the inner cavity of the cuvette. The outer contour shape and size of the floating ring are designed to be compatible with the inner wall of the cuvette. In this way, when the water body to be measured in the cuvette rises or falls, the floating ring can float up and down with the water body to be measured and scrape the inner wall of the cuvette to prevent algae from adhering to the inner wall of the cuvette and affecting the next round of measurement.

[0036] In some embodiments of the present application, in order to achieve effective collection of excited fluorescence and reference light, a converging lens, a bandpass filter, a measuring photodiode and a reference photodiode may be provided in the optical detection system; wherein the converging lens may be arranged below the cuvette to collect the excited fluorescence; the bandpass filter may be installed below the converging lens to filter out stray light and ambient light other than the desired fluorescence; the measuring photodiode may be installed below the bandpass filter to convert the fluorescence passing through the bandpass filter into a measuring electrical signal; the reference photodiode may be installed on the light source fixture and adjacent to the reference light source to collect the reference light formed after the light is split by the spectrometer and convert it into a reference electrical signal.

[0037] In some embodiments of the present application, an amplifier, an analog-to-digital converter, and a processor may be provided in the signal processing unit; wherein the amplifier is used to amplify the measurement electrical signal and the reference electrical signal; the analog-to-digital converter is used to perform analog-to-digital conversion on the measurement electrical signal and the reference electrical signal after the amplification process, and correspondingly generate measurement data and reference data; the processor controls the LED light sources of different wavelengths to start in turn, and performs synchronous constant current driving on multiple LED light sources of the same wavelength, so that the working conditions of multiple LED light sources of the same wavelength are the same, and they decay synchronously, so as to ensure the accuracy of the final optimization result. At the same time, the processor receives the measurement data and reference data output by the analog-to-digital converter to calculate the optimized fluorescence kinetic curve data.

[0038] Compared with the prior art, the advantages and positive effects of the present invention are mainly reflected in:

[0039] 1. In the process of measuring the fluorescence kinetic curve of algae chlorophyll, the present invention monitors the stability of multiple measurements of the light source and the attenuation characteristics after long-term use by adding a reference light path, and uses the light source intensity information measured by the reference light path to correct the collected fluorescence kinetic curve, thereby solving the problem of changes in fluorescence excitation efficiency caused by light source attenuation after long-term use, so that the optimized fluorescence kinetic curve will not be affected by light source fluctuations and light source attenuation, and can well maintain the accuracy of the measurement results.

[0040] 2. The algal chlorophyll fluorescence kinetics measurement device of the present invention can automatically complete the whole process of sampling the water body to be measured, exciting the fluorescence kinetics signal, detecting the fluorescence kinetics signal, collecting and processing the reference signal, and plotting and optimizing the fluorescence kinetics curve, improving the speed and stability of algal fluorescence kinetics measurement.

[0041] 3. By adding a floating ring in the cuvette of the sampling system, when the water body to be measured in the cuvette is drained, due to the buoyancy effect, the floating ring also descends. While the floating ring descends, it can scrape off the algae adhering to the inner wall of the cuvette and discharge them with the water body to be measured from the cuvette. Thus, it can avoid the problem that the algal sample used in this round of measurement adheres to the inner wall of the cuvette and affects the algal measurement of the next round of samples. Subsequently, the measurement device can be reused without cleaning, which can well meet the requirements of long-term in-situ measurement of marine algae.

[0042] After reading the detailed description of the embodiments of the present invention in conjunction with the drawings, other features and advantages of the present invention will become clearer. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. For those skilled in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0044] Figure 1 It is a schematic diagram of the overall structure of an embodiment of the algal chlorophyll fluorescence kinetics measurement device proposed by the present invention;

[0045] Figure 2 is Figure 1 a schematic diagram of the structure of an embodiment of the light source fixing member in

[0046] Figure 3 is Figure 1 a sectional view of the algal chlorophyll fluorescence kinetics measurement device shown in

[0047] Figure 4 is Figure 1 a top view of the algal chlorophyll fluorescence kinetics measurement device shown in

[0048] Figure 5 is Figure 4 a sectional view in the F-F direction of

[0049] Figure 6 It is a fluorescence kinetics curve graph of marine algae excited by an LED light source with a wavelength of 440 nm;

[0050] Figure 7 It is a kinetic curve of marine algal fluorescence excited by an LED light source with a wavelength of 470 nm;

[0051] Figure 8 It is a kinetic curve of marine algal fluorescence excited by an LED light source with a wavelength of 520 nm. Specific embodiments

[0052] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the protection scope of the present invention.

