CN109060734B - Fluorescence imaging visual rapid screening system and method for zearalenone - Google Patents
Fluorescence imaging visual rapid screening system and method for zearalenone Download PDFInfo
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- CN109060734B CN109060734B CN201810628805.9A CN201810628805A CN109060734B CN 109060734 B CN109060734 B CN 109060734B CN 201810628805 A CN201810628805 A CN 201810628805A CN 109060734 B CN109060734 B CN 109060734B
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention belongs to the technical field of food detection, and particularly relates to a fluorescence imaging visual rapid screening system and method for zearalenone. The screening system comprises a light intensity regulation and control module, a camera bellows and a computer; the camera bellows comprises an EMCCD camera, a time sequence controllable multi-wavelength LED ultraviolet annular light source module and a sample cup from top to bottom in sequence, and a photoelectric detector is arranged near the sample cup; the computer is respectively and electrically connected with the EMCCD camera and the light intensity regulation and control module; the light intensity regulation and control module is electrically connected with the light source module and the photoelectric detector respectively. The screening system can be used for rapidly screening zearalenone, and specifically comprises the following steps: pretreating a sample, debugging an instrument, collecting a fluorescent color image, measuring the content of zearalenone by high performance liquid chromatography, establishing and optimizing a mathematical model, and measuring the content of zearalenone. The light source modules are densely arranged at equal intervals, and each sequence of LEDs is independently controlled, so that the uniform stability of illumination is improved, and the detection speed and the prediction precision are improved.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a fluorescence imaging visual rapid screening system and method for zearalenone.
Background
According to the food and oil agriculture organization (FAO) statistics of the United nations, 25 percent of the grain and oil crops are polluted by mycotoxin on average in the world. Under the influence of factors such as climate and storage, China is one of the most serious countries polluted by mycotoxin in the world, and the grain safety faces serious challenges. The mycotoxin is a secondary metabolite produced by fungi, and mainly comprises aflatoxin, zearalenone, deoxynivalenol, ochratoxin, fumonisin and the like. Mycotoxins have strong toxicity and carcinogenicity, can pollute all kinds of edible and feeding agricultural products, particularly corn, wheat, rice, barley, millet, oat and other grain crops, and seriously harm the health of people and livestock.
Mycotoxin pollution seriously threatens the food safety of China, but the traditional food safety management system lacks effective preventive technical means and rapid detection means, for example, the method for determining the zearalenone in the food safety national standard food of GB 5009.209-2016 adopts a liquid chromatography, a fluorescence photometry or a solid-phase extraction column purification liquid chromatography-mass spectrometry to determine the zearalenone. The required equipment is complex, the steps are complex, the detection period is long, the field rapid detection is difficult to realize, and the requirements of rapid real-time detection in the food circulation and processing processes cannot be met. The rapid detection method of zearalenone, for example, the methods such as ELISA, colloidal gold test paper, immunoaffinity column and the like are adopted to improve the detection speed and the detection precision, and the detection methods all need the extraction, purification and other treatments of organic solvents such as acetonitrile or methanol, and the like, and have complicated steps and time consumption.
The method has the advantages that the rapid identification of the grain pollution degree is realized by using the optical signal difference of normal grain materials and fungal-polluted materials, and the Aspergillus flavus pollution can be effectively identified by obtaining a fluorescence spectrum by using a high-resolution spectrometer; related patents disclose identification and sorting equipment and a sorting method for vomitoxin, an aflatoxin detection and sorting device, and a rapid detection method for aflatoxin content in brown rice based on FT-NIR technology, wherein mycotoxin pollution can be rapidly detected through fluorescence difference or near infrared spectrum characterization, but the grain particle mycotoxin is unevenly distributed (sometimes internally distributed), and the fluorescence capacity generated by ultraviolet light excitation in a mode of combining the mycotoxin and grain components is different, so that the misjudgment rate of the existing fluorescence detection method is higher; the method for evaluating the pollution level of the food mycotoxin by using the near infrared spectrum point measurement has the problem of partial completeness, so that the stability of the evaluation result is poor. If the visual distribution evaluation of the mycotoxins in the grains is realized by adopting a green analysis technology, the detection speed and accuracy are greatly improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a fluorescence imaging visual rapid screening system and method for zearalenone, which can be used for overcoming the problems of low conventional physicochemical detection speed and unfriendly chemical reagents to the environment, overcoming the problem of high measurement misjudgment rate caused by uneven mycotoxin distribution, overcoming the problem of poor adaptability and stability of spectral detection point measurement, and remarkably improving the detection speed and the detection reliability of zearalenone in grains.
