Algae chlorophyll fluorescence kinetics measuring device

Abstract

The invention discloses an algae chlorophyll fluorescence kinetics measuring device, and belongs to the technical field of water quality detection. Comprising a sample introduction system, a light source system for exciting algae to generate fluorescence, a reference light path for generating reference light, an optical detection system for converting the fluorescence and the reference light into electric signals, and a signal processing unit for generating measurement data and reference data according to the electric signals. The signal processing unit is used for generating fluorescence dynamic curve data Er (n) according to measured data, generating stable reference data fR (n) without light source fluctuation after performing discrete Fourier transform, low-pass filtering and inverse Fourier transform on reference data, and then calculating optimized fluorescence dynamic curve data ER (n) = Er (n) / fR (n). According to the method, the problem of fluorescence excitation efficiency change caused by light source attenuation after long-term use can be solved, so that the optimized fluorescence dynamic curve is not influenced by light source fluctuation and light source attenuation.

Description

Technical Field

[0001] This invention belongs to the field of water quality testing technology, specifically, it relates to a fluorescence dynamics measurement device for detecting chlorophyll in algae in water. Background Art

[0002] Marine algae form the foundation of the marine food chain. They primarily absorb carbon dioxide and produce oxygen through photosynthesis, making them major oxygen producers in the Earth's biosphere and important pathways for marine carbon sinks. Therefore, studying the primary productivity of marine algae is crucial for understanding climate change and the function of marine ecosystems.

[0003] By measuring the fluorescence kinetics of marine algae, information on their photosynthetic efficiency and light energy conversion rate can be obtained, thereby estimating the primary productivity of marine phytoplankton. This provides important data for studying global biogeochemical cycles, marine ecosystems, and global climate change. Simultaneously, it can also be used to monitor and provide early warning of water pollution; when pollution occurs, the fluorescence signal of algae changes, thus enabling the tracing of pollution sources.

[0004] Currently, the common method for measuring the fluorescence kinetic curves of marine algae is to illuminate seawater samples with a light source of a specific wavelength to excite the algae in the seawater samples to produce fluorescence; to use a photoelectric conversion device to receive the excitation fluorescence and convert it into an electrical signal that is proportional to the fluorescence intensity; and then to fit the collected electrical signal to generate a fluorescence kinetic curve for subsequent analysis of the photosynthesis and primary productivity of marine algae.

[0005] The main problem with the above-mentioned fluorescence kinetic curve measurement method is that the light source often fluctuates during use and attenuates after long-term use. Both light source fluctuation and attenuation will cause changes in fluorescence excitation efficiency, which will lead to deviations in measurement results and affect the accuracy of marine ecological analysis. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, this invention proposes a device for measuring the chlorophyll fluorescence dynamics of algae, in order to reduce the impact of light source fluctuations or light source attenuation on the measurement results.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A device for measuring the fluorescence dynamics of algal chlorophyll, comprising:

[0009] The sampling system is used to introduce the water body to be tested;

[0010] A light source system is used to emit measurement light to excite algae in the water body to be tested to produce fluorescence.

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

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

[0013] The signal processing unit is used to perform analog-to-digital conversion on the measured electrical signal, generate measurement data, and fit and 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 execute the following process:

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

[0015] The spectrum data is filtered to remove the frequencies of noise signals;

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

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

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

[0019]

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

[0021] The calculation formula for filtering the spectrum data is as follows:

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

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

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

[0025]

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

[0027] The measurement data is smoothed using the smooth function;

[0028] The smoothed data is fitted with a regression model to generate fluorescence kinetic curves.

[0029] The residual points were identified and removed based on the fluorescence kinetic curves, resulting in the processed fluorescence kinetic curve data E. r (n).