[0053] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "upper", "lower", "front", "rear", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed or operated in a specific orientation, and therefore should not be construed as a limitation of the present invention.

[0054] In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, the terms "installation", "connection", and "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection, or the internal connection of components. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations. In the description of the embodiments, specific features, structures, materials, or characteristics can be combined in a suitable manner in any one or more embodiments or examples.

[0055] In the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0056] As Figure 1 shown, the algal chlorophyll fluorescence kinetic measurement device of this embodiment mainly includes a sampling system 100, a light source system 200, a reference optical path 300, an optical detection system 400, a signal processing unit and other components.

[0057] The sampling system 100 is used to introduce the water body to be measured. As Figure 3 shown, it mainly includes a cuvette 110, a flow pump 120, a pipeline 130 for entering and leaving the water body to be measured, a floating ring 140 and other components.

[0058] Among them, the cuvette 110 serves as a sample flow cell and can be made of a transparent material such as quartz glass for accommodating the water body to be measured. In some embodiments, the cuvette 110 can be designed in a cylindrical shape with the bottom surface sealed to form a flat window 111 and the top narrowed to form an exhaust passage 112. A sample inlet and outlet 113 is provided on the bottom surface or the side wall adjacent to the bottom surface of the cuvette 110. The sample inlet and outlet 113 is externally connected to a pipeline 130, and a flow pump 120 is installed on the pipeline 130. The flow pump 120 quantitatively pumps the water body to be measured (such as seawater, etc.) from the outside into the cuvette 110 for fluorescence kinetics measurement of algae chlorophyll in the water body, or pumps out the water body to be measured after the measurement from the cuvette 110.

[0059] In some embodiments, a solenoid valve can be installed on the pipeline 130. By controlling the opening or closing of the solenoid valve and coordinating with the forward and reverse rotation adjustment of the flow pump 120, automatic sampling and automatic discharging of the water body to be measured can be achieved.

[0060] In this embodiment, the sample inlet and outlet 113 is provided at the bottom of the cuvette 110. On the one hand, during sampling, the air in the cuvette 110 can be discharged to the outside through the exhaust passage 112. On the other hand, during sample discharging, it can ensure that all the water body to be measured in the cuvette 110 is completely discharged to avoid the influence of residual samples on the next round of measurement.

[0061] Meanwhile, a floating ring 140 is installed in the inner cavity of the cuvette 110, and the outer contour shape and size of the floating ring 140 are designed to be adapted to the inner wall of the cuvette 110. Thus, during sampling, the floating ring 140 can float as the liquid level in the cuvette 110 rises, thereby pushing the gas in the cuvette 110 upward and discharging it to the outside through the exhaust passage 112 at the top of the cuvette 110. In this way, the problem that the gas in the cuvette 110 dissolves in the water body to be measured and affects the accuracy of the measurement results can be solved. During sample discharging, the floating ring 140 descends as the liquid level in the cuvette 110 drops. While the floating ring 140 descends, it can scrape off the attachments such as algae adhering to the inner wall of the cuvette 110. While ensuring the cleanliness of the inner cavity of the cuvette 110, the problem that the residual attachments affect the next round of algae measurement results can be solved.

[0062] Since the floating ring 140 can automatically clean the inner cavity of the cuvette 110 after each round of measurement, the measuring device can be repeatedly used and perform multiple measurement tests after being put into the sea, thus being able to well meet the actual needs of long-term in-situ monitoring of algae in the ocean.

[0063] The light source system 200 is used to emit measurement light to irradiate the water body to be measured in the cuvette 110 to excite the algae in the water body to produce fluorescence.

