A method and system for non-destructive determination of chlorophyll content

Abstract

The present invention relates to a non-destructive determination method and system for chlorophyll content, including: establishing an empirical conversion model of fluorescence images and chlorophyll content; calculating the specific leaf weight of the leaf by measuring the leaf area and dry weight, and taking the value of the specific leaf weight as the value of parameter B; extracting the chlorophyll solution of the same leaf, diluting the chlorophyll solution into different concentration gradients, and using the same leaf cross-section fluorescence imaging device to obtain the fluorescence images of the chlorophyll solutions with different concentration gradients; obtaining the basic fluorescence yield after digitization, inputting the concentration gradient of the chlorophyll solution and the basic fluorescence yield into the empirical conversion model for fitting, and obtaining the value of parameter M; inputting the basic fluorescence yield of the fluorescence image of the leaf to be measured into the empirical conversion model after determining the parameters to be determined, and obtaining the chlorophyll concentration of the leaf to be measured. The empirical conversion model is established based on the chlorophyll fluorescence imaging technology and the traditional chlorophyll content extraction method to realize the non-destructive determination of the chlorophyll content of any plant.

Description

Technical Field

[0001] The present invention relates to a method and system for non-destructive determination of chlorophyll content, belonging to the technical field of plant photosynthesis measurement. Background Art

[0002] Photosynthesis is the material basis for the survival and growth of all life on earth. Chlorophyll is the most important pigment in photosynthesis and is the uniquely active group in the reaction centers of photosystem II (PSII) and photosystem I (PSI) of the photosynthesis mechanism. Together with other pigment molecules, it absorbs and utilizes light energy for photosynthesis. The chlorophyll content is closely related to the photosynthetic ability of plants and further affects the growth and development of plants. Chlorophyll fluorescence is the fluorescence emitted by chlorophyll molecules in the plant photosynthesis mechanism after being excited by light. It not only contains the structural information of the photosynthetic mechanism but also reflects the dynamic information of the photosynthetic process. Almost all changes in the photosynthetic process can be reflected by chlorophyll fluorescence. The chlorophyll fluorescence technology has the characteristics of in vivo measurement, simplicity, rapidity, reliability, etc. and has been widely used internationally. The chlorophyll content of plant leaves and its distribution in the mesophyll tissue directly reflect the light energy absorption and utilization and CO2 fixation ability at the leaf level. According to Figure 1 the time course of the fast fluorescence kinetics curve, i.e., the OJIP curve, as shown, within the first 30 us (before point O) after plants absorb light energy, only very weak fluorescence is generated by chlorophyll, which is called the basic fluorescence yield, and its intensity is related to the chlorophyll content. Then, the photochemical reaction is initiated, and the fluorescence gradually increases, accompanied by fluorescence quenching. Therefore, the fluorescence yield at point O directly reflects the chlorophyll content and its distribution inside the leaf.

[0003] Common chlorophyll content determination methods can be roughly divided into three categories: The most widely used is the solvent extraction method. After extracting the leaves with ethanol or acetone, the absorbance is measured by colorimetry, and the chlorophyll content is calculated through a formula. The second is the optical instrument determination method, which uses a chlorophyll meter to measure the relative chlorophyll content of the leaves, such as the SPAD-502 or CCI-200 chlorophyll meter. The third is the estimation based on spectral data. A relationship model is established between the remote sensing data of a hyperspectral spectrometer and the solvent extraction method to estimate the chlorophyll content. The solvent extraction method obtains the contents of chlorophyll a, chlorophyll b, and carotenoids by extracting and colorimetrically analyzing chloroplast pigments, with the highest accuracy. However, it requires destructive sampling, the process is cumbersome, and the extract is easily photo-oxidized, resulting in measurement errors. The optical instrument method using a chlorophyll meter is fast and convenient to measure and does not require destroying plant samples. However, due to the differences in the optical properties of leaves, the irregular distribution of palisade tissue and spongy tissue inside the leaves and their different light absorption and transmission properties, as well as the differences in the ratio of chlorophyll a and chlorophyll b, the measurement results of different species with the same chlorophyll content will vary greatly. The determination based on spectral data is mostly used for the prediction of agricultural remote sensing, and the estimation error at the leaf level is relatively large. Summary of the Invention

[0004] In view of the above problems, the object of the present invention is to provide a non-destructive method and system for measuring chlorophyll content, which establishes an empirical conversion model based on the chlorophyll fluorescence imaging technology of the leaf cross-section and the traditional chlorophyll content extraction method, and verifies it with different plants to achieve non-destructive measurement of the chlorophyll content of any plant.

