A method for correcting fluorescence images to obtain amplification fluorescence results and an amplification detection system

Abstract

The present invention belongs to the field of in vitro diagnostic technology, and particularly relates to a method for correcting fluorescence images to obtain amplification fluorescence results and an amplification detection system. The technical scheme is: a method for correcting fluorescence images to obtain amplification fluorescence results, an array optical detector obtains multiple fluorescence images at different times during the amplification process, and obtains a saturation / null value binary state associated image from at least one of the fluorescence images based on a first threshold value, and calculates at least one of the fluorescence images and the associated image using a first calculation method to obtain a corrected amplification reference image, and divides the amplification reference image into multiple ROI areas using a segmentation algorithm, and calculates all pixel values ​​of each ROI area of ​​the amplification reference image using a second calculation method, and uses all pixel values ​​of each ROI area as the amplification fluorescence result reference value of at least one of the fluorescence images at the corresponding moment. The present invention provides a method for correcting fluorescence images to obtain amplification fluorescence results and an amplification detection system.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to a method for correcting a fluorescence image to obtain an amplification fluorescence result and an amplification detection system. Background Art

[0002] As a detection technology that uses in vitro samples as the object, in vitro diagnostic technology has the advantages of little damage to the main body and low probability of contamination of the main body during the detection process. Therefore, it is widely used in scenarios such as disease diagnosis, forensic identification, component determination, and seed identification and cultivation in agriculture. Among them, the amplification fluorescence quantitative method using nucleic acid fragments as the detection object has become the gold standard for disease diagnosis. Domestic and foreign companies have also designed different systems to perform amplification detection, which include two types of systems: constant temperature and variable temperature. By quantitatively acquiring the fluorescence value in the reaction well, it can accurately determine whether the sample contains the target gene fragment or can quantify the original copy number of the target gene fragment in the sample. This technology has also played an important role in the detection of respiratory diseases such as influenza A and influenza B in the past.

[0003] Most of the fluorescence detection systems designed at present are single-point or scanning type detection systems, in which optical fiber is usually used as the transmission medium in the single-point detection scheme. The emission light source projects light of a specific wavelength into the sample tube through the optical fiber. The target gene fragment is amplified in the sample tube, resulting in the separation of the fluorescent agent and the quencher, so that the emission light is absorbed by the fluorescent agent and converted into fluorescence of another wavelength, and then transmitted to the receiving end through the optical fiber. The receiving end here can be a photosensitive end of the type of photodiode or photomultiplier tube, which can convert the change of fluorescence amount into the change of strength of the electrical signal value, and then can be processed to obtain a fluorescence amplification curve. The target gene fragment can be qualitatively or quantitatively analyzed by analyzing the fluorescence amplification curve. This detection system can be seen in the schemes of US invention patent application US20070098594A1 and Chinese invention patent CN105675574B. This method can only transmit and illuminate a small area in the sample tube through the optical fiber, so the fluorescence detection result obtained is only the value of the small area. There is a risk that the detection result may deviate from the true result. In addition, the sample tube and the detection end need to correspond or the two can be driven to produce relative motion to complete the detection, which makes the overall design more complicated. Another is a scanning type detection scheme. The scheme authorized by the Chinese invention patent CN101698823B integrates the transmitter and the receiver in the same module, and uses mechanical motion to drive the module to scan along a specific scanning path to complete the final sample detection. In this scheme, scanning needs to be performed one by one, so the detection efficiency of the system is low. In order to improve efficiency, EP1303747B1 authorizes a linear scanning scheme, in which the detection sensor is configured to linearly arrange multiple photosensitive units, and in order to make the detection result more accurate, a linear scanning scheme tilted at a predetermined acute angle is proposed. Although the efficiency is improved to a certain extent, the signal obtained by this scanning scheme has more blank areas and noise signal areas, and requires a more complex algorithm to remove noise and the algorithm design of the corresponding reaction hole position signal. In the early stage of the development of fluorescent quantitative PCR, there is another technical route such as the scheme authorized by the European invention patent EP1681558B1, which sets the transmitter and the receiver to an array type, and the light source is delivered in the form of a field. The array type receiving element can also focus and obtain image information in multiple sample tube holes. In order to ensure the image quality, the incident angle of the emitted light source projected into the sample tube is limited and optimized in this scheme, and the emission enters the sample tube at the optimal incident angle to complete the excitation of fluorescence. This design can only achieve an efficient design in hardware to obtain fluorescence images in multiple sample tubes as a whole. However, in fact, there are still many problems in subsequent applications. These problems include background noise, crosstalk, and the need to correct image results. If these problems cannot be solved, the accuracy of the sample detection results will be low. This is also related to the fact that fluorescence detection is easily affected by interference due to the weak intensity of fluorescence itself. In order to adapt to the observation needs of the human eye, existing cameras will introduce various correction methods, making it very difficult to obtain multiple relatively accurate amplified fluorescence curve results simultaneously through image processing.