The invention provides a fluorescence imaging visual rapid screening system for zearalenone, which is specifically carried out according to the following scheme:
a fluorescence imaging visual rapid screening system for zearalenone comprises a light intensity regulation and control module, a dark box and a computer; the camera bellows sequentially comprises an EMCCD camera, a time sequence controllable multi-wavelength LED ultraviolet annular light source module and a sample cup from top to bottom; the EMCCD camera is arranged at the top of the camera bellows, and a lens of the EMCCD camera can extend into the camera bellows; the time sequence controllable multi-wavelength LED ultraviolet annular light source module is fixed at the middle lower part of the dark box through a lifting device; the sample cup is arranged at the bottom of the dark box, and a photoelectric detector is arranged near the sample cup;
the light intensity regulation and control module is positioned outside the camera bellows and is respectively and electrically connected with the time sequence controllable multi-wavelength LED ultraviolet annular light source module and the photoelectric detector, acquisition control processing software is installed in the computer, and the computer is respectively and electrically connected with the EMCCD camera and the light intensity regulation and control module;
the time sequence controllable multi-wavelength LED ultraviolet annular light source module is annular and comprises a plurality of sequence LED light source groups and a contour indicating lamp group; the sequence LED light source groups are sequentially distributed on the whole ring at equal intervals and are positioned in the central position between the inner ring and the outer ring of the ring; the sequence LED light source group comprises a first sequence LED, a second sequence LED and a third sequence LED which are arranged at equal intervals; the outline indicating lamp group comprises 4 or 6 outline indicating lamps which are uniformly distributed along the circumferential direction and is arranged close to the inner ring of the circular ring;
the light intensity regulating module comprises a constant-voltage constant-current controller, an optocoupler regulator a for independently regulating the illumination intensity of the first sequence of LEDs, an optocoupler regulator b for independently regulating the illumination intensity of the second sequence of LEDs and an optocoupler regulator c for independently regulating the illumination intensity of the third sequence of LEDs; the optical coupling regulator a, the optical coupling regulator b and the optical coupling regulator c are connected in parallel to form an optical coupling regulator group, and the optical coupling regulator group is connected with the constant-voltage constant-current controller in series.
Preferably, the outer diameters of the first sequence of LEDs, the second sequence of LEDs and the third sequence of LEDs are all 1.6 mm; the excitation wavelength of the first sequence of LEDs is 236nm, the excitation wavelength of the second sequence of LEDs is 274nm, and the excitation wavelength of the third sequence of LEDs is 365 nm;
preferably, the center of the sample cup is arranged on a connecting line of a focusing center line of the EMCCD camera and a center line of the time sequence controllable multi-wavelength LED ultraviolet annular light source module; the vertical distance between the sequential controllable multi-wavelength LED ultraviolet annular light source module and the sample cup is 30-45 cm; the photoelectric detector is arranged in a uniform illumination area of the time sequence controllable multi-wavelength LED ultraviolet annular light source module;
preferably, the response band of the photodetector is 200-700 nm;
preferably, the EMCCD camera adopts an EMCCD chip with the resolution of 512 pixels by 512 pixels, a photosensitive device in the EMCCD camera is manufactured by an EX2 coating technology, and the time marking of the EMCCD camera is carried out by adopting an FPGA; the computer adopts a USB2.0 or 3.0 computer interface;
preferably, the acquisition control processing software adopts Visual Studio programming to realize that the development environments of the acquisition control processing software are a computer system Windows 7 flagship edition (64 bits) and a Visual Studio 2010 flagship edition, and the developed executable file of the acquisition control processing software can be installed in a computer or an industrial personal computer conforming to the lowest configuration.
The invention also provides a fluorescence imaging visual rapid screening method for zearalenone, which is realized by the following steps:
s1, preprocessing a grain sample:
obtaining a grain sample containing zearalenone naturally polluted by fusarium in batches, and crushing the grain sample into a grain powder sample by a crusher; dividing a grain powder sample into a plurality of sample samples; flatly paving a plurality of sample samples in the center of a sample cup, and placing the sample cup at a cross point of an image acquisition area of an EMCCD camera;
s2, debugging an instrument:
determining the luminous intensity under the condition of the optimal fluorescent color image through the definition, saturation and brightness of the fluorescent color image of the sample, wherein the luminous intensity is the standard value of the three sequence LED light source modules; the photoelectric detector acquires the luminous intensity of the LED light source modules of the three sequences and feeds the luminous intensity back to the computer, if the luminous intensity is not a standard value, the current intensity is increased or decreased through the light intensity regulation module, and the luminous intensity of the LED light source modules of the three sequences is automatically adjusted to be the standard value; then, standardizing acquisition parameters of the EMCCD camera, and setting the parameters as an optimization mode of the zearalenone, wherein the optimization mode comprises exposure time, the size of an area of interest, a field angle, a linkage trigger signal and an object distance;
s3, collecting a fluorescent color image:
controlling the sequential controllable multi-wavelength LED ultraviolet annular light source module to emit exciting light with different wavelengths through acquisition control processing software; sequentially lightening all the first sequence LEDs, all the second sequence LEDs and all the third sequence LEDs, exciting the sample after each lightening, and simultaneously collecting corresponding sequence fluorescent color images by an EMCCD camera; r, G and B color components are respectively extracted from the three collected sequence fluorescent color images;
s4, determining the content of the zearalenone by using a high performance liquid chromatography:
respectively measuring the content of zearalenone in the grain powder sample by adopting a high performance liquid chromatography on a plurality of sample samples collected by the fluorescent color image, and taking the measurement result as a reference value;
s5, establishing a mathematical model:
respectively taking average values of R, G and B color components of the obtained multiple sample samples and high performance liquid chromatography measurement results as the color components and the high performance liquid chromatography measurement values of the grain powder samples;
establishing a mathematical model between R, G and B color components of the grain powder sample and a high performance liquid chromatography measured value by adopting one or a combination mode of stepwise multi-linear regression, local weighted linear regression, weighted least square method and partial least square method, specifically:
CZEN＝a1×R1+b1×G1+c1×B1+a2×R2+b2×G2+c2×B2+a3×R3+b3×G3+c3×B3+d；
in the formula CZENFor an image space position f(x,y)The content of zearalenone in the lower grain, a1、a2、a3The values are respectively the image space location f(x,y)Lower R component regression coefficient, R1、R2、R3Respectively, red component values extracted from the fluorescent color image, b1、b2、b3The values are respectively the image space location f(x,y)Lower G component regression coefficient, G1、G2、G3Green component value extracted from fluorescent color image, c1、c2、c3Value of image space location f(x,y)Lower B component regression coefficient, B1、B2、B3The value of the blue component extracted from the fluorescent color image is d, and the value of d is a correction value of a mathematical regression model;
s6, optimizing a mathematical model:
verifying and evaluating the performance of the established zearalenone mathematical model by using an independent sample set and further optimizing the model;
s7, determining the content of zearalenone in the grain sample to be detected:
after the grain sample to be detected is processed in the steps S2 and S3, R, G and B color components of three sequence fluorescent color images are extracted, the zearalenone content of each spatial site of the color images is calculated by utilizing the zearalenone mathematical model optimally established in the step S6, and the zearalenone content of each spatial site in the grain sample is expressed by pseudo-color images.
