阅读说明：本技术 一种植物中光合色素种类的识别方法 (Method for identifying variety of photochromic substances in plant ) 是由 魏凯华 苏向龙 于 2021-07-19 设计创作，主要内容包括：本发明公开了一种植物中光合色素种类的识别方法,通过获取不同波段下特定光合色素的飞秒激光强度-多光子吸收荧光强度的曲线,确立其多光子吸收阶数,比较不同光合色素的多光子吸收阶数及对应波长,最终实现植物光合色素种类的有效识别。本发明是一种快速有效的实时检测方法,不但解决了传统化学方法需要离体检测、有损检测的问题,更避免了目前光学无损检测中不同光合色素线性吸收光谱相互叠加的困境。(The invention discloses a method for identifying the variety of photosynthetic pigments in plants, which is characterized in that a curve of femtosecond laser intensity-multiphoton absorption fluorescence intensity of a specific photosynthetic pigment under different wavebands is obtained, a multiphoton absorption order of the curve is determined, the multiphoton absorption orders and corresponding wavelengths of different photosynthetic pigments are compared, and finally, the effective identification of the variety of the photosynthetic pigments in plants is realized. The invention is a rapid and effective real-time detection method, which not only solves the problems that the traditional chemical method needs in-vitro detection and destructive detection, but also avoids the dilemma that the linear absorption spectra of different photosynthetic pigments are mutually superposed in the current optical nondestructive detection.)

1. A method for identifying the type of photosynthetic pigment in a plant is characterized by comprising the following steps: the method comprises the following specific steps:

step one, building a detection and identification device: arranging a focusing lens, a cuvette and a fluorescence detector side by side along the optical axis direction of the femtosecond laser, wherein the cuvette is positioned between the focusing lens and the fluorescence detector and is positioned on the focus of the focusing lens;

step two, identification of chlorophyll a: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in the range of 1291-1326 nm, irradiating laser emitted by the laser onto a cuvette containing a plant photosynthetic pigment sample to be detected after the laser is focused by a focusing lens, and detecting the fluorescence intensity generated by the photosynthetic pigment sample to be detected after the laser is irradiated by a fluorescence detector; wherein, the plant photosynthetic pigment sample to be detected is one of chlorophyll a, chlorophyll b, lutein or beta-carotene; then, the power of a laser light source is changed to obtain the fluorescence intensity under different powers, curve fitting is carried out, if the slope of a fitting curve is 2, a 2-photon absorption effect is generated, the fact that the plant photosynthetic pigment sample to be detected is chlorophyll a is identified, and if not, the next step is executed;

step three, identification of lutein: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in the range of 1336-1350 nm, irradiating laser emitted by the laser onto a cuvette containing a plant photosynthetic pigment sample to be detected after the laser is focused by a focusing lens, and detecting the fluorescence intensity of the photosynthetic pigment sample to be detected, which is generated by laser irradiation, by a fluorescence detector; then, the power of a laser light source is changed to obtain fluorescence intensity under different powers, curve fitting is carried out, if the slope of a fitting curve is 4, a 4-photon absorption effect is generated, the photosynthetic pigment sample to be detected is identified to be lutein, and if not, the next step is executed;

step four, identification of chlorophyll b: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in the range of 1936-1988 nm, irradiating laser emitted by the laser onto a cuvette containing a plant photosynthetic pigment sample to be detected after the laser is focused by a focusing lens, and detecting the fluorescence intensity generated by the photosynthetic pigment sample to be detected after the laser is irradiated by a fluorescence detector; then, the power of a laser light source is changed to obtain the fluorescence intensity under different powers, curve fitting is carried out, if the slope of a fitting curve is 4, a 4-photon absorption effect is generated, the photosynthetic pigment sample to be detected is identified to be chlorophyll b, and if not, the next step is executed;

step five, identification of beta-carotene: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in the range of 2201-2250 nm, irradiating laser emitted by the laser onto a cuvette containing a plant photosynthetic pigment sample to be detected after the laser is focused by a focusing lens, and detecting the fluorescence intensity generated by the photosynthetic pigment sample to be detected after the laser is irradiated by a fluorescence detector; and then, obtaining the fluorescence intensity under different powers by changing the power of the laser light source, performing curve fitting, and if the slope of the fitted curve is 5, generating a 5-photon absorption effect, and identifying that the photosynthetic pigment sample to be detected is beta-carotene.

Technical Field

The invention belongs to the technical field of detection, and particularly relates to a method for identifying the type of a photochromic substance in a plant.