[0004] It is precisely for the above reasons that there are relatively few efficient detection systems that use field-type light source projection and array-type sensors to obtain regional images to simultaneously obtain amplification fluorescence results in multiple sample tubes. In order to achieve efficient and accurate amplification results, it is necessary to design a correction scheme to correct the image before calculating the amplification results of the area of ​​interest. This is also a solution that urgently needs to be researched and developed. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the object of the present invention is to provide a method for correcting fluorescence images to obtain amplification fluorescence results and an amplification detection system.

[0006] The technical solution adopted by the present invention is:

[0007] A method for correcting a fluorescence image to obtain an amplification fluorescence result, wherein an array optical detector obtains a plurality of fluorescence images at different moments in an amplification process, obtains a saturation / null value binary state associated image from at least one of the fluorescence images based on a first threshold value, calculates at least one of the fluorescence images and the associated image using a first calculation method to obtain a corrected amplification reference image, divides the amplification reference image into a plurality of ROI regions using a segmentation algorithm, calculates all pixel values ​​of each ROI region of the amplification reference image using a second calculation method, and uses all pixel values ​​of each ROI region as the amplification fluorescence result reference value of at least one of the fluorescence images at a corresponding moment.

[0008] As a preferred solution of the present invention, the first operation method is the Hough circle method, and the amplified reference image is divided into a plurality of circular ROI areas by the first operation method.

[0009] As a preferred solution of the present invention, the first threshold is a non-fixed value that changes adaptively according to the multiple fluorescence images corresponding to different moments.

[0010] As a preferred solution of the present invention, the first operation method is a multiplication operation, and at least one of the fluorescence images and the associated image are multiplied to obtain a corrected amplified reference image.

[0011] As a preferred solution of the present invention, the second operation method is one of an RGB method, an HSL method, a grayscale method and a cumulative calculation method.

[0012] As a preferred solution of the present invention, the segmentation algorithm is an area equalization method, by which the amplified reference image is divided into a plurality of quadrilateral ROI regions of equal area.

[0013] As a preferred solution of the present invention, the multiple fluorescence images are all original images without gain.

[0014] An amplification detection system comprises an emission source capable of generating an excitation source of at least one wavelength, and a biological detection consumable comprising a plurality of well units; each well unit in the biological detection consumable comprises a fluorescent agent, a quencher and a fragment to be detected; the biological detection consumable can be subjected to a constant temperature or variable temperature amplification cycle, and cooperates with an array-type detector to obtain a plurality of fluorescence images corresponding to different moments; and further comprises a processing module, which can obtain amplification fluorescence results by correcting the fluorescence image.

[0015] As a preferred solution of the present invention, a microlens unit is further configured above each well unit.

[0016] A computer-readable medium comprises the above-mentioned method for correcting fluorescence images and obtaining amplified fluorescence results.