Preferably, in step S2, the exposure time is 20ms, the diameter of the region of interest is 8cm, the field angle is 24 °, the linkage trigger signal is 1-1-1, and the object distance is 55 cm.
Preferably, in step S5, the mathematical model is built by using spotting modeling, and the spatial distribution of samples in a tiled batch is predicted when testing the samples.
Preferably, in step S7, the pseudo-color image expression sets 64 linear transitions from green to red to represent the contamination level of zearalenone in the grain sample.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention discloses a visual rapid screening system and a visual rapid screening method for zearalenone by fluorescence imaging, wherein a multi-wavelength ultraviolet LED is used as an excitation source to obtain a fluorescent color image of a grain powder sample, a chemical reagent is not needed in the detection process, and the problems of low speed and unfriendliness of the chemical reagent to the environment in the conventional physicochemical detection are solved; and in addition, the method is more convenient and fast in detection speed, and the use cost and the maintenance cost are far lower than those of high performance liquid chromatography and solid phase extraction column purification liquid chromatography-mass spectrometry.
(2) The invention overcomes the problem of poor adaptability and stability of point measurement caused by uneven distribution of mycotoxin, and solves the fundamental problems of uneven surface distribution and distribution inside and outside due to mycotoxin pollution in the conventional online color sorting method for detecting the mycotoxin of the grain or oil crops.
(3) According to the time sequence controllable multi-wavelength LED ultraviolet annular light source module, the annular equal-interval dense arrangement is adopted, the light intensity of each sequence of LED is independently controlled through the light intensity regulation and control module, the uniformity and the stability of light are improved, and the prediction precision of a zearalenone mathematical model is ensured by the excitation of the standardized ultraviolet LED.
(4) The invention controls the illumination intensity of each sequence of LEDs by the feedback of the photoelectric detector, eliminates the light intensity change caused by the service life of a light source or the disturbance of an external circuit, and improves the stability of the original signal of the fluorescent color image.
Drawings
Fig. 1 shows a schematic structural diagram of a fluorescence imaging zearalenone visual rapid screening system;
FIG. 2 is a schematic diagram showing an arrangement structure of a time-sequence controllable multi-wavelength LED ultraviolet annular light source module;
in the figure, 1-a time sequence controllable multi-wavelength LED ultraviolet annular light source module, 2-an EMCCD camera, 3-a light intensity regulation and control module, 4-a dark box, 5-a computer, 6-a photoelectric detector, 7-a fluorescent color image acquisition special sample cup, 8-a first sequence LED, 9-a second sequence LED, 10-a third sequence LED and 11-a contour indicating lamp.
FIG. 3 shows a timing control flow chart of the LED light source module;
fig. 4 shows a flow chart for establishing a fluorescence imaging zearalenone visual rapid screening method.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments, but not all embodiments, of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The following description will explain embodiments of the present invention in further detail with reference to the accompanying drawings.