Background

Plant photosynthesis is the most basic substance and energy metabolism in nature, and it converts light energy into chemical energy and inorganic matter into organic matter, and it is self-evident that among many elements affecting photosynthesis efficiency, photosynthetic pigments such as chlorophyll (chlorophyll a, chlorophyll b), beta-carotene, lutein, etc. play a key role.

Most of the methods for early detection of photosynthetic pigment species involve chemical extraction, and have obvious defects that damage detection and in-vitro detection are caused, and a large amount of plants are required for extraction of photosynthetic pigments, so that the photosynthetic pigments can cause great damage to crops. In order to solve the problem of destructive detection, an optical method is used for solving the problem, but the linear absorption spectra of different photosynthetic pigments are overlapped and difficult to separate effectively.

The nonlinear multi-photon absorption effect is a third-order optical nonlinearity generated in the process of the action of strong laser and a medium. Under the irradiation of high-intensity incident light, particles in a ground state in a medium can simultaneously absorb a plurality of photons to jump to an excited state, finally jump to a metastable state through non-radiation, and emit fluorescence, and the excited state information of the medium is supplemented by a nonlinear multi-photon absorption effect. Therefore, the problem of "spectral overlap" can be solved by using the principle that multiphoton absorption characteristics of different pigments are different.

Disclosure of Invention

The invention aims to provide a quick and efficient identification method for the variety of photopigments in plants aiming at the problems of destructive detection, low efficiency and the like of the existing method.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention relates to a method for identifying the type of photosynthetic pigments in plants, which comprises the following steps:

step one, building a detection and identification device: and arranging the focusing lens, the cuvette and the fluorescence detector side by side along the optical axis direction of the femtosecond laser, wherein the cuvette is positioned between the focusing lens and the fluorescence detector and is positioned on the focus of the focusing lens.

Step two, identification of chlorophyll a: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in the range of 1291-1326 nm, irradiating laser emitted by the laser onto a cuvette containing a plant photosynthetic pigment sample to be detected after the laser is focused by a focusing lens, and detecting the fluorescence intensity generated by the photosynthetic pigment sample to be detected after the laser is irradiated by a fluorescence detector; wherein, the plant photosynthetic pigment sample to be detected is one of chlorophyll a, chlorophyll b, lutein or beta-carotene; and then, obtaining fluorescence intensity under different powers by changing the power of the laser light source, performing curve fitting, generating a 2-photon absorption effect if the slope of the fitted curve is 2, identifying that the photosynthetic pigment sample to be detected is chlorophyll a, and otherwise, executing the next step.

Step three, identification of lutein: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in the range of 1336-1350 nm, irradiating laser emitted by the laser onto a cuvette containing a plant photosynthetic pigment sample to be detected after the laser is focused by a focusing lens, and detecting the fluorescence intensity of the photosynthetic pigment sample to be detected, which is generated by laser irradiation, by a fluorescence detector; and then, obtaining fluorescence intensity under different powers by changing the power of the laser light source, performing curve fitting, generating a 4-photon absorption effect if the slope of the fitted curve is 4, identifying that the photosynthetic pigment sample to be detected is lutein, and otherwise, executing the next step.

Step four, identification of chlorophyll b: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in the range of 1936-1988 nm, irradiating laser emitted by the laser onto a cuvette containing a plant photosynthetic pigment sample to be detected after the laser is focused by a focusing lens, and detecting the fluorescence intensity generated by the photosynthetic pigment sample to be detected after the laser is irradiated by a fluorescence detector; and then, obtaining fluorescence intensity under different powers by changing the power of the laser light source, performing curve fitting, generating a 4-photon absorption effect if the slope of the fitted curve is 4, identifying that the photosynthetic pigment sample to be detected is chlorophyll b, and otherwise, executing the next step.

Step five, identification of beta-carotene: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in the range of 2201-2250 nm, irradiating laser emitted by the laser onto a cuvette containing a plant photosynthetic pigment sample to be detected after the laser is focused by a focusing lens, and detecting the fluorescence intensity generated by the photosynthetic pigment sample to be detected after the laser is irradiated by a fluorescence detector; and then, obtaining the fluorescence intensity under different powers by changing the power of the laser light source, performing curve fitting, and if the slope of the fitted curve is 5, generating a 5-photon absorption effect, and identifying that the photosynthetic pigment sample to be detected is beta-carotene.

The invention has the beneficial effects that:

the invention realizes the rapid and effective identification of different types of photosynthetic pigments by acquiring the curve of femtosecond laser intensity-multiphoton absorption fluorescence intensity of specific photosynthetic pigments at different wave bands, is a real-time detection method, solves the problems of in-vitro detection and destructive detection required by the traditional chemical method, and solves the problem of mutual superposition of linear absorption spectra of different photosynthetic pigments in the current optical nondestructive detection,

drawings

FIG. 1 is a flow chart of the identification of the type of plant photosynthetic pigments according to the present invention;

fig. 2 is a schematic diagram of the detection and identification device constructed by the invention.

