阅读说明：本技术 基于空间外差差分拉曼光谱技术的近海沉积物中微塑料的快速检测系统与检测方法 (Rapid detection system and detection method for micro-plastic in offshore sediments based on spatial heterodyne difference Raman spectroscopy ) 是由 薛庆生 卢继涛 杨柏 王楠 栾晓宁 王福鹏 李千 于 2020-06-15 设计创作，主要内容包括：本发明涉及基于空间外差差分拉曼光谱技术的近海沉积物中微塑料的快速检测系统与检测方法,该检测系统的视觉光源辅以显微单元和荧光成像单元可使微塑料的检测过程更加可视化；荧光成像单元与拉曼检测系统的联合应用,通过三维位移台的辅助作用,使拉曼检测系统可快速寻找感兴趣的区域,从而自动识别微塑料荧光信号,大大提升检测效率；同时将拉曼光谱技术与标准的光学显微镜相耦合,使所采集到的拉曼信号通过显微单元回到空间外差拉曼光谱仪,实现同时获取高通量、宽波段、高分辨率的拉曼光谱；通过可调谐激光器对目标样品点的信号进行二次采集,再通过差分计算和多重约束迭代算法,得到纯净的拉曼光谱信号。(The invention relates to a rapid detection system and a detection method of micro-plastic in offshore sediments based on a spatial heterodyne difference Raman spectroscopy technology, wherein a visual light source of the detection system is assisted by a microscope unit and a fluorescence imaging unit so that the detection process of the micro-plastic can be more visualized; the combined application of the fluorescence imaging unit and the Raman detection system enables the Raman detection system to quickly search an interested area through the auxiliary action of the three-dimensional displacement table, so that a micro-plastic fluorescence signal is automatically identified, and the detection efficiency is greatly improved; simultaneously, the Raman spectrum technology is coupled with a standard optical microscope, so that the collected Raman signals return to the spatial heterodyne Raman spectrometer through the microscope unit, and the Raman spectrum with high flux, wide waveband and high resolution is obtained simultaneously; and performing secondary acquisition on signals of the target sample point through a tunable laser, and obtaining pure Raman spectrum signals through differential calculation and a multiple constraint iterative algorithm.)

1. The system is characterized by comprising a three-dimensional electric displacement table, a visual light source unit, a microscope unit, a tunable laser, a spliced grating type spatial heterodyne Raman spectrometer, a fluorescence imaging unit and a data acquisition and storage unit;

the three-dimensional electric displacement table is provided with a sample table, a microscope unit, a non-polarization beam splitting cube and a fluorescence imaging unit are sequentially arranged above the sample table, and the fluorescence imaging unit is connected with the data acquisition and storage unit;

the visual light source unit is used for providing illumination for the detection system, and parallel light generated by the visual light source unit is incident to a visual field area observed by the microscope unit;

the fluorescence signal of the sample marked by fluorescence on the sample platform is transmitted by the microscope unit and the non-polarization beam splitting cube and enters the fluorescence imaging unit for fluorescence imaging, the fluorescence imaging unit transmits the fluorescence image to the data acquisition and storage unit for storage, and quantitative analysis is carried out on the micro-plastic in the sample in the data acquisition and storage unit;

laser emitted by a tunable laser is subjected to beam expanding collimation by a beam expanding and collimating lens group, reflected by a dichroic mirror, reflected to a microscopic unit by a non-polarizing beam splitting cube, focused on the surface of a sample on a sample table, and then backscattered to generate a Raman signal, and the Raman signal is reflected by the microscopic unit and the non-polarizing beam splitting cube, then transmitted to a converging lens and a coupling lens by the transmission of the dichroic mirror, and finally transmitted to a spliced grating type spatial heterodyne Raman spectrometer; the tunable laser sequentially emits two lasers with different wavelengths to realize secondary collection of the Raman spectrum of a sample point, the Raman spectrum information of the sample is acquired in the spliced grating type spatial heterodyne Raman spectrometer, the spliced grating type spatial heterodyne Raman spectrometer transmits the Raman spectrum information to the data collection and storage unit for storage, and the pure Raman spectrum of the sample is obtained in the data collection and storage unit.

2. The system for rapidly detecting micro-plastics in offshore sediments based on the spatial heterodyne differential Raman spectroscopy of claim 1, wherein the tunable laser is an external cavity tunable laser, and the output wavelengths of the tunable laser are 780nm and 782nm, respectively.