[0030] In some embodiments of this application, in order to prevent the amplitude of the optimized fluorescence dynamics curve from being excessively attenuated and affecting the accurate judgment of the overall waveform, the energy of the reference light emitted by the reference optical path can 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 this application, the algal chlorophyll fluorescence kinetics measuring device is further provided with a light source fixing component, which is configured to have a hollow cavity. The sample introduction system is installed in the cavity. A light source mounting hole is formed on the light source fixing component, penetrating the cavity. The light source system can be configured to include several light sources, which are installed in the light source mounting hole, with the light-emitting surface of the light source facing the sample introduction system in the cavity, so as to ensure that as much light source energy as possible can act on the water body to be tested in the sample introduction system. At the same time, the inner wall of the cavity is vacuum aluminum electroplated to form a reflective surface with a reflection efficiency of over 90%, so as to enhance the strong excitation of transient fluorescence, thereby closing the photosynthetic reaction center of algae and blocking the photosynthetic electron transport chain to obtain a more accurate fluorescence kinetics curve.

[0032] In some embodiments of this application, a cuvette made of transparent material can be provided in the sample introduction system to contain the water to be tested. The cuvette is installed in the cavity of the light source fixture. To detect multiple algae in the water to be tested, multiple LED light sources for exciting diatoms 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 can be provided in the light source system. Multiple LED light sources for the same purpose can be installed in light source mounting holes on the same layer of the light source fixture to irradiate the water to be tested in the same layer of the cuvette from different directions, thereby improving the excitation efficiency of the fluorescence signal. Simultaneously, the reference light path can be designed with an identical reference light source configured for 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 fixture, 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 and generate the reference light.

[0033] In some embodiments of this application, two LED light sources with a wavelength of 440 nm and two LED light sources with a wavelength of 470 nm can be configured to excite diatoms and green algae to produce fluorescence; two LED light sources with a wavelength of 520 nm can be configured to excite cryptophytes to produce fluorescence; two LED light sources with a wavelength of 645 nm can be configured to excite cyanobacteria to produce fluorescence; four reference light sources are set in the reference optical path, namely one LED light source with a wavelength of 440 nm, one LED light source with a wavelength of 470 nm, one LED light source with a wavelength of 520 nm, and one LED light source with a wavelength of 645 nm. The system utilizes 45nm LED light sources. Specifically, three 440nm and three 470nm LED light sources are mounted in different mounting holes on the same layer of the light source fixture, with the two wavelengths arranged alternately and evenly around the cuvette. Similarly, three 520nm and three 645nm LED light sources are mounted in different mounting holes on a different layer of the light source fixture, also arranged alternately and evenly around the cuvette. This arrangement prevents light disturbance caused by direct illumination from two LED light sources of a specific wavelength simultaneously illuminating the water sample in the cuvette radially.

[0034] In some embodiments of this application, in order to achieve automatic sampling and discharge of the water to be tested, a flow pump can be installed in the measuring device and placed outside the light source fixing component. The pipeline connected to the flow pump is connected to the bottom of the cuvette to pump the water to be tested into or completely pump out of the cuvette.

[0035] In some embodiments of this 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 match the inner wall of the cuvette. In this way, when the water to be tested in the cuvette rises or falls, the floating ring can float up and down with the water to be tested, scraping 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 this application, to achieve effective collection of excitation fluorescence and reference light, a converging lens, a bandpass filter, a measurement photodiode, and a reference photodiode can be provided in the optical detection system. The converging lens can be positioned below the cuvette to collect the excitation fluorescence; the bandpass filter can be installed below the converging lens to filter out stray light and ambient light other than the desired fluorescence; the measurement photodiode can be installed below the bandpass filter to convert the fluorescence passing through the bandpass filter into a measurement electrical signal; and the reference photodiode can be installed on the light source fixture and adjacent to the reference light source to collect the reference light formed after being split by the beam splitter and convert it into a reference electrical signal.

[0037] In some embodiments of this application, an amplifier, an analog-to-digital converter (ADC), and a processor can be included in the signal processing unit. The amplifier amplifies the measurement electrical signal and the reference electrical signal. The ADC performs analog-to-digital conversion on the amplified measurement and reference electrical signals, generating corresponding measurement data and reference data. The processor controls the sequential activation of LED light sources of different wavelengths and synchronously drives multiple LED light sources of the same wavelength with constant current, ensuring that the operating conditions of the multiple LED light sources of the same wavelength are the same and that they decay synchronously, thereby guaranteeing the accuracy of the final optimization result. Simultaneously, the processor receives the measurement data and reference data output from the ADC 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 algal chlorophyll, this invention adds a reference optical path to monitor the stability of the light source after multiple measurements and the attenuation characteristics after long-term use. The light source intensity information measured by the reference optical path is used to correct the collected fluorescence kinetic curve. This solves the problem of fluorescence excitation efficiency changes caused by light source attenuation after long-term use. The optimized fluorescence kinetic curve is not affected by light source fluctuations and light source attenuation, and can maintain the accuracy of the measurement results.