[0017] The beneficial effects of the present invention are:

[0018] 1. The present invention uses fluorescence images at different times as a reference, obtains associated images from at least one of the fluorescence images using a first threshold, and calculates the associated images and at least one of the fluorescence images using a first calculation method to obtain a corrected amplification reference image, and divides the amplification reference image into multiple ROI areas (regions of interest) based on the analysis object, and performs pixel-level calculations in a single ROI area to finally obtain the amplification fluorescence result in the area, thereby completing the processing of all fluorescence images and obtaining amplification fluorescence curves in multiple ROI areas. In this way, the fitting of fluorescence amplification curves in all sample tubes can be performed efficiently and accurately, overcoming the problem of circuit noise, background interference, and fluorescence crosstalk of adjacent or adjacent sample tubes when using the original image, especially the low-intensity fluorescence image, which has a more serious influence, thereby distorting the amplification result or even having a false negative problem. Setting the first threshold as a non-fixed value can adapt to the influence of various noise signals at different times, so that the fluorescence images at different times can be adaptively and efficiently calibrated.

[0019] 2. The first operation method of the present invention is a multiplication operation, which can quickly perform image calibration without using complex operations such as convolution. Then, the correlation values ​​of each pixel in all areas of the multiple ROI areas are processed by an operation method such as RGB method, HSL method, grayscale method or cumulative calculation method, and finally the amplified fluorescence results at the corresponding time are obtained. By processing multiple fluorescence images at different times, the fluorescence curves of the amplification process of all ROI areas can be finally obtained, so that the quantitative or qualitative detection results of all units to be tested can be efficiently completed. The area equal division method or Hough circle method can be used to obtain different types of ROI areas, which can be adapted to different detection scenarios. The use of the original image without gain for the fluorescence image can achieve a more thorough elimination of the influence of noise during the correction process. The first threshold is less affected by noise and the correction result is more accurate.

[0020] 3. The present invention also protects a computer-readable medium, which contains the method of the present invention. The present invention also protects an amplification detection system, which can achieve efficient batch acquisition of fluorescence quantitative curves, and the result has high accuracy, and is suitable for fast and efficient amplification detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a diagram of the amplification detection system provided by the present invention when the carrier platform is pulled out;

[0022] Figure 2 is a diagram of the amplification detection system provided by the present invention when the carrier platform is retracted;

[0023] Figure 3 It is an optical principle diagram of amplification detection provided by the present invention;

[0024] Figure 4 It is the associated image result obtained by the gain fluorescence detection image provided by the present invention;

[0025] Figure 5 It is the associated image result obtained by the gain-free fluorescence detection image provided by the present invention;

[0026] Figure 6 A result diagram obtained by processing multiple pixel values ​​using different processing methods provided by the present invention;

[0027] Figure 7 It is a stability verification diagram of whether or not the associated image operation is provided by the present invention and the multi-pixel operation results obtained by different algorithms;

[0028] Figure 8 It is a circular ROI area map obtained by the first method provided by the present invention;

[0029] Fig. 9 is a quadrilateral ROI area map obtained by the second method provided by the present invention;

[0030] Fig.10 It is a verification diagram of different well units of the present invention after correcting images with the same fluorescence intensity;

[0031] Fig.11 is a verification curve after correcting images of different fluorescence intensities for different well units of the present invention;

[0032] Fig.12 It is a linearization verification diagram of fluorescence intensity in multiple detection well units at different positions provided by the present invention;

[0033] Fig.13 It is the statistical result of the linearization verification of fluorescence intensity in multiple detection well units at different positions provided by the present invention;

[0034] Fig.14The first method provided by the present invention is used to obtain a process diagram of amplification fluorescence results in multiple ROI regions;

[0035] Fig.15 This is a process diagram of obtaining amplification fluorescence results in multiple ROI regions using the second method provided by the present invention.