3. The system for rapidly detecting the micro-plastics in the offshore sediments based on the spatial heterodyne differential Raman spectroscopy of claim 1, wherein the first and second spliced gratings in the spliced grating type spatial heterodyne Raman spectrometer each comprise n sub-gratings, wherein n is greater than or equal to 2, the scribing direction and the dispersion direction of each sub-grating are the same, and each sub-grating is sequentially arranged along the scribing direction of the spliced grating; splicing the sub-gratings of n different scribed lines into a spliced grating;

further preferably, the first spliced grating and the second spliced grating in the spliced grating type spatial heterodyne raman spectrometer each include 2 sub-gratings.

4. The system for rapidly detecting micro-plastics in offshore sediments based on the spatial heterodyne differential Raman spectroscopy of claim 1, wherein the fluorescence imaging unit comprises a second aperture diaphragm, a double cemented lens, a front cut-off filter and a CCD detector,

and the transmitted fluorescence signal passing through the non-polarization beam splitting cube is filtered by a second aperture diaphragm, a double-cemented lens and a front cut-off filter in the fluorescence imaging unit in sequence, and finally fluorescence imaging is carried out in a CCD detector.

5. The system for rapidly detecting micro-plastics in offshore sediments based on the spatial heterodyne differential Raman spectroscopy of claim 1, wherein the microscope unit performs microscopic imaging by using a microscope objective with a numerical aperture of 0.6 and a magnification of 40 times.

6. The system for rapidly detecting micro-plastics in offshore sediments based on the spatial heterodyne differential Raman spectroscopy of claim 1, wherein the calculation precision of the three-dimensional motorized displacement stage is 0.1 μm.

7. The system for rapidly detecting micro-plastics in offshore sediments based on the spatial heterodyne difference Raman spectroscopy technology as claimed in claim 1, wherein the visual light source unit comprises a visual light source, a converging collimating lens group and a beam steering cage cube; a first small aperture diaphragm is arranged in the convergence collimating lens group;

after light beams emitted by the visual light source pass through the convergence collimating lens group and the first aperture diaphragm, the light beams pass through the light beam steering cage cube in a parallel light beam mode, and the light beams irradiate a visual field area observed by the microscope unit.

8. Method for the detection of micro-plastics in offshore sediments, based on a rapid detection system according to any one of claims 1 to 7, characterized in that it comprises the following steps:

(1) collecting a sediment sample;

(2) drying the sediment sample to constant weight, and soaking the sediment sample in hydrogen peroxide to remove natural organic matters in the sediment;

(3) extracting low-density micro-plastic and high-density micro-plastic particles by a double-density separation method;

(4) dyeing the supernatant liquid containing the low-density micro-plastic and the high-density micro-plastic particles separated in the step (3), and then filtering and drying to obtain a sample to be detected;

(5) placing a sample to be detected on a sample stage of a three-dimensional electric displacement stage in a detection system for micro-plastics in offshore sediments based on a spatial heterodyne difference Raman spectroscopy technology to observe, realizing focusing detection through the three-dimensional electric displacement stage, carrying out fluorescence imaging on the fluorescence-labeled micro-plastics by using a fluorescence imaging unit, and collecting and storing fluorescence images by using a data collecting and storing unit for quantitative analysis;

(6) the tunable laser respectively emits two lasers with different wavelengths, the lasers are focused on the surface of a sample after passing through the beam expanding collimating lens group, the dichroic mirror, the non-polarization beam splitting cube and the microscopic unit, Raman signals generated by backscattering of the sample are transmitted to the spliced grating type spatial heterodyne Raman spectrometer through the microscopic unit, reflection of the non-polarization beam splitting cube, transmission of the dichroic mirror, the convergent lens and the coupling lens, two original Raman spectrograms are obtained, and the two original Raman spectrograms are transmitted to the data acquisition and storage unit for storage; and in the data acquisition and storage unit, carrying out differential calculation on the two obtained original Raman spectrograms to obtain a differential Raman spectrum, and restoring the differential Raman spectrum by using a multiple constraint iterative algorithm to obtain a pure Raman spectrum.