[0040] 2. The algal chlorophyll fluorescence dynamics measurement device of the present invention can automatically complete the entire process of sampling the water body to be tested, excitation of fluorescence dynamics signal, detection of fluorescence dynamics signal, acquisition and processing of reference signal, and plotting and optimization of fluorescence dynamics curve, thereby improving the speed and stability of algal fluorescence dynamics measurement.

[0041] 3. This invention adds a floating ring to the cuvette in the sample introduction system. When the water to be tested is discharged from the cuvette, the floating ring also descends due to buoyancy. As the floating ring descends, it can scrape off the algae adhering to the inner wall of the cuvette and discharge it with the water to be tested. This avoids the problem that the algae sample used in the current measurement will adhere to the inner wall of the cuvette and affect the algae measurement of the next sample. As a result, the measuring device can be reused repeatedly without cleaning, which can well meet the needs of long-term in-situ measurement of marine algae.

[0042] Other features and advantages of the present invention will become clearer after reading the detailed description of the embodiments of the present invention in conjunction with the accompanying drawings. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort.

[0044] Figure 1 This is a schematic diagram of the overall structure of an embodiment of the algal chlorophyll fluorescence dynamics measurement device proposed in this invention;

[0045] Figure 2 yes Figure 1 A schematic diagram of one embodiment of the light source fixing component;

[0046] Figure 3 yes Figure 1 A cross-sectional view of the algal chlorophyll fluorescence dynamics measurement device shown;

[0047] Figure 4 yes Figure 1 Top view of the algal chlorophyll fluorescence dynamics measurement device shown;

[0048] Figure 5 yes Figure 4 FF section view;

[0049] Figure 6 This is a graph showing the fluorescence dynamics of marine algae excited by an LED light source with a wavelength of 440 nm.

[0050] Figure 7 This is a graph showing the fluorescence dynamics of marine algae excited by an LED light source with a wavelength of 470 nm.

[0051] Figure 8 This is a graph showing the fluorescence dynamics of marine algae excited by an LED light source with a wavelength of 520 nm. Detailed Implementation

[0052] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0053] In the description of this invention, it should be understood that the terms "upper", "lower", "front", "rear", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed or operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0054] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, an integral connection, or internal communication between components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances. In the description of embodiments, specific features, structures, materials, or characteristics can be combined in any suitable manner in one or more embodiments or examples.

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

[0056] like Figure 1 As shown, the algal chlorophyll fluorescence dynamics measurement device of this embodiment mainly includes a sample introduction 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 sample introduction system 100 is used to introduce the water body to be tested, such as... Figure 3 As shown, it mainly includes components such as a cuvette 110, a flow pump 120, a pipe 130 for entering and exiting the water to be tested, and a floating ring 140.

[0058] The cuvette 110, serving as a sample flow cell, can be made of a transparent material, such as quartz glass, to contain the water to be tested. In some embodiments, the cuvette 110 can be designed as a cylinder, with a sealed bottom forming a planar window 111 and a tapered top forming an exhaust passage 112. A sample inlet / outlet 113 is provided on the bottom surface or the side wall adjacent to the bottom surface of the cuvette 110. A pipe 130 is connected to the sample inlet / outlet 113, and a flow pump 120 is installed on the pipe 130. The flow pump 120 quantitatively pumps the water to be tested (e.g., seawater) into the cuvette 110 for fluorescence kinetic measurement of algal chlorophyll in the water, or pumps the water to be tested out of the cuvette 110 after the measurement is completed.

[0059] In some embodiments, a solenoid valve can be installed on the pipeline 130. By controlling the solenoid valve to open or close, and in conjunction with the forward and reverse rotation adjustment of the flow pump 120, automatic sampling and automatic discharge of the water to be tested can be achieved.