[0005] To achieve the above object, the present invention proposes the following technical solutions: A non-destructive method for measuring chlorophyll content, comprising the following steps: establishing an empirical conversion model between the fluorescence image and the chlorophyll content according to the correlation between the basic fluorescence generated by chlorophyll under weak light and the chlorophyll content, the empirical conversion model including undetermined parameter M and parameter B; calculating the specific leaf weight of the leaf by measuring the leaf area and dry weight of the leaf, and taking the value of the specific leaf weight as the value of parameter B; extracting the chlorophyll solution of the same leaf, diluting the chlorophyll solution into different concentration gradients, and using the fluorescence imaging device of the same leaf cross-section to obtain the fluorescence images of the chlorophyll solutions with different concentration gradients, and digitizing to obtain the basic fluorescence yield F; inputting the concentration gradient of the chlorophyll solution and the basic fluorescence yield F into the empirical conversion model for fitting to obtain the value of parameter M; inputting the basic fluorescence yield F of the leaf to be measured into the empirical conversion model after determining the undetermined parameter to obtain the chlorophyll concentration of the leaf to be measured.

[0006] Further, the calculation formula of the empirical conversion model is:

[0007] C = -lg(1 - F / M) / B

[0008] Wherein, C is the chlorophyll content, F is the basic fluorescence yield; M is the instrument characteristic constant, and B is the leaf characteristic constant.

[0009] Further, the calculation formula of parameter B is:

[0010]

[0011] Wherein, B is the leaf characteristic constant, the leaf characteristic constant takes the value of the specific leaf weight, m is the dry weight, and SL is the leaf area.

[0012] Further, the calculation method of the concentration of the chlorophyll solution is: testing the absorbance of the chlorophyll solution by a spectrophotometer, and calculating the concentration of the chlorophyll solution according to the Lambert-Beer law through the absorbance.

[0013] Further, the measurement method of the fluorescence images of the chlorophyll solutions with different concentrations is: placing the chlorophyll solutions with different concentration gradients on the fluorescence imaging device of the leaf cross-section, and irradiating with short-pulse strong light to obtain the solution fluorescence images.

[0014] Further, the short-pulse intense light uses blue laser with a wavelength of 400nm - 500nm, and the light intensity is not less than 200000 μmolm -2 s -1 , and the pulse width is not greater than 30 us.

[0015] The present invention also discloses a non-destructive chlorophyll content measurement system for the non-destructive chlorophyll content measurement method of any one of the above, including: a leaf chamber, a laser light source, a CCD image acquisition device, and a data processing module; the leaf chamber is used to place the leaf; the laser light source is used to generate laser and irradiate the chlorophyll solution and the leaf with the generated laser; the CCD image acquisition device is used to acquire the fluorescence images of the chlorophyll solution and the cross-section of the leaf; the data processing module is used to process the acquired fluorescence images to obtain the basic fluorescence yield F.

[0016] Further, a dichroic mirror is arranged between the laser light source and the CCD image acquisition device. The laser generated by the laser light source is reflected by the dichroic mirror onto the chlorophyll solution or the cross-section of the leaf. The fluorescence generated by the chlorophyll solution or the cross-section of the leaf reaches the dichroic mirror through the microscope objective lens and is projected onto the CCD image acquisition device by the dichroic mirror.

[0017] Further, the CCD image acquisition device uses a high-sensitivity black-and-white CCD image acquisition device. A short-wave cut-off filter is arranged in front of the CCD image acquisition device to only acquire the chlorophyll fluorescence of the chlorophyll solution or the cross-section of the leaf in the range of 680 - 750 nm, avoiding the contamination of incident light or ambient stray light.