Detailed Description

The invention will be further explained with reference to the drawings.

As shown in fig. 1, a method for identifying the type of photosynthetic pigment in a plant comprises the following specific steps:

step one, building a detection and identification device: as shown in fig. 2, the focusing lens 2, the cuvette 3, and the fluorescence detector 4 are arranged side by side along the optical axis direction of the femtosecond laser, and the cuvette 3 is located between the focusing lens 2 and the fluorescence detector 4 and at the focal point of the focusing lens 2.

Step two, identification of chlorophyll a: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in a range of 1291-1326 nm (1310 nm is adopted in the embodiment), irradiating laser emitted by the laser onto a cuvette 3 containing a plant photosynthetic pigment sample to be detected after being focused by a focusing lens 2, and detecting the fluorescence intensity generated by the photosynthetic pigment sample to be detected after being irradiated by the laser by a fluorescence detector 4; wherein, the plant photosynthetic pigment sample to be detected is one of chlorophyll a, chlorophyll b, lutein or beta-carotene; and then, obtaining fluorescence intensities under different powers by changing the power of a laser light source, performing curve fitting, if the number of multiphoton absorption is 2 (a straight line with a slope of 2 can be fitted), identifying that the photosynthetic pigment sample to be detected is chlorophyll a (only chlorophyll a can absorb 2 photons for four plant photosynthetic pigments of chlorophyll a, chlorophyll b, lutein and beta-carotene when the emergent wavelength of the laser light source is within the range of 1291-1326 nm, and therefore identifying the chlorophyll a), and otherwise, executing the next step.

Step three, identification of lutein: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in the range of 1336-1350 nm (1340 nm is adopted in the embodiment), irradiating laser emitted by the laser onto a cuvette 3 containing a plant photosynthetic pigment sample to be detected after being focused by a focusing lens 2, and detecting the fluorescence intensity generated by the plant photosynthetic pigment sample to be detected after being irradiated by the laser by a fluorescence detector 4; and then, obtaining fluorescence intensities under different powers by changing the power of a laser light source, performing curve fitting, and if the multiphoton absorption number is 4 (a straight line with a slope of 4 can be fitted), identifying that the plant photosynthetic pigment sample to be detected is lutein (only 4 photons can be absorbed by the lutein for the four plant photosynthetic pigments of chlorophyll a, chlorophyll b, lutein and beta-carotene when the emission wavelength of the laser light source is in the range of 1336-1350 nm, and thus identifying the lutein), otherwise, executing the next step.

Step four, identification of chlorophyll b: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in the range of 1936-1988 nm (1950 nm is adopted in the embodiment), irradiating laser emitted by the laser onto a cuvette 3 containing a plant photosynthetic pigment sample to be detected after being focused by a focusing lens 2, and detecting the fluorescence intensity generated by the plant photosynthetic pigment sample to be detected after being irradiated by the laser by a fluorescence detector 4; and then, obtaining fluorescence intensities under different powers by changing the power of a laser light source, performing curve fitting, and if the number of multiphoton absorption is 4 (a straight line with a slope of 4 can be fitted), identifying that the plant photosynthetic pigment sample to be detected is chlorophyll b (only chlorophyll b can absorb 4 photons for four plant photosynthetic pigments of chlorophyll a, chlorophyll b, lutein and beta-carotene when the emission wavelength of the laser light source is in the range of 1936-1988 nm, and identifying the chlorophyll b), otherwise, executing the next step.

Step five, identification of beta-carotene: enabling the emergent wavelength of a femtosecond laser with tunable wavelength and power to be in the range of 2201-2250 nm (2230 nm is adopted in the embodiment), irradiating laser emitted by the laser onto a cuvette 3 containing a plant photosynthetic pigment sample to be detected after being focused by a focusing lens 2, and detecting the fluorescence intensity generated by the plant photosynthetic pigment sample to be detected after being irradiated by the laser by a fluorescence detector 4; and then, obtaining fluorescence intensities under different powers by changing the power of the laser light source, performing curve fitting, and if the number of multiphoton absorption is 5 (a straight line with a slope of 5 can be fitted), identifying that the plant photosynthetic pigment sample to be detected is beta-carotene (when the emission wavelength of the laser light source is in the range of 2201-2250 nm, only the beta-carotene can absorb 5 photons for four plant photosynthetic pigments of chlorophyll a, chlorophyll b, lutein and beta-carotene, so that the beta-carotene is identified).