[0060] In this embodiment, the sample inlet / outlet 113 is located at the bottom of the cuvette 110. On the one hand, when the sample is introduced, the air in the cuvette 110 can be discharged to the outside through the exhaust passage 112; on the other hand, when the sample is discharged, it can ensure that all the water to be tested in the cuvette 110 is drained, so as to avoid the residual sample affecting the next round of measurement.

[0061] Simultaneously, a floating ring 140 is installed inside the cuvette 110, and the outer contour shape and size of the floating ring 140 are designed to fit the inner wall of the cuvette 110. Therefore, during sample injection, the floating ring 140 rises with the increase in liquid level in the cuvette 110, pushing the gas in the cuvette 110 upwards and discharging it to the outside through the exhaust passage 112 at the top of the cuvette 110. This solves the problem of the gas in the cuvette 110 dissolving in the water being tested, thus affecting the accuracy of the measurement results. During sample discharge, the floating ring 140 descends with the decrease in liquid level in the cuvette 110. As the floating ring 140 descends, it scrapes off algae and other deposits adhering to the inner wall of the cuvette 110, ensuring the cleanliness of the cuvette 110's inner cavity and preventing residual deposits from affecting the algae measurement results in subsequent rounds.

[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 reused repeatedly after being deployed into the sea to perform multiple measurement experiments, thus well meeting 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 illuminate the water body to be tested in the cuvette 110, so as to excite the algae in the water body to produce fluorescence.

[0064] Because seawater contains a diverse range of algae, including but not limited to diatoms, green algae, cryptophytes, and cyanobacteria, this embodiment can incorporate multiple wavelengths of light sources in the light source system 200 to excite different types of algae in the test water to produce fluorescence, generating fluorescence kinetic curves for different algae. For example, LED light sources can be set to excite diatoms and green algae to produce fluorescence, LED light sources can be set to excite cryptophytes to produce fluorescence, and LED light sources can be set to excite cyanobacteria to produce fluorescence, etc.

[0065] In some embodiments, two or more LED light sources with a wavelength of 440 nm can be used to excite marine diatoms and green algae to produce fluorescence; two or more LED light sources with a wavelength of 470 nm can also be used to excite marine diatoms and green algae to produce fluorescence with higher efficiency. Both 440 nm and 470 nm wavelength LED light sources respond well to diatoms and green algae, but their response kinetic curves are different, which can complement each other and make the measurement results more accurate.

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

[0067] Multiple LED light sources of each wavelength are set up. On the one hand, this can increase the excitation energy of the measurement light. On the other hand, LED light sources of the same wavelength can be configured to illuminate the water body to be tested in the same layer of the cuvette 110 from different directions. This can improve the excitation efficiency of the fluorescence signal and facilitate the effective acquisition and accurate detection of the fluorescence signal.

[0068] To simplify the device structure and reduce the device size, this embodiment provides two LED light sources for each wavelength. The two LED light sources of the same wavelength are identical in specifications and are arranged around the outer periphery of the cuvette 110 to irradiate the water to be tested in the cuvette 110 from different directions.

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

[0070] In some embodiments, when the light source system 200 is equipped with LED light sources of multiple wavelengths, a reference light source with a corresponding wavelength can also be set in the reference light path 300. In conjunction with the beam splitter, a reference light with the same wavelength but lower energy than the measurement light can be generated.

[0071] For example, for a light source system 200 with four specifications of LEDs: 440nm, 470nm, 520nm, and 645nm, four LED light sources of the same specification can also be set in the reference optical path 300. That is, three LED light sources are configured in the measuring device for each wavelength. One of them serves as a reference light source and, together with a beam splitter, generates reference light. The other two serve as measuring light sources and illuminate the water body to be tested in the cuvette 110 to excite the algae in the water body to produce fluorescence.

[0072] To ensure reliable installation of all LED light sources, this embodiment designs a light source fixing component 600, combined with... Figures 1 to 5 As shown, it is used to assemble an LED light source and a cuvette 110.