[0036] In the figure, 100-biological detection consumables; 200-carrying platform; 201-receiving part; 202-heat sink; 300-detection module; 400-main body; 401-first heat dissipation component; 402-second heat dissipation component; 11-light source; 12-first lens; 13-second lens; 14-light source filter; 21-receiver; 22-fluorescent lens; 23-fluorescent filter; 31-dichroic mirror; 32-reflector; 33-Fresnel lens; 34-microlens. DETAILED DESCRIPTION

[0037] In order to make the purpose, 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 and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0038] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0039] As summarized above, the currently used optical fiber fluorescence detection system has a small detection area, which is only focused on a small area in the sample tube. However, PCR amplification occurs at different locations of the solution in the entire sample tube, and the amplification efficiency at different locations is also different. This difference will also cause the local detection results to not reflect the actual amplification results in the sample tube, and there may be deviations in the amplification data, which will seriously lead to problems such as false negatives. Most non-optical fiber detection systems use a scanning detection scheme, where the transmitter and / or the receiver are configured on a mechanical drive mechanism, and the amplification results in multiple sample tubes arranged in an array are detected one by one or row by row. Although this type of detection has a larger detection range than optical fiber, in order to match more accurate fluorescence reception results, it is generally necessary to control the speed of mechanical scanning, and when the linearly arranged sensor unit performs linear scanning, there are more irrelevant areas with a large focus area, which requires a relatively complex algorithm to eliminate noise interference and needs to perform a combination of scanning results and precise positioning of the hole position. These problems require the design of a new detection system and processing algorithm to obtain efficient and accurate fluorescence detection results.

[0040] Figure 1 and Figure 2: is a diagram of the amplification detection system provided by the present invention. The amplification detection system includes a main body 400, which may include a main body frame obtained by processing several structural frames, and basically presents a cubic appearance. In addition, a circuit board and a detection module 300 are configured in the uppermost area, and a first heat dissipation component 401 is configured on the main body 400 to dissipate heat for the detection module 300 and / or the circuit board in the upper area to ensure the reliable operation of the amplification system. Here, the detection module 300 is configured as a fixed structure, wherein the light source module includes a field projection light source, and the receiving module includes a receiver of an array-type layout photosensitive unit. There are no mechanical movement or adjustment components in the light source module and the receiving module, so that the reliability of the whole machine is higher. The carrier 200 includes a receiving part 201 with multiple sample receiving well units in an array-type layout, and the number of well units included can be 48, 64, 96, 192, 288 and 384, etc. The receiving part 201 can be configured with a thermal cycle heating source, which can include several Peltier elements with heating and cooling functions in a variable temperature type PCR system, and can include several thermal resistors or other heat generating units in a constant temperature type PCR system. A radiator 202 may be provided at the other end relative to the receiving part 201 to dissipate the waste heat generated by the heating element to ensure the reliable operation of the heat source. The receiving part 201 may receive a whole biological detection consumable 100 with multiple well units arranged in an array, or a biological detection consumable 100 with multiple well units arranged in a single row, and all the well units of the entire receiving part 201 are filled with a multi-row layout. In a more general case, the biological detection consumable 100 is an independent individual type, which can be arranged one by one in the well units of the receiving part 201. Here, the well units of the receiving part 201 may be partially vacant to form a state in which only some of the well units contain biological detection consumables 100. In this embodiment, the receiving part 201 and the radiator 202 can be simultaneously driven to extend out of the range of the main body 400 to perform the addition of the biological detection consumable 100. In other scenarios, the carrier 200 may not move, and the receiving part 201 may include a consumable bracket that can be separated from it. When it is necessary to add biological detection consumables 100, the consumables holder is driven to move outside the area of ​​the main body 400. After the addition of the biological detection consumables 100 is completed, the carrier 200 is driven back into the main body 400. At this time, the air duct around the radiator 202 and the second heat dissipation component 402 at the bottom of the main body 400 can form a heat dissipation channel that runs through the front and back of the bottom of the main body 400. In this design, different heat dissipation components at the top and bottom of the main body 400 can dissipate different types of waste heat in a timely and effective manner, and there will be no problem of insufficient heat dissipation components causing heat dissipation dead corners or too long air ducts causing greater pollution risks.