9. The method for detecting micro-plastics in offshore sediments based on the rapid detection system of claim 8, wherein in the step (6), the difference calculation is performed on the obtained two original raman spectrograms to obtain the differential raman spectrum, and the differential raman spectrum is restored by using the multiple constraint iterative algorithm to obtain the pure raman spectrum, which comprises the following steps:

A. and carrying out differential calculation on the two obtained original Raman spectrograms to obtain a differential Raman spectrum, and utilizing a deconvolution method to require a recovered Raman spectrum R: since the raman spectrum shifts with the frequency of the excitation light, the process of shifting is represented by the convolution of a fixed raman spectrum r (v) with the corresponding function at different frequencies, i.e.:

in the formula (V), V₁Indicating the frequency of one of the laser beams emitted by the tuneable laser, R (v, v)₁) Representing a frequency v₁Obtaining a pure Raman signal in the original Raman spectrum;

in the formula (VI), v₂The frequency of the other laser light emitted by the tunable laser, R (v, v)₂) Representing a frequency v₂Obtaining a pure Raman signal in the original Raman spectrum;

through difference calculation, the difference spectrum D (V) can be obtained by the formulas (V) and (VI):

D(v)＝S₁-S₂＝R(v,v₁)-R(v,v₂) (IV)，

in the formula (IV), S₁Representing a frequency v₁The raw Raman spectrum obtained below, S₂Representing a frequency v₂The raw raman spectrum obtained below, the difference spectrum d (v) is expressed as:

order (v, v)₁)-(v,v₂) As is apparent from formulas (V), (VI), and (VII), the difference spectrum d (V) is represented by:

the formula (VIII) is expressed in a matrix form, namely:

D＝H·R (IX)，

in formula (IX), D is a differential spectrum and is a known one-dimensional column vector; h is a convolution kernel matrix which is a square matrix related to two laser frequencies; r is the Raman spectrum to be recovered, and the direct deconvolution signal recovery method is shown as the formula (X):

in formula (IX), superscript T denotes transpose, superscript-1 denotes inverse matrix;

B. the multiple constraint iterative algorithm is utilized to inhibit the amplification of noise by deconvolution and oscillation caused by Raman peak asymmetry, and the method specifically comprises the following steps:

adding a smooth constraint operator Q, a constraint operator N with negative energy and a constraint operator P with positive energy into a multiple constraint iterative algorithm,

the smooth constraint operator Q is used for inhibiting the over-fast fluctuation of data in the restoration result, playing a smooth effect and minimizing the square sum of the second order differential of the processing result;

a constraint operator N of negative energy, for controlling the spectrum result recovered to have no negative number, and its action makes the square sum of negative numbers in the processing result minimum;

the positive energy constraint operator P is used for inhibiting oscillation caused by spectrum peak asymmetry and minimizing the sum of squares of all data in a processing result;

the matrix form of the three constraint operators is shown as (XI), (XII), (XIII):

in the formula (XII), if the k-th digit in the processing result is negative, a_k1, otherwise a_k＝0；

In formula (XIII), P is an identity matrix at the beginning of the iteration of the algorithm;

the multiple constraint mechanism is considered by adopting the angle of multi-objective optimization, wherein the constraint target is as follows:

the optimization function f is:

the optimization function f derives R and makes it equal to zero, resulting in formula (XVI):

R_k＝(H^TH+αQ^TQ+βN^TN+γP^TP)^-1·(H^TD) (XVI)，

in the formula (XVI), α, β and gamma are all parameters of the algorithm, and are used for setting the relative strength R of the smooth constraint operator Q, the constraint operator N with negative energy and the constraint operator P with positive energy respectively_kThe method is used for recovering the obtained pure Raman spectrum through a multiple constraint iterative algorithm.

10. The method for detecting micro-plastics in offshore sediments based on the rapid detection system of claim 8, wherein in the step (4), the supernatant containing the micro-plastics is dyed by a Nile red fluorescent probe solution.

Technical Field

The invention relates to a system and a method for rapidly detecting micro-plastics in offshore sediments based on a spatial heterodyne difference Raman spectroscopy technology, and belongs to the field of detection of micro-plastics by adopting the Raman spectroscopy technology.

Background

Microplastic is a plastic film, fiber, particle and chip with an effective diameter of less than 5 mm. The gel can be produced by large plastic under the condition of environmental factors (light, heat, radiation and the like), and can also be plastic microbeads added in face washing agents, toothpaste and the like, wherein the quantity of the latter plastic microbeads is far more than that of the former plastic microbeads. The widespread presence of micro-plastics in marine environments has been well documented over the past decade from the earliest suggestion of micro-plastic concepts in 2004 to the development of research. The micro plastic is relatively stable in property and can exist in the environment for a long time, but the surface physical and chemical properties of the micro plastic can be changed under the action of sunlight, wind power, waves and the like; the micro plastic has small size, large specific surface area and strong hydrophobicity, is an ideal carrier for a plurality of hydrophobic organic pollutants and heavy metals, is easy to be eaten by plankton, fish and the like by mistake, can be retained in a living body for a long time, is transferred and enriched in a food net, and threatens the safety of the ecological environment.