[0073] like Figure 2 As shown, the light source holder 600 in this embodiment can be designed as a hollow, multi-tiered cylindrical structure. The hollow cavity 620 is axially penetrating and used to mount the cuvette 110. The diameter of the cylinder in the middle part of the light source holder 600 can be slightly larger than the diameter of the cylinders at both ends. Multiple radially penetrating light source mounting holes 610 are formed on it, extending into the hollow cavity 620, for mounting the measuring light source 210 in the light source system 200 and the reference light source 310 in the reference system 300. Figure 5 As shown.

[0074] In cases where LED light sources with multiple wavelengths are configured, all LED light sources of the same wavelength or for the same purpose (including the measuring light source 210 and the reference light source 310) can be installed in the light source mounting holes 610 on the same layer of the light source fixture 600 to irradiate the water body to be tested in the same layer of the cuvette 110 from different directions.

[0075] For example, for LED light sources configured with four specifications of 440nm, 470nm, 520nm and 645nm, upper and lower layers of light source mounting holes 610 can be opened on the light source fixing component 600. The upper layer of light source mounting holes 610 is used to install LED light sources with wavelengths of 440nm and 470nm, and the lower layer of light source mounting holes 610 is used to install LED light sources with wavelengths of 520nm and 645nm.

[0076] The four wavelengths of LED light sources are distributed in two layers, upper and lower, instead of being arranged in a single layer, to reduce the diameter of the light source fixture 600 and thus the overall size of the measuring device. If the four wavelengths of LED light sources were arranged in four layers, the height of the light source fixture 600 would increase, causing the fluorescence excited by the upper LED light source to be too far from the optical detection system 400 located below the cuvette 110, affecting detection efficiency. Therefore, in this embodiment, the four wavelengths of LED light sources are arranged in two layers, positioned slightly below the center of the light source fixture 600 and close to the optical detection system 400, to ensure efficient fluorescence detection.

[0077] For each wavelength of LED light source, including two measurement light sources 210 and one reference light source 310, six upper-layer light source mounting holes 610 can be opened and arranged in a circumferentially evenly spaced manner. Three 440nm wavelength LED light sources and three 470nm wavelength LED light sources are alternately mounted in the six upper-layer light source mounting holes 610, with the light-emitting surfaces all facing the contrast cuvette 110. This avoids the situation where two measurement light sources 210 of the same wavelength are distributed on the same diameter, forming a beam-to-beam relationship.

[0078] Similarly, six mounting holes 610 can be made in the lower layer, arranged in a circumferentially spaced manner. Three 520nm wavelength LED light sources and three 645nm wavelength LED light sources are alternately installed in the six mounting holes 610 in the lower layer, and the light-emitting surfaces of all of them face the cuvette 110.

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

[0080] In addition, the inner wall of the hollow cavity 620 of the light source fixture 600 can be vacuum-plated with aluminum to form a reflective surface with a reflectivity of over 90%, for example, the reflectivity of the reflective surface can reach 94%. In this way, when the measuring light emitted by the measuring light source 210 passes through the water body to be tested in the cuvette 110 and shines on the reflective surface of the hollow cavity 620, most of the measuring light will be reflected back to the water body to be tested for re-excitation. This can enhance the strong excitation of transient fluorescence, close the photosynthetic reaction center of algae, block the photosynthetic electron transport chain, and thus obtain a more accurate fluorescence kinetic 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, such as Figure 5 As shown. The collimating lens 320 can be mounted in front of the light-emitting surface of the reference light source 310, converging the diverging light emitted from the reference light source 310 into a parallel beam, which is then directed towards the beam splitter 330. The beam splitter 330 separates a portion of the energy of the light as reference light, which is then directed towards the optical detection system 400 to monitor the stability of the light source after multiple measurements and its attenuation characteristics after long-term use.

[0082] In some embodiments, the beam splitter 330 can be mounted at a 45° angle in front of the collimating lens 320 to change the propagation direction of the reference light, making it easier for the optical detection system 400 to collect the light.