[0018] Further, the data processing module converts the fluorescence images acquired by the CCD image acquisition device into grayscale images, selects several cross-sections in the grayscale images, calculates the average value of the pixel points of each cross-section, and after standardization, it is the basic fluorescence yield F.

[0019] Due to the above technical solutions adopted by the present invention, it has the following advantages:

[0020] 1. The present invention establishes an empirical conversion model based on the chlorophyll fluorescence imaging technology of the leaf cross-section and the traditional chlorophyll content extraction method, and verifies it through different plants, realizing the non-destructive measurement of the chlorophyll content of any plant.

[0021] 2. The present invention uses a high-light-intensity short-pulse laser light source to avoid fluorescence quenching, making the measurement of the basic fluorescence yield F more accurate, and thus can accurately estimate the chlorophyll content.

[0022] 3. Based on the leaf cross-section fluorescence imaging device, the present invention can also obtain the differences in photosynthetic capacity and chlorophyll distribution in different mesophyll tissues inside plant leaves, and realize the correlation analysis of leaf chlorophyll content and photosynthetic performance. The operation of the present invention is simple and the measurement is reliable, and it has broad application prospects in the research fields of plant physiology, botany, ecology, etc. Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the fast fluorescence kinetics OJIP curve;

[0024] Figure 2 It is a schematic diagram of the non-destructive chlorophyll content measurement system in an embodiment of the present invention;

[0025] Figure 3 It is a schematic diagram of the relationship between chlorophyll content in different gradient solutions and the basic fluorescence yield F in an embodiment of the present invention;

[0026] Figure 4 It is a schematic diagram of the relationship between the multiple of the estimated value and the measured value of different plant chlorophyll solutions and the specific leaf weight in an embodiment of the present invention.

[0027] Reference Signs:

[0028] 1 - leaf chamber; 2 - laser light source; 3 - CCD image acquisition device; 4 - data processing module; 5 - laser; 6 - fluorescence; 7 - dichroic mirror; 8 - microscope objective; 9 - filter. Detailed Embodiments

[0029] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail through specific embodiments. However, it should be understood that the provision of the specific embodiments is only for better understanding of the present invention, and they should not be construed as limitations to the present invention. In the description of the present invention, it should be understood that the terms used are only for the purpose of description and cannot be construed as indicating or implying relative importance.

[0030] In order to solve the problems in the prior art such as the need for destructive sampling, cumbersome processes, and the susceptibility of extraction solutions to photooxidation resulting in measurement errors, the present invention proposes a non-destructive method and system for measuring chlorophyll content. A fluorescence imaging device for the leaf cross-section is selected, and based on the measurement parameters of the selected device, an empirical conversion model between the fluorescence image and the chlorophyll content is established; the values of the undetermined parameters in the empirical conversion model are calculated, and according to the obtained parameter values and the chlorophyll content conversion model, the fluorescence image of any leaf is measured on the fluorescence imaging device for the leaf cross-section, and after digitization, the basic fluorescence yield F is obtained, and the chlorophyll content of any leaf is calculated. It establishes an empirical conversion model based on the chlorophyll fluorescence imaging technology of the leaf cross-section and the traditional chlorophyll content extraction method, and is verified by different plants to achieve non-destructive measurement of the chlorophyll content of any plant. The following will elaborate on the solution of the present invention in detail with reference to the accompanying drawings through examples.

[0031] Example 1:

[0032] There is a direct relationship between chlorophyll content and the photosynthetic performance of leaves. Some studies have also correlated chlorophyll fluorescence parameters with chlorophyll content, and selected one or a group of parameters with the best correlation from a series of fluorescence parameters as indicators of chlorophyll content. However, these parameters cannot exclude fluorescence quenching and cannot accurately reflect the true level of chlorophyll content. If the measurement of leaf photosynthetic performance and chlorophyll content can be completed synchronously, it can not only achieve non-destructive measurement of leaf chlorophyll content, but also conduct correlation analysis with photosynthetic performance, combined with non-invasive measurement of light absorption, photosynthetic efficiency, and carbon assimilation at the leaf level, which is of great significance for deeply understanding the physiological and ecological mechanisms of plant light response and adaptation.