Example 1
As shown in fig. 1, 2 and 3, a fluorescence imaging zearalenone visual rapid screening system comprises a light intensity regulation and control module 3, a dark box 4 and a computer 5; the camera bellows 4 comprises an EMCCD camera 2, a time sequence controllable multi-wavelength LED ultraviolet annular light source module 1 and a sample cup 7 from top to bottom in sequence; the EMCCD camera 2 is arranged at the top of the camera bellows 4, and the lens of the EMCCD camera 2 can extend into the camera bellows 4; the sequential controllable multi-wavelength LED ultraviolet annular light source module 1 is fixed at the middle lower part of the dark box 4 by a bracket, and the bracket moves up and down through a slideway; the sample cup 7 is arranged at the bottom of the dark box 4, the photoelectric detector 6 is arranged near the sample cup 7, and the photoelectric detector 6 is arranged in a uniform illumination area of the sequential controllable multi-wavelength LED ultraviolet annular light source module 1; the vertical distance between the sequential controllable multi-wavelength LED ultraviolet annular light source module 1 and the sample cup 7 is 30-45 cm;
when a grain sample fluorescence image is collected, the sample is placed in a sample cup 7, the center of the sample cup 7 is placed on a connecting line of a focusing center line of an EMCCD camera 2 and a center line of a time sequence controllable multi-wavelength LED ultraviolet annular light source module 1, and a central point is marked in an image collection area of a camera bellows 4;
the light intensity regulation and control module 3 is positioned outside the camera bellows 4 and is respectively and electrically connected with the time sequence controllable multi-wavelength LED ultraviolet annular light source module 1 and the photoelectric detector 6, acquisition control processing software is installed in the computer 5, and the computer 5 is respectively and electrically connected with the EMCCD camera 2 and the light intensity regulation and control module 3;
the time sequence controllable multi-wavelength LED ultraviolet annular light source module 1 is annular and comprises a plurality of sequence LED light source groups and a contour indicating lamp group; the sequence LED light source groups are sequentially distributed on the whole ring at equal intervals and are positioned in the central position between the inner ring and the outer ring of the ring; the sequence LED light source group comprises a first sequence LED 8, a second sequence LED 9 and a third sequence LED 10 which are arranged at equal intervals; the outline-indicating lamp group comprises 4 or 6 outline-indicating lamps 11 uniformly distributed along the circumferential direction and is arranged close to the inner ring of the circular ring.
The outer diameters of the first sequence LED 8, the second sequence LED 9 and the third sequence LED 10 are all 1.6 mm; the excitation wavelength of the first sequence LED 8 is 236nm, and 418nm fluorescence is generated by excitation; the excitation wavelength of the second sequence LED 9 is 274nm, and the excitation generates 440nm fluorescence; the excitation wavelength of the third sequence LED 10 is 365nm, and the third sequence LED is excited to generate 450nm fluorescence; the LED excitation wavelengths of the three sequences are determined by a large number of tests through optimization, the LEDs of the three sequences have high directivity, the light-emitting angle is 6-20 degrees, and the LED light source modules of each sequence form uniform illumination in the image acquisition area; in order to eliminate the interference of external light such as ambient light, the time sequence controllable multi-wavelength LED ultraviolet annular light source module 1 is arranged in the dark box 4, and the dark box 4 is closed during detection; the LED light source modules of each sequence independently control the light intensity through a light intensity regulation module 3;
the outline-showing indicating lamp 11 is a white light LED, and the outline-showing indicating lamp group is called a white light LED light source module; before the fluorescent image of the sample is collected, the illumination range and the collected area of the grain sample are determined by naked eyes, and the grain sample is confirmed to be arranged in the image collecting area;
the EMCCD camera 2 adopts an EMCCD chip with the resolution of 512 pixels by 512 pixels, and a photosensitive device of the EMCCD camera 2 is manufactured by an EX2 coating technology, so that the response range of quantum efficiency is expanded, and the influence of the EMCCD refrigeration temperature on reducing dark noise is controlled; the EMCCD camera 2 adopts an FPGA to perform time marking and accurate timing, and the EMCCD camera 2 adopts a USB2.0/3.0 computer interface to improve the data transmission speed; a 400nm long-wave pass filter is configured on a lens of the EMCCD camera 2, so that excitation light and stray light are prevented from entering the EMCCD camera 2; the EMCCD camera 2 is selected and determined through repeated tests, the content of toxin in the grain sample is high and low, the fluorescent reaction is different under the excitation of ultraviolet light, the sensitization sensitivity of the conventional CCD is poor, the signal to noise ratio of the obtained fluorescent image is low, the EMCCD camera 2 can improve the intensity of the fluorescent detection signal of the zearalenone in the grain sample, and a lower detection limit is obtained, and the peak quantum efficiency of the selected EMCCD is more than 90%.
The light intensity regulating module 3 comprises a constant-voltage constant-current controller, an optocoupler regulator a for independently regulating the illumination intensity of the first sequence of LEDs, an optocoupler regulator b for independently regulating the illumination intensity of the second sequence of LEDs and an optocoupler regulator for independently regulating the illumination intensity of the third sequence of LEDs; the optical coupling regulator a, the optical coupling regulator b and the optical coupling regulator c are connected in parallel to form an optical coupling regulator group, and the optical coupling regulator group is connected with the constant-voltage constant-current controller in series; the constant voltage and constant current controller adjusts the illumination intensity of the corresponding sequence LED through an optical coupling adjuster a, an optical coupling adjuster b or an optical coupling adjuster c, and in addition, the constant voltage and constant current controller supplies power for the outline indicating lamp set (white light LED light source module) in a constant voltage mode;
the light intensity regulating module 3 is electrically connected with the sequential controllable multi-wavelength LED ultraviolet annular light source module 1, and can ensure that the LED light source modules in each sequence keep stable illumination intensity; the light source fluctuates along with the change of the electricity utilization environment, and the light intensity regulating module 3 regulates the illumination intensity of the sequence ultraviolet LED light source group according to the feedback signal of the photoelectric detector 6; the photoelectric detector 6 obtains the luminous intensity of each sequence of LED light source modules, compares the luminous intensity with the luminous intensity when a mathematical model is established, and transmits an instruction to the optical coupling regulator a, the optical coupling regulator b or the optical coupling regulator c through the constant-voltage constant-current controller by acquiring, controlling and processing software when the difference exists, so as to adjust the current of each sequence of LED light source modules which are correspondingly controlled, thereby automatically adjusting the illumination intensity of each sequence of LED light source modules and ensuring the intensity stability of each sequence of LED light source modules; the mathematical model of zearalenone in grains is established on the basis of light source intensity calibration, and the stable and high-precision detection result needs to be adjusted in real time.