[0064] Since there are diverse types of algae in seawater, including but not limited to diatoms, green algae, cryptophytes, cyanobacteria, etc., in order to analyze the photosynthesis efficiency and light energy conversion rate of different algae, in this embodiment, light sources of multiple wavelengths can be arranged in the light source system 200 to excite different types of algae in the water to be measured to generate fluorescence and generate fluorescence kinetic curves of different algae. For example, LED light sources for exciting diatoms and green algae to generate fluorescence, LED light sources for exciting cryptophytes to generate fluorescence, LED light sources for exciting cyanobacteria to generate fluorescence, etc. can be set.

[0065] In some embodiments, two or more LED light sources with a wavelength of 440 nm can be set to excite marine diatoms and green algae to generate fluorescence; two or more LED light sources with a wavelength of 470 nm can also be set to excite marine diatoms and green algae to generate fluorescence with higher efficiency. The LED light sources with wavelengths of 440 nm and 470 nm have good responses to diatoms and green algae, but the kinetic curves of the responses are different and can complement each other, making the measurement results more accurate.

[0066] Two or more LED light sources with a wavelength of 520 nm can be set to excite marine cryptophytes to generate fluorescence; two or more LED light sources with a wavelength of 645 nm can be set to excite marine cyanobacteria to generate fluorescence.

[0067] Multiple LED light sources of each wavelength are set. On the one hand, it can increase the excitation energy of the measurement light. On the other hand, LED light sources of the same wavelength can be configured to irradiate the water to be measured on the same layer in the cuvette 110 from different directions. Thereby, the excitation efficiency of the fluorescence signal can be improved, facilitating the effective collection and accurate detection of the fluorescence signal.

[0068] For the consideration of simplifying the device structure and reducing the device volume, in this embodiment, two LED light sources of each of the above wavelengths are set. The two LED light sources of the same wavelength have the same specifications and are arranged around the outer periphery of the cuvette 110 to irradiate the water to be measured in the cuvette 110 from different directions.

[0069] The reference optical path 300 is used to generate a reference light with the same wavelength as the measurement light emitted by the light source system 200 but with lower energy than the measurement light, and runs synchronously with the light source system 200 to achieve the purpose of synchronous attenuation with the light source system 200.

[0070] In certain embodiments, for the case where there are LED light sources of multiple wavelengths set in the light source system 200, reference light sources with corresponding wavelengths can also be respectively set in the reference optical path 300, and together with the beam splitter, generate reference light with the same wavelength but lower energy than the measurement light.

[0071] For example, for the light source system 200 equipped with four types of LEDs with specifications of 440 nm, 470 nm, 520 nm, and 645 nm, four LEDs of the same specifications can also be set in the reference optical path 300. That is, three LEDs of each wavelength are respectively configured in the measuring device, one of which is used as the reference light source, and together with the beam splitter, it generates reference light; the other two are used as measuring light sources to irradiate the water sample to be measured in the cuvette 110 to excite the algae in the water to produce fluorescence.

[0072] To reliably install all the LED light sources, in this embodiment, a light source fixing member 600 is designed, as shown in combination with Figures 1 to 5 to be used for assembling the LED light source and the cuvette 110.

[0073] As Figure 2 shown, the light source fixing member 600 of this embodiment can be designed as a hollow multi-stage cylinder structure, and the hollow cavity 620 penetrates axially for installing the cuvette 110. The diameter of the cylinder in the middle part of the light source fixing member 600 can be slightly larger than the diameters of the cylinders at its two ends, and a plurality of light source mounting holes 610 radially penetrating into the hollow cavity 620 are formed thereon for installing the measuring light source 210 in the light source system 200 and the reference light source 310 in the reference system 300, as shown in combination with Figure 5 shown.

[0074] For the case of being equipped with multiple wavelengths of LED light sources, all the LED light sources (including the measuring light source 210 and the reference light source 310) with the same wavelength or the same purpose can be installed in the light source mounting holes 610 on the same layer of the light source fixing member 600 to irradiate the water sample to be measured on the same layer in the cuvette 110 from different directions.