[0033] This example discloses a non-destructive method for measuring chlorophyll content, including the following steps:

[0034] S1 Based on the measurement parameters of the fluorescence imaging device for the leaf cross-section, an empirical conversion model between the fluorescence image and the chlorophyll content is established. The empirical conversion model includes undetermined parameter M and parameter B;

[0035] Select a fluorescence imaging device for the leaf cross-section, and based on the measurement parameters of the selected fluorescence imaging device for the leaf cross-section, establish an empirical conversion model between the fluorescence F image and the chlorophyll content. Its calculation formula is:

[0036] C = -lg(1 - F / M) / B

[0037] Where C is the chlorophyll content, F is the basic fluorescence yield; M is the instrument characteristic constant, and B is the leaf characteristic constant. Among them, parameters M and B are the parameters to be measured.

[0038] S2 Calculate the specific leaf weight of the leaf by measuring the leaf area and dry weight of the leaf, and use the value of the specific leaf weight as the value of parameter B.

[0039] Collect fresh leaves, measure the leaf area of the leaves, dry the leaves at 65 °C until a constant weight is reached, weigh the dry weight, calculate the specific leaf weight, and obtain the parameter B of this empirical conversion model. The calculation formula for the parameter B is as follows:

[0040]

[0041] Among them, B is the leaf characteristic constant. The leaf characteristic constant takes the value of the specific leaf weight, m is the dry weight, SL is the leaf area. Taking Ficus benjamina as an example, the value of B is 0.52.

[0042] S3 Extract the chlorophyll solution from the leaves of the same part of the same plant, dilute the chlorophyll solution into different concentration gradients, and use the same leaf cross-section fluorescence imaging device to obtain the fluorescence images of the chlorophyll solutions with different concentration gradients.

[0043] Extract chlorophyll solution: Take fresh leaves, cut about 0.2 g of fresh leaves, avoid the leaf veins, measure the leaf area and then cut them into pieces. Add 25 ml of 95% ethanol, place in the dark for 24 hours, and soak until the leaves turn white. Dilute the completely extracted chlorophyll solution into different concentration gradients (0.025, 0.05, 0.1, 0.2, 1 v / v), measure the absorbance at 665 nm and 649 nm on a spectrophotometer, and calculate the actual chlorophyll content according to the formula. The calculation formula is as follows: Pa = 13.95A 665 - 6.88A 649

[0044] Pb = 24.96A 649 - 7.32A 665

[0045] C real (mg / m 2 FW) = (Pa + Pb) * V / SS

[0046] Among them, Pa is the chlorophyll a content, Pb is the chlorophyll b content, A665 and A649 are the absorbances at 665 nm and 649 nm, V is the volume of the extraction solution, SS is the leaf area used for extraction, and C real is the measured chlorophyll concentration.

[0047] Place the chlorophyll solutions with different concentration gradients in step S3 on the leaf cross-section fluorescence imaging device, irradiate with short-pulse strong light, obtain the image of the basic fluorescence yield F of the solution, and fit the actual chlorophyll content measured by colorimetry with the basic fluorescence yield F to obtain the value of the parameter M. The specific process is as follows:

[0048] The concentration of the chlorophyll solution is calculated by testing the absorbance of the chlorophyll solution with a spectrophotometer. According to the Lambert-Beer law, the concentration of the chlorophyll solution is calculated by the absorbance. The calculation formula is:

[0049]

[0050] Where A is the absorbance, I0 is the incident light, and I T is the transmitted light, ε is the extinction coefficient, C is the chlorophyll concentration, and L is the optical path.