[0083] Since the reference light is used to correct the fluorescence kinetic curve, its energy cannot be too high. In some embodiments, the energy of the reference light can be configured to be less than 10% of the energy of the measurement light. This can eliminate the influence of light source fluctuations and attenuation on the measurement results, and prevent excessive attenuation of the fluorescence kinetic curve amplitude, which would affect the clear observation and accurate analysis of the overall waveform change characteristics.

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

[0085] The optical detection system 400 is used on the one hand to collect the fluorescence excited by the measuring light irradiating the water body to be tested, and convert it into a corresponding measuring 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, combined with Figure 3 , Figure 5 As shown, a converging lens 410, a bandpass filter 420, a measurement photodiode 430, and a reference photodiode 440 can be provided in the optical inspection system 400.

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

[0088] The bandpass filter 420 can be positioned below the converging lens 410 to filter out stray light and ambient light other than the desired fluorescence before directing it toward the measurement photodiode 430 for photoelectric conversion. This converts fluorescence signals of different intensities into measurement electrical signals of different magnitudes, which are then used to generate fluorescence kinetic curves.

[0089] In this embodiment, a bandpass filter 420 with a bandpass range between 400nm and 680nm can be selected to filter out excitation light and ambient light other than algal excitation fluorescence.

[0090] In some embodiments, a fixing base 460 and an optical fixing member 450 may be further provided in the optical detection system 400, such as... Figure 3 As shown. The photodiode 430 for measurement can be mounted in the fixed base 460. A circular groove 461 can be formed on the top surface of the fixed base 460, and an internal thread is formed on the inner wall of the circular groove 461. The converging lens 410 and the bandpass filter 420 are mounted on the optical fixture 450, and an external thread is formed on the outer wall of the optical fixture 450. The optical fixture 450 is mounted in the circular groove 461 of the fixed base 460, and the fixed base 460 and the optical fixture 450 are assembled and fixed by a threaded connection.

[0091] A retaining groove 462 can be formed around the outer perimeter of the circular groove 461 on the top surface of the fixed base 460, such as... Figure 3 As shown, the bottom of the light source fixing component 600 is installed in the slot 462 for positioning. Assembly holes 630 and 463 can be respectively made at corresponding positions in the overlapping area of ​​the light source fixing component 600 and the fixing base 460, as shown. Figure 1 , Figure 2 As shown, the light source fixture 600 is installed on the fixed base 460 and located above the fixed base 460 by means of screw connection.

[0092] To reduce the diameter of the light source fixture 600, the flow pump 120 can be installed outside the light source fixture 600, such as... Figure 3 As shown. A through hole 640 is formed in the light source fixing component 600, as... Figure 2 As shown, the pipeline 130 that enters and exits the water body to be tested is led out from the position of the cuvette 110 through the through hole 640 and the light source fixing part 600 is then assembled with the flow pump 120.

[0093] The reference photodiode 440 can be mounted on the light source fixture 600, combined with Figure 1 , Figure 5 As shown, it is used to receive the reference light separated by the beam splitter 330, and generate an analog electrical signal that changes accordingly based on the intensity change of the reference light, i.e., the reference electrical signal, to be used to correct the fluorescence kinetic curve.

[0094] In some embodiments, such as Figure 2 As shown, a blind hole 650 can be formed on the light source fixture 600. The blind hole 650 can be formed next to and communicate with the light source mounting hole 610 where the reference light source 310 is installed. A reference photodiode 440 is installed in the blind hole 650, allowing the reference photodiode 440 to receive the reference light separated by the beam splitter 330. By ensuring that the blind hole 650 does not communicate with the hollow cavity 620 of the light source fixture 600, the reference photodiode 440 can be prevented from erroneously receiving measurement light or exciting fluorescence, thus affecting the accurate acquisition of the reference light.

[0095] Of course, the blind hole 650 can also be replaced by opening a through hole in the light source fixing component 600 and installing a light-blocking plate in the through hole, which can also achieve the purpose of accurately collecting reference light.

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

[0097] In some embodiments, the measurement photodiode 430 and the reference photodiode 440 may be avalanche photodiodes, which are connected to the signal processing unit via a signal shielding line for signal amplification, analog-to-digital conversion, fluorescence dynamics curve fitting, and optimization.