[0041] Figure 3It is an optical principle diagram of amplification detection provided by the present invention. After the addition of biological detection consumables 100 is completed, the amplification detection system can enable the detection sample in the biological detection consumables 100 to perform constant temperature or variable temperature type amplification. During the amplification process, the detection module 300 can perform fluorescence detection to obtain multiple corresponding fluorescence images. In this embodiment, the light source 11 can be a light source 11 of the type of LED or halogen gas lamp. The light source 11 can be formed by a projection of a lens group, which can include a first lens 12 and a second lens 13 to form a projection light source with a certain expansion angle field. The light source filter 14 can convert the light source 11 into a specific projection light of a certain wavelength type. It is shown here that the emission light of FAM channel 494nm, HEX channel 533nm and CY5 channel 650nm, etc. can be generated. The emission light can penetrate the dichroic mirror 31 and then be projected to the biological detection consumables 100 by the reflector 32. In this embodiment, in order to ensure that the incident angle and the amount of incident light of the emitted light finally projected into the well unit of the biological detection consumable 100 are maximized, a Fresnel lens 33 is also included between the reflector 32 and the biological detection consumable 100 for uniform light and path correction of the emitted light. Furthermore, a microlens array composed of a plurality of microlenses 34 is also included between the Fresnel lens 33 and the biological detection consumable 100, which is similar to the microlens array arranged to enhance the electron capture performance of the electron potential well of the image sensor in image detection, and can achieve effects such as focusing of the emitted light. The emitted light of the present invention is a field projection type, so it can cover all the biological detection consumables 100 on the entire receiving part 201, and the well unit contains a fluorescent agent, a quencher, and a fragment to be detected, etc. In the well unit containing the target fragment, more fluorescent agents are bound to the successfully amplified target fragment over time, and then the emitted light can excite the fluorescent agent to produce fluorescence. As the number of amplified fragments increases, the intensity of the excited fluorescence will also increase, while the well unit that does not contain the target fragment can hardly excite fluorescence, and the fluorescence intensity is only maintained at a very low level. The excited fluorescence can be transmitted in the reverse direction of the emitted light and reflected by the reflector 32 to the dichroic mirror 31, while the fluorescence cannot pass through the dichroic mirror 31 and is reflected to the receiver 21. In order to ensure the quality of the fluorescence image, a fluorescence filter 23 and a fluorescence lens 22 are also configured. Here, the receiver 21 includes a plurality of array-type CCD or CMOS type sensor units, which can obtain fluorescence images of all well units in the biological detection consumables 100. Such an arrangement only needs to ensure that the focus of the receiver 21 is below the liquid level in the biological detection consumables 100, so as to achieve accurate capture of the fluorescence image in the entire well unit, and also eliminates the problem of reduced reliability caused by the need to configure a mechanical transmission mechanism in the scanning scheme.

[0042] Figure 4 and Figure 5It is the result of the associated image obtained by the fluorescence detection image with gain and without gain provided by the present invention. Although the arrayed receiver 21 can cooperate with the field projection light source 11 to efficiently obtain the fluorescence information in the well unit of all biological detection consumables 100, the image obtained by this scheme contains a large number of blank interference areas, so the fluorescence image needs to be corrected to obtain a more reliable detection result. This embodiment first compares the results of the associated image obtained by the corrected image containing gain and the original image without gain correction. Figure 4 The associated image is obtained by using the fluorescence image with gain correction. At present, many industrial cameras will perform gain correction to meet the needs of the human eye for images with visible light wavelengths in order to meet the resolution requirements of the human eye. For example, at least one of the gain methods, such as gamma correction, white balance adjustment, image gain, brightness, and sharpness adjustment, is adaptively corrected. The results of using these gain-corrected fluorescence images to obtain the associated image are as follows: Figure 4 As shown in Fig. b, when this image is used as a reference, the noise data in the fluorescence image will also be amplified synchronously. Therefore, the first threshold value for obtaining the associated image cannot be used as a standard for distinguishing fluorescence data from noise data. Especially in this kind of fluorescence detection with relatively weak light intensity, the difference between some noise data and the fluorescence intensity data obtained in the well unit is relatively small. Therefore, the associated image obtained by using the first threshold value of the gain-corrected fluorescence image as the standard will have more noise pollution points. Figure 5 The diagram illustrates the associated images acquired based on the original image without gain. Since the image without gain does not introduce any gain correction, the original difference between the noise data and the fluorescence data is retained. At this time, the first threshold is obtained based on the fluorescence result in the well unit. In some embodiments, the fluorescence values ​​of several well unit positions set in a specific area in the fluorescence image can be used as the generation standard of the first threshold, or the fluorescence values ​​in one or more well units corresponding to the weakest fluorescence result in the fluorescence image can be used as the generation standard of the first threshold.