At the present stage, the detection of the marine micro-plastic is not mature, the false detection rate is high, and a standard analysis and evaluation method system is not formed, so that the urgent need of the marine field for quickly and accurately detecting the micro-plastic can not be met increasingly.

Chinese patent document CN111122634A discloses a method for identifying nano plastic particles in an aqueous solution based on a scanning electron microscope-raman technique, which realizes target identification and in-situ identification of nano plastic particles by setting and optimizing parameters of a scanning electron microscope and a confocal micro-raman spectrometer containing white light, and comprises: the method comprises the steps of sequentially filtering an aqueous solution to be detected by polyethersulfone filter membranes of 10 micrometers and 1 micrometer in a grading manner, uniformly dripping the aqueous solution onto a clean silicon chip at room temperature, naturally drying the aqueous solution in the air, placing the aqueous solution on a sample table in a vacuum cavity of a scanning electron microscope, setting corresponding test parameters to obtain a scanning electron microscope image, automatically transferring a detection sample to a Raman spectrum, keeping the same position, setting appropriate test parameters to obtain a Raman image, and carrying out image analysis and judgment. However, interference of background fluorescence in Raman detection in the method can cause certain errors to the detection result; the method is time-consuming for fully inspecting the image in the scanning electron microscope image, and limits the quantity of micro-plastics which can be analyzed within a certain time, thereby causing lower working efficiency.

The existing method for detecting the micro-plastics has the problems of high false detection rate, long time consumption, incomplete acquired information and the like, cannot meet the requirements of rapid and accurate detection of the micro-plastics, cannot simultaneously acquire Raman spectrum information with high luminous flux, wide waveband and high resolution, and cannot accurately analyze the micro-plastics with complex components. Therefore, a rapid and accurate detection system capable of simultaneously acquiring spectral information with high luminous flux, wide band and high resolution and realizing the micro-plastic is urgently needed to promote the development of the micro-plastic detection technology in the marine field.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a rapid detection system for micro-plastic in offshore sediments based on a spatial heterodyne difference Raman spectroscopy technology, wherein in the detection system, a tunable laser is used for carrying out secondary acquisition on signals of a target sample point, and a difference Raman technology and a multiple constraint iterative algorithm are introduced to process two acquired original Raman spectrums so as to obtain a pure Raman signal, so that the aim of eliminating fluorescence interference in the micro-plastic detection process is fulfilled, and the accurate detection of chemical components of the micro-plastic can be realized; the fluorescent imaging unit can enable the fluorescent dyed micro-plastic to become more visible in the detection process, and has the advantage of rapid and accurate quantitative analysis; the spliced grating type spatial heterodyne Raman spectrometer has the advantages of high luminous flux, wide waveband, high resolution and the like, and is favorable for further improving the detection limit and realizing accurate measurement of complex components.

The invention also provides a method for detecting micro-plastics in offshore sediments based on the rapid detection system.

Interpretation of terms:

1. double density separation method: firstly, the dried sediment sample is fluidized and pre-extracted by using a saturated NaCl solution, and then, the NaI solution is used for centrifugal flotation, so that the aim of simultaneously and efficiently extracting common polymer and high-density polymer micro plastic from the sediment can be fulfilled. And the sample amount of sediment can be reduced by adopting the saturated NaCl solution to cause fluidization pre-extraction, and the problem of high analysis cost caused by using a large amount of NaI solution is effectively solved.

The technical scheme of the invention is as follows:

the system comprises a three-dimensional electric displacement table, a visual light source unit, a microscopic unit, a tunable laser, a spliced grating type spatial heterodyne Raman spectrometer, a fluorescence imaging unit and a data acquisition and storage unit;

the three-dimensional electric displacement table is provided with a sample table, a microscope unit, a non-polarization beam splitting cube and a fluorescence imaging unit are sequentially arranged above the sample table, and the fluorescence imaging unit is connected with the data acquisition and storage unit;

the visual light source unit is used for providing illumination for the detection system, and parallel light generated by the visual light source unit is incident to a visual field area observed by the microscope unit;

the fluorescence signal of the sample marked by fluorescence on the sample platform is transmitted by the microscope unit and the non-polarization beam splitting cube and enters the fluorescence imaging unit for fluorescence imaging, the fluorescence imaging unit transmits the fluorescence image to the data acquisition and storage unit for storage, and quantitative analysis is carried out on the micro-plastic in the sample in the data acquisition and storage unit;