[0075] For example, for the case of being equipped with four types of LED light sources with specifications of 440 nm, 470 nm, 520 nm, and 645 nm, upper and lower layers of light source mounting holes 610 can be opened on the light source fixing member 600. The upper-layer light source mounting holes 610 are used for installing the LED light sources with wavelengths of 440 nm and 470 nm, and the lower-layer light source mounting holes 610 are used for installing the LED light sources with wavelengths of 520 nm and 645 nm.

[0076] The four-wavelength LED light sources are distributed in the upper and lower layers instead of on the same layer, in order to reduce the diameter of the light source fixture 600 and thus reduce the overall size of the measuring device. If the four-wavelength LED light sources are distributed in four layers, the height of the light source fixture 600 will increase, resulting in too long a distance between the fluorescence excited by the LED light sources in the upper layer and the optical detection system 400 below the cuvette 110, which will affect the detection efficiency. Therefore, in this embodiment, the four-wavelength LED light sources are distributed in two layers and are located in the middle and lower part of the light source fixture 600, close to the optical detection system 400, to ensure the detection efficiency of the excited fluorescence.

[0077] For the case where each wavelength of LED light source includes two measuring light sources 210 and one reference light source 310, six upper-layer light source mounting holes 610 can be opened and arranged in a circumferential equally spaced manner. Three 440-nm wavelength LED light sources and three 470-nm wavelength LED light sources are alternately installed in the six upper-layer light source mounting holes 610, and the light-emitting surfaces are all directly opposite the cuvette 110, so as to avoid having two measuring light sources 210 of the same wavelength distributed on the same diameter and forming a counter-irradiation relationship.

[0078] Similarly, six lower-layer light source mounting holes 610 can also be opened and arranged in a circumferential equally spaced manner. Three 520-nm wavelength LED light sources and three 645-nm wavelength LED light sources are alternately installed in the six lower-layer light source mounting holes 610, and the light-emitting surfaces are all facing the cuvette 110.

[0079] A collimating lens 220 can be added to each light source mounting hole 610 where a measuring light source 210 is installed, as Figure 3 shown, and it is arranged in front of the light-emitting surface of the measuring light source 210, used to collimate the measuring light emitted by the measuring light source 210 to form a parallel light beam to irradiate the water sample to be measured in the cuvette 110, so as to achieve the purpose of improving the fluorescence excitation efficiency.

[0080] In addition, the inner wall of the hollow cavity 620 of the light source fixture 600 can be subjected to vacuum aluminum electroplating treatment to form a reflecting surface on the inner wall of the hollow cavity 620, with a light reflection efficiency of over 90%, for example, the light reflection efficiency of the reflecting surface can be configured to reach 94%. In this way, when the measuring light emitted by the measuring light source 210 passes through the water sample to be measured in the cuvette 110 and irradiates the reflecting surface of the hollow cavity 620, most of the measuring light will be reflected back to the water sample to be measured for re-excitation, thereby enhancing the strong excitation of transient fluorescence, closing the photosynthetic reaction center of algae, blocking the photosynthetic electron transport chain, and thus obtaining a more accurate fluorescence kinetics curve.

[0081] In each light source mounting hole 610 where a reference light source 310 is installed, a collimating lens 320 and a beam splitter 330 can be respectively arranged, as Figure 5 shown. Among them, the collimating lens 320 can be installed in front of the light-emitting surface of the reference light source 310 to converge the divergent light rays emitted by the reference light source 310 into parallel light beams and direct them towards the beam splitter 330. A part of the light rays with separated energy is used as the reference light and directed towards the optical detection system 400 to monitor the stability of multiple measurements of the light source and the attenuation characteristics after long-term use.

[0082] In some embodiments, the beam splitter 330 can be installed at an angle of 45° in front of the collimating lens 320 to change the propagation direction of the reference light, facilitating the acquisition by the optical detection system 400.

[0083] Since the reference light is used to correct the fluorescence kinetic curve, its energy cannot be too large. In some embodiments, the energy of the reference light can be configured to be less than 10% of the energy of the measurement light. In this way, it can not only eliminate the influence of light source fluctuations and light source attenuation on the measurement results, but also prevent the amplitude of the fluorescence kinetic curve from attenuating excessively, affecting the clear observation and accurate analysis of the overall waveform change characteristics.