[0098] In some embodiments, the signal processing unit may include functional circuits or electronic components such as amplifiers, analog-to-digital converters, and processors. The amplifier amplifies the measurement signal output from the measurement photodiode 430 and the reference signal output from the reference photodiode 440, respectively, and then sends them to the analog-to-digital converter for analog-to-digital conversion, thereby generating measurement data and reference data. These data are then sent to the processor for fitting and optimization of the fluorescence dynamics curve, ultimately outputting the optimized measurement result.

[0099] The following combination Figures 1 to 5 The working principle of the algal chlorophyll fluorescence dynamics measurement device in this embodiment is explained in detail.

[0100] First, the processor controls the solenoid valve installed on the pipeline 130 to open, starting the flow pump 120 and causing it to rotate forward, pumping a fixed amount of the water to be tested, such as seawater, into the cuvette 110. During this process, the floating ring 140 rises, discharging 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 drives the measurement light source 210 and the reference light source 310 of one wavelength with constant current, controlling the measurement light source 210 and the reference light source 310 of that wavelength to operate with the same working cycle and scintillation frequency, thereby exciting algae corresponding to that wavelength to produce fluorescence.

[0102] The photodiode 430 for measurement and the photodiode 440 for reference receive excitation fluorescence and reference light respectively, generating measurement electrical signals and reference electrical signals. After signal amplification and analog-to-digital conversion by amplifiers and analog-to-digital converters, measurement data and reference data are output.

[0103] The processor receives 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 smooth the received measurement data, as shown in the following formula:

[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] Where y(i) is the received measurement data; yy(i) is the smoothed measurement data; n is the number of measurement data; and the window width of the moving average filter is an odd number.

[0112] The processor generates a fluorescence kinetic curve from the smoothed measurement data by fitting a regression model. That is, a fitted curve is formed on a coordinate system where the horizontal axis represents time and the vertical axis represents the voltage value corresponding to the fluorescence intensity. Figures 6 to 8 As shown.

[0113] The processor identifies residual points in the measurement data, such as data points far from the fluorescence kinetic curve, classifying them as residual points or outliers and removing them 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, as shown in the following formula:

[0115]

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

[0117] The processor processes the generated spectrum data F r (k) Perform filtering, for example, a first-order low-pass filter, to remove the frequencies of the noise signal and obtain the filtered spectrum data F. R (k), the formula is as follows:

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

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

[0120] The processor processes the noise-filtered spectral data F R (k) Perform an inverse Fourier transform to generate stable reference data f after removing light source fluctuations. R (n), the formula is as follows:

[0121]

[0122] The processor uses the processed fluorescence kinetics data E r (n) and stable reference data f after removing light source fluctuations R (n), calculate the optimized fluorescence kinetic curve data E R (n), 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, but the amplitude is reduced. However, the influence of light source fluctuation and light source attenuation has been removed, and it can be used for subsequent analysis of marine algal photosynthesis processes and primary productivity.

[0125] The processor shuts down the measurement light source and reference light source at the current wavelength, and then turns on the measurement light source and reference light source at other wavelengths in turn, and performs constant current drive to obtain fluorescence dynamics curves under the excitation of measurement light at other wavelengths and optimize them.

[0126] After the measurement is completed, the processor opens the solenoid valve and controls the flow pump 120 to reverse, discharging the measured water from the cuvette 110. During this period, the floating ring 140 descends, scraping off the adhering substances on the inner wall of the cuvette 110 and discharging them along with the water to prevent residual substances from affecting the next round of measurement.

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

[0128] Of course, the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. An algae chlorophyll fluorescence dynamics measurement device, characterized in that: include: A sample introduction system, which is used to introduce the water to be tested; A light source system, which is used to emit measurement light to excite the algae in the water body to be measured to produce fluorescence; A reference light 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; 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; A signal processing unit is used to perform analog-to-digital conversion on the measured electrical signal, generate measurement data, and fit and 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 process: 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 inverse Fourier transform on the filtered spectrum data to generate stable reference data f without light source fluctuations. R (n); 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 a complex exponential function; N is the sequence length; j is the imaginary unit; The calculation formula for filtering the spectrum data is: F R (k)=α*F r (k)+(1-α)F R (k-1); Among them, F R (k) is the spectrum data after filtering; 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 signal processing unit processes the measurement data generated after analog-to-digital conversion as follows: Using a smooth function to smooth the measured data; The smoothed data were fitted with a regression model to generate a fluorescence kinetic curve; According to the fluorescence kinetics curve, the residual value points are determined and eliminated to obtain the processed fluorescence kinetics curve data E r (n).