The response wave band of the photoelectric detector 6 is 200-700nm, and the illumination intensity of the LED light source modules under each sequence is adjusted through the acquisition control processing software self-adaptive control algorithm, so that the stability of the light intensity of the LED light source modules in each sequence is ensured; the adaptive control algorithm is a light intensity change and current magnitude calculation equation and sends an adjustment instruction to the light intensity control module 3.
Acquisition control processing software installed in the computer 5 is realized by adopting Visual Studio programming, and information interaction with the camera is realized by calling a software development kit provided by an EMCCD camera 2 supplier; the acquisition control processing software integrates various control instructions of the EMCCD camera 2 and the light intensity regulation and control module 3; the display interface of the acquisition control processing software has the functions of displaying the acquired fluorescent color image in real time, setting parameters of a built-in camera, regulating and controlling light intensity instructions, correcting the image, extracting image data, calculating a model of toxin content, generating a pseudo-color image, storing and issuing data and the like; the development environment of the acquisition control processing software is a Windows 7 flagship edition (64 bits) and a Visual Studio 2010 flagship edition of a computer system, and an executable file of the developed acquisition control processing software can be installed on a computer or an industrial personal computer which conforms to the lowest configuration, so that the acquisition control processing software has better portability and compatibility.
The acquisition control processing software is used for feeding back a light intensity adjusting instruction of the time sequence controllable multi-wavelength LED ultraviolet annular light source module 1 and controlling the time sequence controllable multi-wavelength LED ultraviolet annular light source module 1 to emit exciting light with different wavelengths; the acquisition control processing software controls to only light all the LEDs 8 of the first sequence 236nm, so that the EMCCD camera 2 acquires a first sequence fluorescent color image; the acquisition control processing software controls to only light the LEDs 9 of the second sequence 274nm, so that the EMCCD camera 2 acquires a fluorescent color image of the second sequence; the acquisition control processing software controls to only light all the 365nm LEDs 10 of the third sequence, so that the EMCCD camera 2 acquires a fluorescence color image of the third sequence; in addition, the acquisition control processing software is also used for calculating the zearalenone content of the grain sample at each spatial site and displaying and storing the result.
Example 2
As shown in fig. 4, a fluorescence imaging zearalenone visual rapid screening method is implemented by using the fluorescence imaging zearalenone visual rapid screening system described in embodiment 1, and the screening method is specifically implemented by the following steps:
s1, preprocessing a grain sample:
firstly, obtaining a grain sample containing zearalenone naturally polluted by fusarium in batches, and crushing the sample by a high-speed crusher (the rotating speed is more than or equal to 12000r/min) to prepare a grain powder sample; dividing a grain powder sample into four sample samples by adopting a quartering method, respectively spreading three parallel sample samples in a sample cup, and placing the center of the sample cup at a cross point of an image acquisition area of an EMCCD camera;
s2, debugging an instrument:
determining the luminous intensity under the condition of the optimal fluorescent color image through the quality parameters of the sample fluorescent color image, such as definition, saturation, brightness and the like, wherein the luminous intensity is the standard value of the three sequence LED light source modules; the photoelectric detector acquires the luminous intensity of the LED light source modules of the three sequences and feeds the luminous intensity back to the computer, if the luminous intensity is not a standard value, the current intensity is increased or decreased through the light intensity regulation module, and the luminous intensity of the LED light source modules of the three sequences is automatically adjusted to be the standard value; then, the acquisition parameters of the EMCCD camera are standardized and set as an optimization mode of the zearalenone, the exposure time is 20ms, the size and the diameter of an interested area are 8cm, the field angle is 24 degrees, the linkage trigger signal is 1-1-1, and the object distance is 55 cm.