[0043] Preferably, the first threshold is a non-fixed value that changes adaptively according to the multiple fluorescence images corresponding to different moments, so that it can adapt to the differences of fluorescence images at different moments, especially in the exponential growth period. The first threshold that adapts to different fluorescence images can more accurately adapt to the changes in the fluorescence amount in the reaction well unit, and can also more accurately adjust the boundary between the well unit and the irrelevant area, so that the associated fluorescence image can more accurately correct the fluorescence image. The first threshold is used as a reference to obtain a saturated\null value binary state associated image from at least one of the fluorescence images. Here, a relatively simple 01 binary state can be used to re-assign values ​​to all pixels in the fluorescence image, and the pixel values ​​above the first threshold are assigned to 1 and the pixel values ​​below the first threshold are assigned to 0 based on the first threshold. After that, the multiple fluorescence images without gain can be subjected to the first operation method, that is, multiplication operation, and at least one of the fluorescence images and the associated image are multiplied to obtain the corrected amplified reference image. The method can divide the amplified reference image into multiple ROI areas by a segmentation algorithm, and all pixel values ​​of each ROI area of ​​the amplified reference image are operated by the second operation method.

[0044] In the fluorescence intensity recognition algorithm, this embodiment experiments with existing methods, selects and retains three methods: RGB method (method 1), HSL method (method 2), and grayscale method (method 3). The processing results of the three methods for the same fluorescence image are as follows: Figure 6 As shown. All three methods have good robustness, and the calculation results will not be affected by factors such as uneven image color distribution and highlights. In terms of calculation speed, the RGB method and the HSL method need to process the pixel values ​​of the three channels, which is time-consuming; while the grayscale method only needs to convert the image into a grayscale image and calculate the average value of the grayscale value, which is relatively fast. From the perspective of accuracy, the grayscale method only considers the grayscale information, and has certain defects in the brightness calculation of color images; the RGB method and the HSL method both consider the color information and can calculate the brightness value more accurately. In summary, the three methods have their own advantages and disadvantages, and the specific application scenarios need to be selected according to the actual situation. If the speed requirement is relatively high, you can choose the grayscale method; if you need more accurate brightness calculation, you can choose the RGB method and the HSL method (currently mainly using the RGB method).

[0045] Figure 7 This is a stability verification diagram of the multi-pixel operation results obtained by different algorithms and the correlation image operation provided by the present invention. The blue curve in the figure represents the original image without gain. Figure 6 The stable curve of pixel fluorescence intensity in the ROI area obtained by the algorithm. The orange curve represents the gain correction map. Figure 6The gray curve represents the stable curve of pixel fluorescence intensity in the ROI region obtained by the cumulative calculation method of pixel values ​​in the region with the original image without gain, and the yellow curve represents the stable curve of pixel fluorescence intensity in the ROI region obtained by the cumulative calculation method of pixel values ​​in the region with the gain correction image. From the stability verification curve, the gray curve has the smallest variation in the uniformity coefficient of the fluorescence result value obtained in different well units. Therefore, the present invention adopts the cumulative calculation method of all pixel values ​​in the ROI region as the most preferred second algorithm to calculate all pixel values ​​in each ROI region of the amplified reference image.