[0084] As an embodiment, the splitting ratio of the beam splitter 330 can be configured as 1:9, that is, 10% of the parallel light beam formed after collimating the light rays emitted by the reference light source 310 is separated as the reference light and transmitted to the optical detection system 400. The remaining 90% of the parallel light beam can be incident on the water body to be measured in the cuvette 110 to further excite the algae in the water body to produce fluorescence.

[0085] The optical detection system 400 is used on the one hand to collect the fluorescence excited by the measurement light irradiating the water body to be measured and convert it into a corresponding measurement electrical signal according to the fluorescence intensity; on the other hand, it is used to collect the reference light output by the reference optical path 300 and convert it into a corresponding reference electrical signal according to the intensity of the reference light.

[0086] In this embodiment, as shown in Figure 3 、 Figure 5 , a converging lens 410, a band-pass filter 420, a measurement photodiode 430, and a reference photodiode 440 can be arranged in the optical detection system 400.

[0087] Among them, as shown in Figure 3 , the converging lens 410 can be arranged below the cuvette 110, for example, below the planar window 111 at the bottom of the cuvette 110, to collect the fluorescence generated by the measurement light exciting the algae in the water body to be measured and transmit it to the band-pass filter 420.

[0088] The band - pass filter 420 can be disposed below the converging lens 410. After filtering out stray light and ambient light other than the required fluorescence, it is directed towards the photodiode 430 for measurement, so as to perform photoelectric conversion, and then convert fluorescence signals of different intensities into measurement electrical signals of different magnitudes, which are used to generate a fluorescence kinetics curve.

[0089] In this embodiment, a band - pass filter 420 with a band - pass range between 400 nm and 680 nm can be selected to filter out the excitation light and ambient light other than the fluorescence excited by algae.

[0090] In some embodiments, a fixed base 460 and an optical fixing member 450 can be further provided in the optical detection system 400, as Figure 3 shown. Among them, the photodiode 430 for measurement can be installed in the fixed base 460. A circular groove 461 can be opened on the top surface of the fixed base 460, and internal threads are formed on the inner wall of the circular groove 461. The converging lens 410 and the band - pass filter 420 are installed on the optical fixing member 450, and external threads are formed on the outer wall of the optical fixing member 450. The optical fixing member 450 is installed in the circular groove 461 of the fixed base 460, and the assembly and fixation of the fixed base 460 and the optical fixing member 450 are realized by means of threaded connection.

[0091] A circular groove 462 can be opened around the periphery of the circular groove 461 on the top surface of the fixed base 460, as Figure 3 shown, and the bottom of the light source fixing member 600 is installed in the circular groove 462 for positioning. Assembly holes 630 and 463 can be respectively opened at corresponding positions in the overlapping area of the light source fixing member 600 and the fixed base 460, as Figure 1 、 Figure 2 shown, and the light source fixing member 600 is installed on the fixed base 460 above the fixed base 460 by means of screw connection.

[0092] In order to reduce the diameter of the light source fixing member 600, the flow pump 120 can be disposed outside the light source fixing member 600, as Figure 3 shown. A through - hole 640 is opened on the light source fixing member 600, as Figure 2 shown. After the pipeline 130 for entering and leaving the water to be measured is led out of the light source fixing member 600 through the through - hole 640 from the position of the cuvette 110, it is assembled with the flow pump 120.

[0093] The reference photodiode 440 can be installed on the light source fixing member 600. Combining Figure 1 、 Figure 5 shown, it is used to receive the reference light separated by the beam splitter 330 and generate an analog electrical signal that changes correspondingly according to the intensity change of the reference light, that is, a reference electrical signal, which is used to correct the fluorescence kinetics curve.

[0094] In some embodiments, as Figure 2 shown, a blind hole 650 may be formed in the light source fixture 600. The blind hole 650 may be formed beside the light source mounting hole 610 where the reference light source 310 is mounted and communicate with the light source mounting hole 610 where the reference light source 310 is mounted. The reference photodiode 440 is installed in the blind hole 650 so that the reference photodiode 440 can receive the reference light separated by the beam splitter 330. The blind hole 650 is arranged not to communicate with the hollow inner cavity 620 of the light source fixture 600, which can prevent the reference photodiode 440 from erroneously receiving the measurement light or the excited fluorescence, affecting the accurate acquisition of the reference light.