S3, collecting a fluorescent color image:
controlling a time sequence controllable multi-wavelength LED ultraviolet annular light source module to emit exciting light with different wavelengths through acquisition control processing software, and determining the wavelength of the exciting light of a first sequence of LEDs to be 236nm, the wavelength of the exciting light of a second sequence of LEDs to be 274nm and the wavelength of the exciting light of a third sequence of LEDs to be 360 nm;
lighting all the first sequence LEDs to excite the sample of the sample, and simultaneously collecting a first sequence fluorescent color image by an EMCCD camera; lighting all the second sequence LEDs to excite the sample, and simultaneously collecting a second sequence fluorescent color image by the EMCCD camera; lighting all the third sequence LEDs to excite the sample, and simultaneously collecting a third sequence fluorescent color image by the EMCCD camera; r, G and B color components are respectively extracted from the three collected sequence fluorescence color images;
s4, determining the content of the zearalenone by using a high performance liquid chromatography:
respectively measuring the content of zearalenone in three parallel sample samples collected by a fluorescent color image by adopting a high performance liquid chromatography, and taking the measurement result as a reference value;
s5, establishing a mathematical model:
the R, G, B color component and the high performance liquid chromatography measurement result obtained from the three parallel sample samples are respectively averaged to be used as the color component and the high performance liquid chromatography measurement value of the grain powder sample;
adopting one or the combination of stepwise multi-linear regression, local weighted linear regression, weighted least square method and partial least square method to establish a mathematical model between the color component of the grain powder sample and the measured value of the high performance liquid chromatography, which specifically comprises the following steps:
CZEN＝a1×R1+b1×G1+c1×B1+a2×R2+b2×G2+c2×B2+a3×R3+b3×G3+c3×B3+d
in the formula CZENFor an image space position f(x,y)The content of zearalenone in the lower grain, a1、a2、a3The values are respectively the image space location f(x,y)Lower R component regression coefficient, R1、R2、R3Respectively, red component values extracted from the fluorescent color image, b1、b2、b3The values are respectively the image space location f(x,y)Lower G component regression coefficient, G1、G2、G3Green component value extracted from fluorescent color image, c1、c2、c3Value of image space location f(x,y)Lower B component regression coefficient, B1、B2、B3The value of the blue component extracted from the fluorescent color image is d, and the value of d is a correction value of a mathematical regression model;
the mathematical model is established by adopting sample application accurate modeling, and when a sample is tested, the spatial distribution of the sample in batches is tiled for prediction;
s6, optimizing a mathematical model:
adding independent grain sample sets, repeating the operations of the steps S1-S4, removing the color components of the samples and the discrete values in the high performance liquid chromatography measured value, verifying and evaluating the performance of the zearalenone mathematical model established in the step S5, and further optimizing a in the mathematical model1、a2、a3、b1、b2、b3、c1、c2、c3The numerical values of d, etc.;
s7, determining the content of zearalenone in the grain sample to be detected:
processing a grain sample to be detected in steps S2 and S3, extracting R, G and B color components of three sequence fluorescent color images, calculating the zearalenone content of each spatial site of the color images by utilizing a zearalenone mathematical model optimally established in S6, and expressing the zearalenone content of each spatial site in the grain sample by using a pseudo-color image;
the pseudo-color image expression sets 64-color linear transition from green (representing 0) to red (representing 64) to represent the pollution degree of the zearalenone, the color linearity of 0 to 64 corresponds to the content of the zearalenone to be 0-2000ppb, when the content of the zearalenone in the grain reaches more than 2000ppb, 64 (red) is also adopted to represent serious pollution, and the content and the hazard of the zearalenone in the grain can be intuitively displayed similarly to a traffic light.
The corn, the wheat, the bean pulp and the rice are successively verified by utilizing the fluorescence imaging visual rapid screening system and the fluorescence imaging visual rapid screening method, the traditional detection method is long in detection time and high in detection cost, the traditional detection method is a point measurement method, the misjudgment rate is high due to the defect that points are used for replacing surfaces, the detection result only represents a detected sample, and the fluorescence imaging visual rapid screening system and the fluorescence imaging visual rapid screening method have substantial progress in the aspects of detection time, detection cost, detection dimension and detection precision.
TABLE 1 comparison of the test results of the screening method of the present invention with those of the conventional method
Experiments prove that the fluorescence imaging visual rapid screening system and method for zearalenone provided by the invention have the advantages that the sequential controllable multi-wavelength LED ultraviolet annular light source modules which are arranged at equal intervals are uniformly illuminated, high-quality fluorescence color images are obtained by controlling the illumination intensity of each sequence of LEDs, and the applicability and accuracy of a zearalenone mathematical model are ensured by controlling a standardized light source.
The invention overcomes the problems of low speed and unfriendly environment of chemical reagents in the conventional physicochemical detection, solves the problem of high misjudgment rate of measurement caused by uneven distribution of mycotoxin, solves the problems of poor adaptability and stability of spectral detection point measurement, obviously improves the detection speed and the detection reliability of zearalenone in grains, and has outstanding substantive characteristics and obvious progress in the aspects of detection capability, detection precision, visual expression and the like compared with the prior art.
Although the embodiments of the present invention have been described in conjunction with the accompanying drawings, those skilled in the art may make various modifications and variations without departing from the spirit and scope of the invention, and such modifications and variations fall within the scope defined by the appended claims.