[0046] Figure 8 and Fig. 9 These are circular / quadrilateral ROI area diagrams obtained by two different methods provided by the present invention. In fact, in fluorescent image processing, many areas are unrelated areas. The well unit of the biological detection consumable 100 is the real ROI area for the area in the image. Therefore, it is very necessary to introduce an ROI area division algorithm or a division basis algorithm into the algorithm. In the embodiment of the present application, at least after the fluorescence image and the associated image are calculated to obtain the amplified reference image, the amplified reference image can be used as the object. Figure 8 The Hough circle method in the amplification reference image defines a specific number of circular ROI areas, where the same number as the well unit number of the biological detection consumable 100 can be used. This method can adapt to the circular cross-section essential characteristics of the consumables in fluorescence detection, and the distance between adjacent well units does not change substantially during processing and manufacturing. The multiple circular ROI areas defined using the Hough circle method can basically and accurately outline the pixel range corresponding to the well unit to be calculated. The algorithm is simple and can more quickly obtain the cumulative calculation results of the pixel values ​​in the ROI area. Fig. 9 The area equal division method is used to divide the amplification reference image into a specific number of quadrilateral ROI areas, where the quadrilateral is optimally a rectangle. In order to reduce the influence of irrelevant results in the quadrilateral area, only some pixels in the quadrilateral ROI area can be selected as fluorescence accumulation characteristic pixels, and then the accumulated pixel value results in the ROI area are used as the amplification fluorescence result reference value of at least one of the fluorescence images at the corresponding moment. Here, the accumulated pixel value results in the ROI area can be directly used, or the accumulated results after algorithm processing can be used. The final amplification fluorescence result at this moment is obtained based on the accumulated pixel value results in the ROI area as the reference value. The fluorescence images of dozens of amplification cycles can be obtained according to the algorithm to obtain the fluorescence amplification curve in the biological related consumables well unit. The algorithm is efficient and the corresponding system is simple and reliable.

[0047] Fig.10The verification diagram after the correction image of the same fluorescence intensity for different well units of the present invention is illustrated. In this embodiment, reagents with the same fluorescence intensity are configured in the well units of 96 biological detection consumables 100, and the fluorescence detection system of the present application is used to obtain the fluorescence image, and the original image without gain correction is used as the reference to obtain the associated image that can be corrected, and then the fluorescence image and the associated image are multiplied to obtain the amplified reference image, and the Hough circle method or the area equal division method is used to obtain the circular ROI area or the quadrilateral ROI area, and all the pixel values ​​of each ROI area of ​​the amplified reference image are cumulatively calculated. The results are shown in the bar graph, the horizontal axis is different biological detection consumables columns, there are a total of 12 columns, each column contains eight well units, and the cumulative results of the pixel values ​​in the ROI area corresponding to different well units are represented as bars of different colors. From the statistical results, it can be seen that the algorithm of the present invention can process and obtain basically consistent cumulative results under the condition of the same fluorescence intensity configuration in different well units. Fig.11 The verification curves after the correction of the image for different fluorescence intensities for different well units of the present invention are shown. Fig.10 Similar to the verification method, different fluorescence intensity reagents are configured in the well unit of the biological detection consumable 100, and the cumulative results of different well units in different columns are obtained by processing using the method of the present invention, as shown in the curve in the figure. Under different fluorescence intensities, the cumulative results of pixel values ​​in the ROI area corresponding to the well units at different positions are also basically stable, and the fluctuation is small with the change of fluorescence intensity, which also shows that the scheme of the present invention can achieve adaptive cumulative result output for different fluorescence intensities.

[0048] Fig.12 Another verification method is shown in Figure 2. Fig.12 In a, a linearly decreasing fluorescence intensity reagent is set in several wells at the secondary edge column. Fig.12 In b, linearly decreasing fluorescence intensity reagents are set in several spaces in the middle two columns, and then the biological detection consumables 100 corresponding to the fluorescent intensity reagents with different linear decreases are placed in the fluorescence detection system of the present application, and the corresponding fluorescent images are taken using the receiving module, and then the pixel value accumulation results in the ROI area are obtained by processing using the method of the present invention. The final results are shown in Tables 1 and 2 below.

[0049] Table 1 shows the cumulative results of pixel values ​​in the ROI area corresponding to the sub-edge column position.