[0095] Of course, it is also possible to adopt the method of forming a through hole in the light source fixture 600 and installing a light shielding plate in the through hole to replace the blind hole 650, which can also achieve the purpose of accurately collecting the reference light.

[0096] For the reference optical path configured with multiple reference light sources 310, a reference photodiode 440 should be respectively configured for each reference light source 310 to receive the reference light of different wavelengths and form multiple paths of reference electrical signals, which are respectively sent to the signal processing unit.

[0097] In some embodiments, the measuring photodiode 430 and the reference photodiode 440 may be avalanche photodiodes, which are connected to the signal processing unit through signal shielding wires for signal amplification, analog-to-digital conversion, fluorescence kinetic curve fitting and optimization, etc.

[0098] In some embodiments, functional circuits or electronic components such as an amplifier, an analog-to-digital converter, and a processor may be arranged in the signal processing unit. Among them, the amplifier is used to respectively amplify the measurement electrical signal output by the measuring photodiode 430 and the reference electrical signal output by the reference photodiode 440, and then send them to the analog-to-digital converter for analog-to-digital conversion, thereby generating measurement data and reference data, which are sent to the processor for fitting to generate a fluorescence kinetic curve and optimization, and finally output the optimized measurement result.

[0099] Next, in combination with Figures 1 to 5 , the specific working principle of the algae chlorophyll fluorescence kinetics measuring device of this embodiment will be elaborated in detail.

[0100] First, the processor controls the solenoid valve installed on the pipeline 130 to open, starts the flow pump 120 and rotates forward to pump a fixed amount of the water body to be measured, such as seawater, into the cuvette 110. During this period, the floating ring 140 floats upward to discharge the air in the cuvette 110 to the outside through the exhaust passage 112.

[0101] The processor controls the flow pump 120 and the solenoid valve to close, and then performs constant current drive on the measurement light source 210 and the reference light source 310 of one of the wavelengths, controlling the measurement light source 210 and the reference light source 310 of this wavelength to operate with the same duty cycle and flashing frequency, and exciting the algae corresponding to this wavelength to generate fluorescence.

[0102] Start the measurement photodiode 430 and the reference photodiode 440 to receive the excited fluorescence and the reference light respectively, generate a measurement electrical signal and a reference electrical signal, and after signal amplification and analog-to-digital conversion processing by the amplifier and the analog-to-digital converter, output the measurement data and the reference data.

[0103] The processor receives the measurement data and performs smoothing processing to remove noise and improve the signal-to-noise ratio.

[0104] In some embodiments, the smooth function can be used to perform smoothing processing on the received measurement data, and the formula is as follows:

[0105] yy(1) = y(1);

[0106] yy(2) = [y(1) + y(2) + y(3)] / 3;

[0107] yy(3) = [y(1) + y(2) + y(3) + y(4) + y(5)] / 5;

[0108] yy(4) = [y(2) + y(3) + y(4) + y(5) + y(6)] / 5;

[0109] ……

[0110] yy(n - 2) = [y(n - 4) + y(n - 3) + y(n - 2) + y(n - 1) + y(n)] / 5;

[0111] Among them, y(i) is the received measurement data; yy(i) is the measurement data after smoothing processing; n is the number of measurement data; the window width of the moving average filter takes an odd number.

[0112] The processor generates a fluorescence kinetic curve from the measurement data after smoothing processing through a fitting regression model, that is, a fitting curve is formed in the coordinate system with the abscissa being time and the ordinate being the voltage value corresponding to the fluorescence intensity, as Figures 6 to 8 shown.

[0113] The processor judges the residual points in the measurement data. For example, the data points far from the fluorescence kinetic curve are judged as residual value points or abnormal points and are excluded to obtain the processed fluorescence kinetic curve data E r (n).

[0114] The processor performs a discrete Fourier transform on the received reference data to generate spectral data, and the formula is as follows:

[0115]

[0116] Where F r (k) is the spectral data generated after the discrete Fourier transform; f r (n) is the reference data, which is a discrete signal; k is the frequency of the frequency-domain signal; n is the sampling point in the time domain; is the complex exponential function; N is the sequence length; j is the imaginary unit.