Claims (10)
1. A fluorescence imaging visual rapid screening system for zearalenone is characterized by comprising a light intensity regulation and control module (3), a dark box (4) and a computer (5); the camera bellows (4) sequentially comprises an EMCCD camera (2), a time sequence controllable multi-wavelength LED ultraviolet annular light source module (1) and a sample cup (7) from top to bottom; the EMCCD camera (2) is arranged at the top of the dark box (4), and a lens of the EMCCD camera (2) can extend into the dark box (4); the sequential controllable multi-wavelength LED ultraviolet annular light source module (1) is fixed at the middle lower part of the dark box (4) through a lifting device; the sample cup (7) is arranged at the bottom of the dark box (4), and a photoelectric detector (6) is arranged near the sample cup (7);
the time sequence controllable multi-wavelength LED ultraviolet annular light source module (1) is annular and comprises a plurality of sequence LED light source groups and a contour indicating lamp group; the sequence LED light source groups are sequentially distributed on the whole ring at equal intervals and are positioned in the central position between the inner ring and the outer ring of the ring; the sequence LED light source group comprises a first sequence LED (8), a second sequence LED (9) and a third sequence LED (10) which are arranged at equal intervals; the outline indicating lamp group comprises 4 or 6 outline indicating lamps (11) which are uniformly distributed along the circumferential direction and are arranged close to the inner ring of the circular ring;
the light intensity regulation and control module (3) is positioned outside the camera bellows (4) and is respectively and electrically connected with the time sequence controllable multi-wavelength LED ultraviolet annular light source module (1) and the photoelectric detector (6), acquisition control processing software is installed in the computer (5), and the computer (5) is respectively and electrically connected with the EMCCD camera (2) and the light intensity regulation and control module (3); the light intensity regulation and control module (3) regulates the illumination intensity of the sequence ultraviolet LED light source module according to the feedback signal of the photoelectric detector (6), and specifically comprises the following steps: the photoelectric detector (6) obtains the luminous intensity of each sequence of LED light source modules, the luminous intensity is compared with the luminous intensity when a mathematical model between the color component of a grain powder sample and a high performance liquid chromatography measured value is established, when a difference exists, the acquisition control processing software transmits an instruction to the optical coupling regulator a, the optical coupling regulator b or the optical coupling regulator c through a constant-voltage constant-current controller through an adaptive control algorithm, the current of each sequence of LED light source modules which are correspondingly controlled is adjusted, the illumination intensity of each sequence of LED light source modules is automatically adjusted, and the intensity stability of each sequence of LED light source modules is ensured; the self-adaptive control algorithm is a light intensity change and current magnitude calculation equation;
the acquisition control processing software integrates various control instructions of the EMCCD camera (2) and the light intensity regulation and control module (3); the display interface of the acquisition control processing software has the functions of displaying the acquired fluorescent color image in real time, setting parameters of a built-in camera, regulating and controlling light intensity instructions, correcting the image, extracting image data, calculating a toxin content model, generating a pseudo-color image and storing and publishing data; the acquisition control processing software utilizes the embedded toxin content calculation model for calculating the zearalenone content of each spatial site of the grain sample and displaying and storing the result, and the zearalenone content of each spatial site in the grain sample is expressed by a pseudo-color image;
the content calculation model is a mathematical model established between the color component of the grain powder sample and the measured value of the high performance liquid chromatography, and specifically comprises the following steps: cZEN=a1×R1+b1×G1+c1×B1+a2×R2+b2×G2+c2×B2+a3×R3+b3×G3+c3×B3+ d; in the formula CZENFor a spatial location of an imagef (x,y) The content of zearalenone in the lower grain, a1、a2、a3Values are respectively image space positionsf (x,y) Lower R component regression coefficient, R1、R2、R3Respectively, red component values extracted from the fluorescent color image, b1、b2、b3Values are respectively image space positionsf (x,y) Lower G component regression coefficient, G1、G2、G3Green component value extracted from fluorescent color image, c1、c2、c3Value of image space locationf (x,y) Lower B component regression coefficient, B1、B2、B3The value of the blue component extracted from the fluorescent color image is d, and the value of d is a correction value of a mathematical regression model; and then verifying and evaluating the performance of the established zearalenone mathematical model by using an independent sample set and further optimizing the model.
2. The fluorescence imaging zearalenone visual rapid screening system according to claim 1, wherein the first sequence LED (8), the second sequence LED (9) and the third sequence LED (10) are all 1.6mm in outer diameter; the excitation wavelength of the first sequence of LEDs (8) is 236nm, the excitation wavelength of the second sequence of LEDs (9) is 274nm, and the excitation wavelength of the third sequence of LEDs (10) is 365 nm.
3. The fluorescence imaging zearalenone visual rapid screening system according to claim 1, wherein the light intensity regulation and control module (3) comprises a constant voltage and constant current controller, an optical coupling regulator a for individually regulating the illumination intensity of the first sequence of LEDs (8), an optical coupling regulator b for individually regulating the illumination intensity of the second sequence of LEDs (9), and an optical coupling regulator c for individually regulating the illumination intensity of the third sequence of LEDs (10); the optical coupling regulator a, the optical coupling regulator b and the optical coupling regulator c are connected in parallel to form an optical coupling regulator group, and the optical coupling regulator group is connected with the constant-voltage constant-current controller in series.
4. The fluorescence imaging zearalenone visual rapid screening system according to claim 1, characterized in that the center of the sample cup (7) is placed on the connection line of the focusing center line of the EMCCD camera (2) and the center line of the time sequence controllable multi-wavelength LED ultraviolet annular light source module (1); the vertical distance between the time sequence controllable multi-wavelength LED ultraviolet annular light source module (1) and the sample cup (7) is 30-45 cm; the photoelectric detector (6) is arranged in a uniform illumination area of the time sequence controllable multi-wavelength LED ultraviolet annular light source module (1); the response wave band of the photoelectric detector (6) is 200-700 nm.