4. The algae chlorophyll fluorescence dynamics measuring device according to claim 1, characterized in that: The energy of the reference light emitted by the reference light path is less than 10% of the energy of the measurement light emitted by the light source system.

5. The algae chlorophyll fluorescence dynamics measuring device according to any one of claims 1 to 4, characterized in that: Also includes: The light source fixing part has a hollow cavity, in which the sample injection system is installed, and the inner wall of the cavity is treated by vacuum aluminum electroplating to form a reflective surface, and the reflective efficiency is more than 90%; a light source installation hole penetrating the cavity is formed on the light source fixing part, and the light source system includes a plurality of light sources, which are installed in the light source installation hole, and the light emitting surface of the light source faces the sample injection system in the cavity.

6. The algae chlorophyll fluorescence dynamics measuring device according to claim 5, characterized in that: The sample injection system comprises a cuvette made of a transparent material, which is used to contain the water body to be tested; the cuvette is installed in the cavity of the light source fixing member; The light source system includes a plurality of LED light sources for stimulating diatomaceous algae and green algae to produce fluorescence, a plurality of LED light sources for stimulating cryptophytes to produce fluorescence, and a plurality of LED light sources for stimulating cyanobacteria to produce fluorescence; a plurality of LED light sources for the same purpose are installed in light source installation holes on the same level of the light source fixing member, and illuminate the water body to be tested on the same level in the cuvette from different directions; The reference light path is 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 part, and a beam splitter is arranged in the light source mounting hole in which the reference light source is installed, and the light emitted by the reference light source is split by the beam splitter to generate the reference light.

7. The algae chlorophyll fluorescence dynamics measuring device according to claim 6, characterized in that: The LED light sources used to excite diatoms and green algae to produce fluorescence include two LED light sources with a wavelength of 440nm and two LED light sources with a wavelength of 470nm; The LED light source used to excite cryptoalgae to produce fluorescence includes two LED light sources with a wavelength of 520nm; The LED light source for exciting cyanobacteria to produce fluorescence includes two LED light sources with a wavelength of 645nm; The reference light path includes 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 440nm and three LED light sources with a wavelength of 470nm are respectively installed in different light source installation holes located on the same layer of the light source fixing member, and the LED light sources of the two wavelengths are staggered and evenly arranged to surround the cuvette; Three LED light sources with a wavelength of 520nm and three LED light sources with a wavelength of 645nm are respectively installed in different light source installation holes on another level of the light source fixing member, and the LED light sources of the two wavelengths are staggered and evenly arranged to surround the cuvette.

8. The algae chlorophyll fluorescence dynamics measuring device according to claim 6, characterized in that: Also includes: A flow 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; The 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.

9. The algae chlorophyll fluorescence dynamics measuring device according to claim 6, characterized in that: The optical detection system comprises: A converging lens, located below the cuvette, for collecting the excited fluorescence; A bandpass filter, located below the converging lens, for filtering out stray light and ambient light except for the desired fluorescence; a measurement photodiode, located below the bandpass filter and used to convert the fluorescence passing through the bandpass filter into a measurement electrical signal; A reference photodiode is mounted on the light source fixture and adjacent to the reference light source, and is used to collect reference light formed after light is split by the beam splitter and convert it into a reference electrical signal.

10. The algae chlorophyll fluorescence dynamics measuring device according to claim 6, characterized in that: The signal processing unit comprises: An amplifier, which is used to amplify the measurement electrical signal and the reference electrical signal; An analog-to-digital converter, which is used to perform analog-to-digital conversion on the amplified measurement electrical signal and the reference electrical signal, and generate measurement data and reference data accordingly; 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; and receives the measurement data and reference data, and calculates the optimized fluorescence kinetic curve data.

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