laser emitted by a tunable laser is subjected to beam expanding collimation by a beam expanding and collimating lens group, reflected by a dichroic mirror, reflected to a microscopic unit by a non-polarizing beam splitting cube, focused on the surface of a sample on a sample table, and then backscattered to generate a Raman signal, and the Raman signal is reflected by the microscopic unit and the non-polarizing beam splitting cube, then transmitted to a converging lens and a coupling lens by the transmission of the dichroic mirror, and finally transmitted to a spliced grating type spatial heterodyne Raman spectrometer; the tunable laser sequentially emits two lasers with different wavelengths to realize secondary collection of the Raman spectrum of a sample point, the Raman spectrum information of the sample is acquired in the spliced grating type spatial heterodyne Raman spectrometer, the spliced grating type spatial heterodyne Raman spectrometer transmits the Raman spectrum information to the data collection and storage unit for storage, and the pure Raman spectrum of the sample is obtained in the data collection and storage unit.

In the detection system provided by the invention, the visual light source unit of the system is assisted with the microscope unit and the fluorescence imaging unit, so that the detection process of the micro-plastic can be more visualized; the combined application of the fluorescence imaging unit and the Raman detection system enables the Raman detection system to quickly search an interested area through the auxiliary action of the three-dimensional displacement table, so that a micro-plastic fluorescence signal is automatically identified, and the detection efficiency is greatly improved; meanwhile, the Raman spectrum technology is coupled with a standard optical microscope, so that the collected Raman signals return to the spatial heterodyne Raman spectrometer through the microscope unit, and the Raman spectrum with high flux, wide waveband and high resolution can be obtained simultaneously.

According to the invention, the tunable laser is an external cavity tunable laser, and the output wavelengths of the tunable laser are 780nm and 782nm respectively. The tunable laser emits two lasers with slightly different wavelengths to realize secondary collection of the Raman spectrum of the sample to be detected, and then a pure Raman spectrum characteristic signal is obtained by applying differential calculation and a multiple constraint iterative algorithm.

According to the preferred embodiment of the invention, the first spliced grating and the second spliced grating in the spliced grating type spatial heterodyne raman spectrometer both comprise n sub-gratings, wherein n is larger than or equal to 2, the scribing direction and the dispersion direction of each sub-grating are the same, and each sub-grating is sequentially arranged along the scribing direction of the spliced grating; splicing the sub-gratings of n different scribed lines into a spliced grating; the spliced grating is adopted to replace a diffraction grating in an interference arm of the traditional spatial heterodyne Raman spectrometer, so that the spatial heterodyne Raman spectrometer is equivalent to an integral spatial heterodyne Raman spectrometer consisting of n subspace heterodyne Raman spectrometers with the same spectral resolution and different spectral detection ranges; because each sub-spectrometer has different Raman spectrum detection ranges, the spectrum detection range of the integral spatial heterodyne Raman spectrometer is expanded, and high-flux, broadband and high-resolution Raman information can be simultaneously acquired.

Further preferably, the first spliced grating and the second spliced grating in the spliced grating type spatial heterodyne raman spectrometer each include 2 sub-gratings. The instrument can be manufactured at 4400cm-¹The resolution within the wave band width reaches 3.37cm-¹。

According to a preferred embodiment of the present invention, the fluorescence imaging unit comprises a second aperture stop, a double cemented lens, a front cut-off filter and a CCD detector,

and the transmitted fluorescence signal passing through the non-polarization beam splitting cube is filtered by a second aperture diaphragm, a double-cemented lens and a front cut-off filter in the fluorescence imaging unit in sequence, and finally fluorescence imaging is carried out in a CCD detector.

Further preferably, the front cut filter is a 525nm filter.

A525 nm optical filter is selected, the fluorescent condition is set to be a green excitation light source, under the green excitation light, the sand basically has no fluorescence, and the interference of the sand in the sample to the detection process can be eliminated.

According to the invention, the microscope unit preferably performs microscopic imaging by using a microscope objective with a numerical aperture of 0.6 and a magnification of 40 times. The detection precision of the sample to be detected reaches the micron level, so that the detection limit of the detection of the micro plastic is improved.

According to the invention, the calculation accuracy of the three-dimensional electric displacement table is preferably 0.1 μm. The three-dimensional electric displacement platform has the functions of automatic positioning and manual interaction observation.

According to the invention, the vision light source unit comprises a vision light source, a convergence collimating lens group and a beam steering cage cube; a first small aperture diaphragm is arranged in the convergence collimating lens group;

after light beams emitted by the visual light source pass through the convergence collimating lens group and the first aperture diaphragm, the light beams pass through the light beam steering cage cube in a parallel light beam mode, and the light beams irradiate a visual field area observed by the microscope unit.