5. The fluorescence imaging zearalenone visual rapid screening system according to claim 1, characterized in that the EMCCD camera (2) adopts an EMCCD chip with a resolution of 512 x 512 pixels, the photosensitive device in the EMCCD camera (2) is made by EX2 coating technology, and the FPGA is used for time marking of the EMCCD camera (2); the EMCCD camera (2) adopts a USB2.0 or 3.0 computer interface.
6. The fluorescence imaging zearalenone Visual rapid screening system according to claim 1, wherein the acquisition control processing software is implemented by Visual Studio programming; the development environment of the acquisition control processing software is 64-bit Windows 7 flagship edition and Visual Studio 2010 flagship edition of a computer system, and the executable file of the developed acquisition control processing software can be installed in a computer or an industrial personal computer which accords with the lowest configuration.
7. The rapid screening method of a rapid screening system according to claim 1, comprising the steps of:
s1, preprocessing the grain sample:
obtaining a grain sample containing zearalenone naturally polluted by fusarium in batches, and crushing the grain sample into a grain powder sample by a crusher; dividing a grain powder sample into a plurality of sample samples; flatly paving a plurality of sample samples in the center of a sample cup, and placing the sample cup at a cross point of an image acquisition area of an EMCCD camera;
s2, debugging the instrument:
determining the luminous intensity under the condition of the optimal fluorescent color image through the definition, saturation and brightness of the fluorescent color image of the sample, wherein the luminous intensity is the standard value of the three sequence LED light source modules; the photoelectric detector acquires the luminous intensity of the LED light source modules of the three sequences and feeds the luminous intensity back to the computer, if the luminous intensity is not a standard value, the current intensity is increased or decreased through the light intensity regulation module, and the luminous intensity of the LED light source modules of the three sequences is automatically adjusted to be the standard value; then, standardizing acquisition parameters of the EMCCD camera, and setting the parameters as an optimization mode of the zearalenone, wherein the optimization mode comprises exposure time, the size of an area of interest, a field angle, a linkage trigger signal and an object distance;
s3, collecting a fluorescent color image:
controlling the sequential controllable multi-wavelength LED ultraviolet annular light source module to emit exciting light with different wavelengths through acquisition control processing software; sequentially lightening all the first sequence LEDs, all the second sequence LEDs and all the third sequence LEDs, exciting the sample after each lightening, and simultaneously collecting corresponding sequence fluorescent color images by an EMCCD camera; r, G and B color components are respectively extracted from the three collected sequence fluorescent color images;
s4, measuring the content of the zearalenone by using a high performance liquid chromatography:
respectively measuring the content of zearalenone in the grain powder sample by adopting a high performance liquid chromatography on a plurality of sample samples collected by the fluorescent color image, and taking the measurement result as a reference value;
s5, establishing a mathematical model:
respectively taking average values of R, G and B color components of the obtained multiple sample samples and high performance liquid chromatography measurement results as the color components and the high performance liquid chromatography measurement values of the grain powder samples;
establishing a mathematical model between R, G and B color components of the grain powder sample and a high performance liquid chromatography measured value by adopting one or a combination mode of stepwise multi-linear regression, local weighted linear regression, weighted least square method and partial least square method, specifically:
CZEN=a1×R1+b1×G1+c1×B1+a2×R2+b2×G2+c2×B2+a3×R3+b3×G3+c3×B3+d；
in the formula CZENFor a spatial location of an imagef (x,y) The content of zearalenone in the lower grain, a1、a2、a3Values are respectively image space positionsf (x,y) Lower R component regression coefficient, R1、R2、R3Respectively, red component values extracted from the fluorescent color image, b1、b2、b3Values are respectively image space positionsf (x,y) Lower G component regression coefficient, G1、G2、G3Green component value extracted from fluorescent color image, c1、c2、c3Value of image space locationf (x,y) Lower B component regression coefficient, B1、B2、B3The value of the blue component extracted from the fluorescent color image is d, and the value of d is a correction value of a mathematical regression model;
s6, optimizing the mathematical model:
verifying and evaluating the performance of the established zearalenone mathematical model by using an independent sample set and further optimizing the model;
s7, determining the content of zearalenone in the grain sample to be detected:
after the grain sample to be detected is processed in the steps S2 and S3, R, G and B color components of three sequence fluorescent color images are extracted, the zearalenone content of each spatial site of the color images is calculated by utilizing the zearalenone mathematical model optimally established in the step S6, and the zearalenone content of each spatial site in the grain sample is expressed by pseudo-color images.
8. The rapid screening method according to claim 7, wherein in step S2, the exposure time is 20ms, the diameter of the region of interest is 8cm, the field angle is 24 °, the linkage trigger signal is 1-1-1, and the object distance is 55 cm.
9. The rapid screening method according to claim 7, wherein in step S5, the mathematical model is established by using spotting modeling, and spatial distribution prediction of tiled sample is performed when testing sample.
10. The rapid screening method according to claim 7, wherein in step S7, the pseudo-color image expression is set to have 64-color linear transitions from green to red to represent the degree of zearalenone contamination in the grain sample.
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