[0117] The processor filters the generated spectral data F r (k), for example, performs a first-order low-pass filtering process to filter out the frequency of the noise signal, and obtains the filtered spectral data F R (k), and the formula is as follows:

[0118] F R (k) = α * F r (k) + (1 - α)F R (k - 1);

[0119] Where α is the filtering coefficient, which takes values between (0, 1).

[0120] The processor performs an inverse Fourier transform on the spectral data F R (k) after removing the noise to generate the stable reference data f R (n) that removes the light source fluctuation, and the formula is as follows:

[0121]

[0122] The processor calculates the optimized fluorescence kinetic curve data E r (n) according to the processed fluorescence kinetic curve data E R (n) and the stable reference data f R (n) that removes the light source fluctuation, and the formula is as follows:

[0123] E R (n) = E r (n) / f R (n).

[0124] Compared with the fluorescence kinetic curve before optimization, the overall waveform of the optimized fluorescence kinetic curve remains unchanged, the amplitude decreases, but the influence of light source fluctuation and light source attenuation is removed, and it can be used for the analysis of the photosynthesis process and primary productivity of marine algae in the later stage.

[0125] The processor turns off the measuring light source and the reference light source of the current wavelength, and then starts the measuring light source and the reference light source of other wavelengths in turn, and drives them at a constant current to obtain and optimize the fluorescence kinetic curve under the excitation of the measuring light of other wavelengths.

[0126] After the measurement is completed, the processor opens the solenoid valve and controls the flow pump 120 to reverse, and discharges the measured water from the cuvette 110. During this period, the floating ring 140 descends, scrapes off the attachments adhering to the inner wall of the cuvette 110, and discharges them together with the water to be tested, so as to prevent the residual attachments from affecting the next round of measurement tests.

[0127] The algae chlorophyll fluorescence dynamics measurement device of this embodiment can automatically complete the entire measurement process, has a fast measurement speed, good stability, and outputs high accuracy measurement results. It can also be reused without manual cleaning, and can well meet the actual needs of long-term in-situ monitoring of the ocean.

[0128] Of course, the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. An apparatus for measuring the fluorescence kinetics of algal chlorophyll, characterized in that, include: A light source fixing part, which has a hollow cavity, the inner wall of which is vacuum aluminum-plated to form a reflective surface, and the reflective efficiency is more than 90%; a light source mounting hole penetrating the cavity is formed on the light source fixing part; The sample introduction system comprises a cuvette made of a transparent material for containing the water body to be tested; the cuvette is installed in the cavity of the light source fixing member, and the top of the cuvette is narrowed to form an exhaust passage; flow A pump, which is located outside the light source fixture and connected to the bottom of the cuvette through a pipeline, and is used to pump the water to be tested into or out of the cuvette; A floating ring is installed in the inner cavity of the cuvette, and the shape and size of its outer contour are adapted to the inner wall of the cuvette. When the water body to be tested in the cuvette rises or falls, the floating ring floats up and down with the water body to be tested, and scrapes the inner wall of the cuvette; A light source system, comprising a light source of multiple wavelengths, for emitting measurement light of different wavelengths to stimulate different types of algae in the water body to produce fluorescence; A reference light path, wherein a reference light source identical to each wavelength of the light source in the light source system is respectively configured, the reference light source is installed in a light source installation hole on the light source fixing member, and a beam splitter is arranged in the light source installation hole where the reference light source is installed, and the light emitted by the reference light source is split by the beam splitter to generate reference light with the same wavelength but energy less than 10% of the energy of the measurement light emitted by the light source system; An optical detection system, which is used to collect the fluorescence and convert it into a measurement electrical signal, collect the reference light and convert it into a reference electrical signal; The signal processing unit is used to control the light sources of different wavelengths to start in turn, and to drive the measurement light source and the reference light source of the same wavelength synchronously with constant current, and to perform analog-to-digital conversion on the measurement electrical signal to generate measurement data, to perform analog-to-digital conversion on the reference electrical signal to generate reference data, and to perform the following processes: Using a smooth function to smooth the measured data; The smoothed measurement data are fitted with a regression model to generate a fluorescence kinetic curve; Determine the data points far from the fluorescence kinetic curve as residual value points or abnormal points, and eliminate them to obtain the processed fluorescence kinetic curve data E r (n); Performing discrete Fourier transform on the reference data to generate spectrum data; Filtering the spectrum data to remove the frequency of the noise signal; Perform an inverse Fourier transform on the filtered spectral data to generate stable reference data f R (n) that removes the light source fluctuations; Calculate the optimized fluorescence kinetic curve data E R (n) = E r (n) / f R (n).