This arrangement is advantageous in that the light beam provided by the visual light source unit can be uniformly and sufficiently irradiated on the visual field area observed by the microscope unit.

According to the invention, the visual light source is a cold white LED array light source. The power of the cold white LED array light source is stable.

The method for detecting the micro-plastics in the offshore sediments based on the rapid detection system comprises the following steps:

(1) collecting a sediment sample;

(2) drying the sediment sample to constant weight, soaking the sediment sample in hydrogen peroxide, removing natural organic matters in the sediment, and eliminating fluorescence interference caused by the natural organic matters;

(3) extracting low-density micro-plastic and high-density micro-plastic particles by a double-density separation method;

(4) dyeing the supernatant liquid containing the low-density micro-plastic and the high-density micro-plastic particles separated in the step (3), and then filtering and drying to obtain a sample to be detected; the micro plastic particles are floated during the double density separation process, wherein the supernatant refers to the mixture of suspended particles (including completely separated low-density and high-density micro plastic particles) and the flotation solution.

(5) Placing a sample to be detected on a sample stage of a three-dimensional electric displacement stage in a detection system for micro-plastics in offshore sediments based on a spatial heterodyne difference Raman spectroscopy technology to observe, realizing focusing detection through the three-dimensional electric displacement stage, carrying out fluorescence imaging on the fluorescence-labeled micro-plastics by using a fluorescence imaging unit, and collecting and storing fluorescence images by using a data collecting and storing unit for quantitative analysis; the quantitative analysis is specifically to utilize a CCD detector to carry out fluorescence imaging on a sample and carry out quantitative analysis on a fluorescence image, the quantitative analysis is to aim at the shape, the size and the quantity of the micro-plastic, and a data acquisition and storage unit is a computer.

(6) The tunable laser respectively emits two lasers with different wavelengths, the lasers are focused on the surface of a sample after passing through the beam expanding collimating lens group, the dichroic mirror, the non-polarization beam splitting cube and the microscopic unit, Raman signals generated by backscattering of the sample are transmitted to the spliced grating type spatial heterodyne Raman spectrometer through the microscopic unit, reflection of the non-polarization beam splitting cube, transmission of the dichroic mirror, the convergent lens and the coupling lens, two original Raman spectrograms are obtained, and the two original Raman spectrograms are transmitted to the data acquisition and storage unit for storage; and in the data acquisition and storage unit, carrying out differential calculation on the two obtained original Raman spectrograms to obtain a differential Raman spectrum, and restoring the differential Raman spectrum by using a multiple constraint iterative algorithm to obtain a pure Raman spectrum.

According to the differential Raman spectrum technology, two lasers with slightly different wavelengths are used for respectively irradiating a sample to obtain two original Raman spectrograms, because the fluorescence background does not move along with the slight change of the wavelengths, but the position of a Raman peak changes obviously, the two spectrograms are subtracted to obtain a differential spectrum, and fluorescence background signals in the differential spectrum are mutually offset, so that the fluorescence interference can be well eliminated. Such as: by v₁And v₂Two lasers with a slight difference in frequency, where S₁And S₂Are the two original Raman spectra measured, F (v, v)₁) And F (v, v)₂) Respectively representing the fluorescence in two spectra, R (v, v)₁) And R (v, v)₂) The pure raman signals in the two spectra, d (v) representing the difference spectrum.

S₁＝F(v,v₁)+R(v,v₁) (I)，

S₂＝F(v,v₂)+R(v,v₂) (II)，

F(v,v₁)≈F(v,v₂) (III)，

D(v)＝S₁-S₂＝R(v,v₁)-R(v,v₂) (IV)，

Due to the fluorescent moiety F (v, v)₁) And F (v, v)₂) Are substantially the same, R (v, v)₁) And R (v, v)₂) Is a function related to the laser frequency, the difference spectrum D is only related to the raman signal and no component of the fluorescence is present.

And aiming at the phenomenon that partial peak values of the differential spectrum are asymmetric due to the existence of noise and the fact that each Raman peak is close to each other, the differential Raman spectrum is effectively restored by adopting a multiple constraint iterative algorithm, the position of the restored Raman peak is basically consistent with that of the original Raman peak, and the purpose of obtaining a pure Raman spectrum signal is achieved.