[0051] In general, plant chlorophyll fluorescence is proportional to light energy absorption. Light energy absorption is equal to the incident light I0 minus the transmitted light I T and reflected light I r , reflected light accounts for about 2% in the microscope system and can be ignored. When measuring the fluorescence of the leaf cross section, the leaf is aligned vertically to the lens and the transmitted light is also close to 0, so:

[0052]

[0053] Assume M′I o is a constant M, εL is a constant B, then:

[0054]

[0055] Since the chlorophyll solution is uniform, the extinction coefficient and optical path length are fixed, but the internal structure of leaves of different species is different, and the leaf thickness, chlorophyll arrangement, etc. lead to differences in leaf light absorption. Therefore, the specific leaf weight is used as the leaf characteristic constant B, and the calculation formula is:

[0056]

[0057] In this embodiment, it is preferred that the short pulse strong light adopts a blue laser with a wavelength of 400nm-500nm and a light intensity of not less than 200000μmol m -2 s -1 The pulse width is no more than 30us. According to the duty cycle, the average light intensity irradiated to the leaves is about 1μmol m -2 s -1 , which can avoid fluorescence quenching and obtain a stable basic fluorescence yield F of chlorophyll solution.

[0058] S4 fits the concentration gradient of the chlorophyll solution and the basic fluorescence yield F of the fluorescence image into the empirical conversion model to obtain the value of the parameter M, such as Figure 3 As shown, M is 93.79.

[0059] S5 inputs the basic fluorescence yield F of the fluorescence image of the leaf to be measured into the empirical conversion model after the undetermined parameters are determined, and obtains the chlorophyll concentration of the leaf to be measured.

[0060] Example 2:

[0061] Based on the same inventive concept, this embodiment discloses a non-destructive chlorophyll content measurement system, as Figure 2 shown, for the non-destructive chlorophyll content measurement method of any one of the above, including: a leaf chamber 1, a laser light source 2, a CCD image acquisition device 3, and a data processing module 4;

[0062] The leaf chamber 1 is used to place the chlorophyll solution and the leaf. Place the cuvette containing the chlorophyll solution or the leaf at the position of the leaf chamber 1 and place it in the dark for 5 minutes;

[0063] The laser light source 2 is used to generate the laser 5 and irradiate the chlorophyll solution with the generated laser 5;

[0064] The CCD image acquisition device 3 is used to acquire the fluorescence image of the chlorophyll solution;

[0065] The data processing module 4 is used to process the acquired fluorescence image to obtain the basic fluorescence yield F. In this embodiment, the data processing module 4 is preferably a computer or other device with a computing function.

[0066] Turn on the CCD image acquisition device 3 and the computer, and set the parameters of the image acquisition device. In this embodiment, the parameters of the image acquisition device are preferably that the CCD exposure time is 600 ms, the gain is 22.5X, and the video shooting option sets the recording time to 12 s. Turn on the laser light source 2, and the CCD image acquisition device 3 starts to acquire the fluorescence image. After the CCD image acquisition device 3 finishes recording, export and save the RGB image sequence to generate a fluorescence image sequence. Use Matlab software in the data processing module 4 to process the RGB image sequence, convert the RGB image sequence into a grayscale image, select several cross-sections in the grayscale image, calculate the average value of the pixel points of each cross-section, and after data standardization, it is the chlorophyll basic fluorescence yield F. In this embodiment, the number of cross-sections is preferably 5. Make a curve of the actual chlorophyll content and the basic fluorescence yield F, and establish an empirical conversion model between the chlorophyll content and the basic fluorescence yield F. Fit to obtain the parameter M and the parameter B value in the empirical conversion model.

[0067] A dichroic mirror 7 is arranged between the laser light source 2 and the CCD image acquisition device 3. The laser 5 generated by the laser light source 2 is reflected onto the chlorophyll solution through the dichroic mirror 7. The fluorescence 6 generated by the chlorophyll solution reaches the dichroic mirror 7 through the microscope objective 8 and is projected onto the CCD image acquisition device 3 by the dichroic mirror 7. The data processing module 4 processes the fluorescence image acquired by the CCD image acquisition device 3.