2. The algae chlorophyll fluorescence dynamics measuring device according to claim 1, characterized in that: The formula for performing discrete Fourier transform on the reference data is: Among them, F r (k) is the spectrum data generated after discrete Fourier transform; f r (n) is the reference data; k is the frequency of the frequency-domain signal; n is the sampling point in the time domain; is the complex exponential function; N is the sequence length; j is the imaginary unit; The calculation formula for filtering the spectrum data is: F R F(k) = α * F r F(k) + (1 - α)F R F(k - 1); Among them, F R (k) is the filtered spectral data; α is the filtering coefficient; The formula for performing inverse Fourier transform on the filtered spectrum data is:

3. The algae chlorophyll fluorescence dynamics measuring device according to claim 1, characterized in that: The light source system includes multiple LED light sources for exciting diatomaceous algae and green algae to produce fluorescence, multiple LED light sources for exciting cryptophytes to produce fluorescence, and multiple LED light sources for exciting cyanobacteria to produce fluorescence; multiple LED light sources for the same purpose are installed in light source installation holes located on the same level of the light source fixing part, and illuminate the water body to be tested on the same level in the cuvette from different directions.

4. The algal chlorophyll fluorescence kinetics measurement device according to claim 3, wherein: The LED light sources for exciting fluorescence in dinoflagellates and green algae include two LED light sources with a wavelength of 440 nm and two LED light sources with a wavelength of 470 nm; The LED light sources for exciting fluorescence in cryptophytes include two LED light sources with a wavelength of 520 nm; The LED light sources for exciting fluorescence in cyanobacteria include two LED light sources with a wavelength of 645 nm; In the reference optical path, there are four reference light sources, namely an LED light source with a wavelength of 440 nm, an LED light source with a wavelength of 470 nm, an LED light source with a wavelength of 520 nm, and an LED light source with a wavelength of 645 nm; Among them, three LED light sources with a wavelength of 440 nm and three LED light sources with a wavelength of 470 nm are respectively installed in different light source mounting holes on the same layer of the light source fixing member, and the two wavelengths of LED light sources are staggered and evenly arranged, surrounding the cuvette for one week; Three LED light sources with a wavelength of 520 nm and three LED light sources with a wavelength of 645 nm are respectively installed in different light source mounting holes on another layer of the light source fixing member, and the two wavelengths of LED light sources are staggered and evenly arranged, surrounding the cuvette for one week.

5. The algal chlorophyll fluorescence kinetics measurement device according to claim 3, wherein The signal processing unit includes: An amplifier for amplifying the measured electrical signal and the reference electrical signal; An analog-to-digital converter for performing analog-to-digital conversion on the amplified measured electrical signal and reference electrical signal, respectively generating measured data and reference data; A processor that controls the sequential startup of LED light sources with different wavelengths and performs synchronous constant current drive on multiple LED light sources with the same wavelength; and receives the measured data and reference data, and calculates the optimized fluorescence kinetics curve data.

6. The algal chlorophyll fluorescence kinetics measurement device according to any one of claims 1 to 5, characterized in that, The optical detection system includes: A converging lens located below the cuvette for collecting the excited fluorescence; A band-pass filter located below the converging lens for filtering out stray light and ambient light other than the required fluorescence; A photodiode for measurement located below the band-pass filter for converting the fluorescence passing through the band-pass filter into a measured electrical signal; A photodiode for reference installed on the light source fixing member and adjacent to the reference light source for collecting the reference light formed by splitting the light through the beam splitter and converting it into a reference electrical signal.

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