According to the preferable method, in the step (6), the difference calculation is performed on the two obtained original raman spectrograms to obtain the difference raman spectrum, the difference raman spectrum is restored by using the multiple constraint iterative algorithm to obtain the pure raman spectrum, and the specific process is as follows:

A. and carrying out differential calculation on the two obtained original Raman spectrograms to obtain a differential Raman spectrum, and utilizing a deconvolution method to require a recovered Raman spectrum R: since the raman spectrum shifts with the frequency of the excitation light, the process of shifting is represented by the convolution of a fixed raman spectrum r (v) with the corresponding function at different frequencies, i.e.:

in the formula (V), V₁Indicating the frequency of one of the laser beams emitted by the tuneable laser, R (v, v)₁) Representing a frequency v₁Obtaining a pure Raman signal in the original Raman spectrum;

in the formula (VI), v₂The frequency of the other laser light emitted by the tunable laser, R (v, v)₂) Representing a frequency v₂Pure draw in raw Raman spectra obtained belowA man signal;

through difference calculation, the difference spectrum D (V) can be obtained by the formulas (V) and (VI):

D(v)＝S₁-S₂＝R(v,v₁)-R(v,v₂) (IV)，

in the formula (IV), S₁Representing a frequency v₁The raw Raman spectrum obtained below, S₂Representing a frequency v₂The raw raman spectrum obtained below, the difference spectrum d (v) is expressed as:

order (v, v)₁)-(v,v₂) As is apparent from formulas (V), (VI), and (VII), the difference spectrum d (V) is represented by:

the formula (VIII) is expressed in a matrix form, namely:

D＝H·R (IX)，

in formula (IX), D is a differential spectrum and is a known one-dimensional column vector; h is a convolution kernel matrix which is a square matrix related to two laser frequencies; r is the Raman spectrum to be recovered, and the direct deconvolution signal recovery method is shown as the formula (X):

in formula (IX), superscript T denotes transpose, superscript-1 denotes inverse matrix;

the method can well recover the Raman spectrum under the condition of no noise interference; however, noise interference exists in the actual detection process, the algorithm has an amplification effect on the noise, and when the positive and negative intensities of a pair of spectrum peaks in the difference spectrum are asymmetric, the recovery result can generate serious oscillation;

B. the multiple constraint iterative algorithm is utilized to inhibit the amplification of noise by deconvolution and oscillation caused by Raman peak asymmetry, and the method specifically comprises the following steps:

adding a smooth constraint operator Q, a constraint operator N with negative energy and a constraint operator P with positive energy into a multiple constraint iterative algorithm,

the smooth constraint operator Q is used for inhibiting the over-fast fluctuation of data in the restoration result, playing a smooth effect and minimizing the square sum of the second order differential of the processing result;

a constraint operator N of negative energy, for controlling the spectrum result recovered to have no negative number, and its action makes the square sum of negative numbers in the processing result minimum;

the positive energy constraint operator P is used for inhibiting oscillation caused by spectrum peak asymmetry and minimizing the sum of squares of all data in a processing result;

the matrix form of the three constraint operators is shown as (XI), (XII), (XIII):

in the formula (XII), if the k-th digit in the processing result is negative, a_k1, otherwise a_k＝0；

In the formula (XIII), the larger the value of the k-th digit in the result of the processing, the larger b_kThe smaller the algorithm iteration is, the P is an identity matrix at the initial time;

q is a second-order difference Laplace transformation matrix, so that the over-fast fluctuation of data in a restoration result is inhibited, and the effect of smoothing is achieved;

n is iteratively evolved on the basis of a zero matrix, when a negative number appears in the last iteration result, 0 on the corresponding position of the diagonal line of the N matrix is replaced by 1, and therefore the restored spectrum result is controlled not to have the negative number;

p is iteratively evolved from the unit matrix, for the position with stronger Raman spectrum signal, the value of the corresponding position of the diagonal line of the P matrix is reduced from 1, the constraint is relaxed, and for the position with weak Raman spectrum signal or no signal, the P matrix constraint is stronger, and the propagation of noise and oscillation is prevented;

the multiple constraint mechanism is considered by adopting the angle of multi-objective optimization, wherein the constraint target is as follows:

the optimization function f is:

the optimization function f derives R and makes it equal to zero, resulting in formula (XVI):

R_k＝(H^TH+αQ^TQ+βN^TN+γP^TP)^-1·(H^TD) (XVI)，

in the formula (XVI), α, β and gamma are all parameters of the algorithm, and are used for setting the relative strength R of the smooth constraint operator Q, the constraint operator N with negative energy and the constraint operator P with positive energy respectively_kThe method is used for recovering the obtained pure Raman spectrum through a multiple constraint iterative algorithm.