[0068] In this embodiment, the CCD image acquisition device 3 is a high-sensitivity black-and-white CCD image acquisition device 3 with a resolution of not less than 1392×1040 pixel and a shooting speed of not less than 30 fps / s. A short-wave cut-off filter 9 is provided in front of the CCD image acquisition device 3, allowing only the chlorophyll fluorescence 6 in the range of 680 - 750 nm to pass through, so as to avoid the interference of incident light and ambient stray light.

[0069] To avoid the contamination of fluorescence 6, in this embodiment, the laser light source 2 uses blue laser with a wavelength of 450 nm ± 50 nm, and high-intensity short pulses are used for irradiation, with the light intensity not less than 200000 μmol m -2 s -1 , the light source is turned on for 1 μs bright + 15 ms dark, and the average light intensity reaching the sample is 1 μmol m -2 s -1 , avoiding the increase of the basal fluorescence yield F caused by fluorescence quenching, so as to obtain a stable F and accurately estimate the chlorophyll content.

[0070] To ensure a long working distance and resolution, an inverted fluorescence microscope can be used for the microscope, with an objective lens magnification of 4X or 10X, which can distinguish the palisade tissue and spongy tissue inside the leaf.

[0071] In order to reduce the physiological activity changes of the leaf after cutting, the measurement time for each leaf to be measured is within 2 minutes.

[0072] Example 3:

[0073] To verify the method in the present invention, in this embodiment, the accuracy of the empirical formula is verified through 4 different plants. The specific verification process is as follows:

[0074] In this embodiment, 4 plants in the greenhouse are selected, namely Schefflera actinophylla, Polyscias balfouriana, Hibiscus rosa-sinensis, and Ficus benjamina. 3 fresh leaves with the same growth part and the same growth vigor are collected from each plant, and the surface dust is wiped clean;

[0075] Take out 1 leaf, weigh the fresh weight, measure the leaf area, dry it to a constant weight in an oven at 65 °C, and calculate the specific leaf weight, which is the B value;

[0076] Take out 1 leaf, completely wrap it with a wet gauze, and perform dark adaptation for 20 min for measuring the fluorescence 6 of the cross-section chlorophyll; take out 1 leaf, cut off the leaf veins, weigh the fresh weight, measure the leaf area, then cut it into pieces, add 25 ml of 95% ethanol and extract for 24 h to extract the chlorophyll solution, and measure the absorbance at 665 nm and 649 nm on a spectrophotometer, and calculate the chlorophyll content C according to the above formula real ;

[0077] Cut the dark-adapted leaves into pieces of 1 cm × 1 cm. Clamp the cut leaves in the middle of the light-shielding sponge, which is pre-wetted, and fix it in the leaf chamber 1. Place it on the stage of the microscope, align the cross-section of the leaf with the light source, and cover it with a wet gauze;

[0078] Turn on the microscope, the CCD image acquisition device 3 and the data processing module 4. Open the image acquisition software, and set the exposure time, gain, image acquisition time interval and duration; turn on the laser light source 2 to generate high-intensity short-pulse measurement light, and adjust the focal length to make the internal tissue of the leaf clear; collect the fluorescence image (fluorescence 6) video of the leaf cross-section, export the RGB image sequence, and process the image using Matlab software. After digitization, obtain the basic fluorescence yield F of the leaf cross-section;

[0079] Calculate the chlorophyll content C according to the empirical conversion model cal ;

[0080] Repeat the above steps to measure the remaining 3 kinds of dark-adapted plant leaves in turn;

[0081] The above measurement results are shown in Table 1. In this embodiment, the measurement results are basically consistent with those obtained by the traditional method, which is reliable; it proves that the technical method and empirical formula in the present invention are reliable.