The multiple constraint iterative algorithm is characterized in that three constraint operators are added on the basis of a deconvolution method to inhibit oscillation caused by noise amplification and Raman peak asymmetry of deconvolution, the multiple constraint iterative algorithm can restore Raman signals without fluorescence backgrounds, meanwhile, the signal-to-noise ratio of the system can be greatly improved, a difference Raman spectrum is effectively restored, the positions of the restored Raman peaks and the original Raman peaks are basically consistent, and the purpose of obtaining pure Raman spectrum signals is achieved.

Preferably, in the step (2), the mass-to-volume ratio of the sediment sample to the hydrogen peroxide is 1:5, the unit g/mL, the volume fraction of the hydrogen peroxide is 30%, and the soaking time of the sediment sample in the hydrogen peroxide is 24 hours.

According to the inventionOptionally, in the step (3), the low-density plastic has a density of less than 1.2 g-cm^-3The high-density plastic is plastic with the density of 1.2 g-cm^-3—1.8g·cm^-3Plastic in between.

Preferably, in step (4), the supernatant containing the microplastic is stained with a nile red fluorescent probe solution.

The invention has the beneficial effects that:

1. the invention discloses a system and a method for rapidly detecting micro-plastics in offshore sediments based on a spatial heterodyne difference Raman spectroscopy technology.

2. The fluorescence imaging unit and the Raman detection system are jointly applied, and the Raman detection system can quickly search an interested area through the auxiliary action of the three-dimensional displacement table, so that a micro-plastic fluorescence signal is automatically identified; meanwhile, the acquired Raman signals are transmitted to the spatial heterodyne Raman spectrometer through the microscope unit, so that high-flux, broadband and high-resolution Raman spectra can be acquired simultaneously.

3. And performing secondary acquisition on signals of the target sample point through a tunable laser emission system, and obtaining pure Raman spectrum signals through differential calculation and a multiple constraint iterative algorithm.

4. The grating splicing type spatial heterodyne Raman spectrum technology provided by the invention is characterized in that n (n is more than or equal to 2) sub-gratings with different reticle densities are spliced to replace the traditional plane grating, each sub-grating corresponds to different measurement wave bands, and the resolution of the full wave band can be improved by n times. And the instrument can be made to be 4400cm by adopting two sub-gratings^-1The resolution within the wave band width reaches 3.37cm^-1And it can be known from the large band overlapping area between the measuring bands corresponding to the 2 sub-gratings that 2cm is to be realized^-1The resolution ratio is only needed to be spliced by 3 sub-gratings, if the number of the sub-gratings is increased, the spectral resolution ratio in the full waveband can be further improved, the advantages of high luminous flux, wide waveband, high resolution ratio and the like can be obtained, and the promotion of ocean information in China is facilitatedThe development of micro-plastic detection technology.

Drawings

Fig. 1 is a schematic structural diagram of a system for rapidly detecting micro-plastics in offshore sediments based on a spatial heterodyne differential raman spectroscopy in embodiment 1 of the present invention.

Fig. 2 is a schematic diagram of a principle of an optical path of the spliced spatial heterodyne raman spectrometer in embodiment 1 of the present invention.

FIG. 3 is a schematic diagram of the method for detecting micro-plastics in offshore sediments based on the rapid detection system in embodiment 2 of the present invention.

FIG. 4 is a fluorescence image for a Polyethylene (PE) sample in example 2 of the present invention.

FIG. 5 is a comparison of the differential Raman spectrum of Polyethylene (PE) in example 2 of the present invention with the standard Raman spectrum of PE.

1. The device comprises a three-dimensional electric displacement platform, a 1a sample platform, a 2, a visual light source, a 2a, a convergence collimating lens group, a 2b, a first small-hole diaphragm, a 2c, a beam steering cage cube, a 3, a microscope unit, a 4, a tunable laser, a 4a, a beam expanding collimating lens group, a 4b, a 45-degree high reflector, a 4c, a dichroic mirror, a 4d, a non-polarizing beam splitting cube, a 4e, a convergence lens, a 4f, a second 45-degree high reflector, a 4g, a coupling lens, a 4h, a fiber coupler, a 5, a spliced grating type spatial heterodyne Raman spectrometer, a 5a, a Raman filter group, a 5b, a beam splitting prism, a 5c-1, a first field broadening prism, a 5c-2, a second field broadening prism, a 5d-1, a first spliced grating, a 5d-2, a second spliced grating, a 5e, lens group imaging, a 5f, a, The device comprises an area array detector 6, a fluorescence imaging unit 6a, a second small aperture diaphragm 6b, a double cemented lens 6c, a front cut-off optical filter 6d, a CCD detector 7 and a data acquisition and storage unit.

Detailed Description

The invention is further described below, but not limited thereto, with reference to the following examples and the accompanying drawings.