[0082] Table 1 Chlorophyll content measurement values of 4 kinds of plant leaves obtained by the methods of the present invention and the prior art

[0083]

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: the specific implementation manners of the present invention can still be modified or equivalently replaced, and any modification or equivalent replacement without departing from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention. The above content is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of changes or replacements within the technical scope disclosed in the present application, and all of them should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A non-destructive method for measuring chlorophyll content, characterized in that, Including the following steps: Based on the measurement parameters of the leaf cross-section fluorescence imaging device, an empirical conversion model between the fluorescence image and the chlorophyll content is established. The empirical conversion model includes undetermined parameter M and parameter B; Calculate the specific leaf weight of the leaf by measuring the leaf area and dry weight of the leaf, and use the value of the specific leaf weight as the value of parameter B; Extract the chlorophyll solution of the same leaf, dilute the chlorophyll solution into different concentration gradients, and use the same leaf cross-section fluorescence imaging device to obtain the fluorescence images of the chlorophyll solutions with different concentration gradients. After digitization, the basic fluorescence yield F is obtained; Input the concentration gradient of the chlorophyll solution and the basic fluorescence yield F of the fluorescence image into the empirical conversion model for fitting to obtain the value of parameter M; Input the basic fluorescence yield F of the fluorescence image of the leaf to be measured into the empirical conversion model after determining the undetermined parameter to obtain the chlorophyll concentration of the leaf to be measured; The calculation formula of the empirical conversion model is: Where C is the chlorophyll content, F is the basic fluorescence yield; M is the instrument characteristic constant, and B is the leaf characteristic constant; The measurement method of the fluorescence images of the chlorophyll solutions with different concentrations is: place the chlorophyll solutions with different concentration gradients on the leaf cross-section fluorescence imaging device, irradiate with short-pulse strong light to obtain the solution fluorescence image, and after digitization, obtain the basic fluorescence yield F of the chlorophyll solution; 2. The non-destructive determination method of chlorophyll content according to claim 1, wherein The calculation formula of parameter B is: Where B is the leaf characteristic constant, the leaf characteristic constant takes the value of the specific leaf weight, M is the dry weight, and SL is the leaf area; 3. The non-destructive determination method of chlorophyll content according to claim 2, wherein, The calculation method of the chlorophyll solution concentration is: test the absorbance of the chlorophyll solution with a spectrophotometer, and calculate the concentration of the chlorophyll solution according to the Lambert-Beer law through the absorbance; 4. The non-destructive determination method of chlorophyll content according to claim 1, characterized in that, The short-pulse intense light is blue laser with a wavelength of 400nm - 500nm, and the light intensity of the short-pulse intense light is not less than 200000 μmol m -2 s -1 , and the pulse width of the short-pulse intense light is not more than 30 μs.

5. A non-destructive chlorophyll content measurement system, characterized in that, For the non-destructive determination method of chlorophyll content as described in any one of claims 1-4, including: a leaf chamber, a laser light source, a CCD image acquisition device, and a data processing module; The leaf chamber is used to place the chlorophyll solution; The laser light source is used to generate laser light and irradiate the chlorophyll solution with the generated laser light to make it generate fluorescence; The CCD image acquisition device is used to collect the fluorescence of the chlorophyll solution to generate a fluorescence image; The data processing module is used to process the collected fluorescence to obtain a fluorescence image; 6. The chlorophyll content non-destructive measurement system according to claim 5, wherein A dichroic mirror is arranged between the laser light source and the CCD image acquisition device. The laser light generated by the laser light source is reflected by the dichroic mirror onto the chlorophyll solution in the leaf chamber. The fluorescence generated by the chlorophyll solution reaches the dichroic mirror through the microscope objective lens and is projected by the dichroic mirror onto the CCD image acquisition device; 7. The chlorophyll content non-destructive measurement system according to claim 6, characterized in that, The CCD image acquisition device adopts a high-sensitivity black-and-white CCD image acquisition device. A short-wave cut-off filter is arranged in front of the CCD image acquisition device, and the collected signal is limited to the chlorophyll fluorescence in the wavelength range of 680-750nm; 8. The chlorophyll content non-destructive measurement system according to claim 5, characterized in that, The data processing module converts the fluorescence image collected by the CCD image acquisition device into a grayscale image, selects several cross-sections in the grayscale image, calculates the average value of the pixel points of each cross-section, and after standardization, obtains the basic fluorescence yield F.

Images