[0050] 1 2 3 4 5 6 7 8 9 10 11 12 1 1 401079 28 17 10 12 21 61 23 13 29 13 2 24 173946 21 26 24 16 24 16 11 21 26 16 3 -233 95066 22 11 20 2 136 17 21 28 8497 12 4 11 50523 2 20 15 1828 11 10 10 18 51791 1 5 11 25935 18 15 23 12 18 14 19 9 70256 9 6 5 9041 1 20 17 16 3 18 17 22 122128 5 7 5 -225 -618 21 27 10 96 -3 17 12 302749 16 8 8 5 -526 -557 -244 -169 -163 -173 -78 -28 490394 17

[0051] Table 2 shows the cumulative results of pixel values ​​in the ROI area corresponding to the positions in the middle two columns.

[0052] 1 2 3 4 5 6 7 8 9 10 11 12 1 0 12 18 21 10 467337 15 375 23 21 34 13 2 18 11 20 27 28 219455 18 8 11 7 31 16 3 -233 105 19 11 16 121276 15905 17 25 21 25 19 4 11 15 11 36 29 66548 61601 6 10 16 38 1 5 19 2 14 15 23 33680 84815 18 15 9 26 8 6 5 915 -5 25 13 15484 156331 19 17 18 10 5 7 10 -219 -737 11 36 0 358896 11 17 6 18 11 8 12 4 -527 -553 -255 -170 525420 -171 -85 -28 -2 17

[0053] By performing linear fitting on the cumulative results in Table 1 and comparing the fitting degree with the true fluorescence intensity linear function, it can be found that the linear fit of the second column is 99.75%, and the linear fit of the 11th column is 99.19%. Similarly, by comparing the fit degree of the cumulative results in Table 2, it can be obtained that the fit degree of the 6th column is 99.91%, and the fit degree of the 7th column is 98.40%. It can be seen that the cumulative value of pixels in the ROI area obtained by the method of the present invention can accurately reflect the changes in the fluorescence intensity of different reaction well units.

[0054] Fig.13 and Fig.12 The method is similar to that of the method of the invention, except that all the well units of the biological detection consumables 100 are grouped into four well units, and the whole is divided into multiple groups. A reagent with a linearly decreasing fluorescence intensity is configured in at least one group, and a fluorescence detection system is used to obtain a fluorescence image. The image is then corrected to divide the ROI identification area, and the cumulative pixel value results in the ROI area are counted. From the statistical results, the cumulative results in different areas can better reflect the linear law, and have little correlation with the specific location of the well unit. It can also be explained that the method of the present invention can more realistically depict the amplified fluorescence results in the biological detection consumables 100, and can more accurately obtain the fluorescence amplification curve of each well unit by batch processing.

[0055] Fig.14 and Fig.15 The present invention provides two different methods to obtain the amplification fluorescence results in multiple ROI areas. First, two different types of detection reagents corresponding to the fluorescence intensity are configured in the biological detection unit, and then the detection system is used to obtain the fluorescence image results. Fig.14 Figure a and Fig.15 As shown in Figure a in the figure, the fluorescence image here is the original image without gain correction, and then the first threshold is used as a reference to process and deduce the binary state associated image from the fluorescence image. In some embodiments, the Hough circle method or the area equal division method can be applied to the original fluorescence image to process it into a preset number of units to be processed, and the fluorescence results in at least part of the units to be processed are integrated, and the first threshold is adaptively determined based on this standard. This is equivalent to directly obtaining the first threshold from the key area corresponding to the fluorescence amplification area in the well unit. This can shield various types of noise generated by circuit noise, background interference and crosstalk, and thus obtain a more accurate associated image. Of course, in order to ensure the accuracy of the associated image, the area with a value of 1 in the associated image can be circularized so that the area can become a circular area that is more in line with the actual situation. Of course, the actual algorithm does not introduce the key area division method here, but directly adopts a fixed position or other adaptive methods to obtain the first threshold corresponding to the image. The final result is as follows Fig.14 Figure b and Fig.15As shown in Figure b. Then, the associated image is multiplied with the fluorescence image to correct various noise effects in irrelevant areas, such as Fig.14 Figure c and Fig.15 As shown in Figure c in the figure, after completing the multiplication operation, the amplified reference image is obtained. At this time, the Hough circle method or the area equal division method can be used again to process the image to be analyzed containing multiple ROI regions, and finally the cumulative result of the pixel values ​​in the ROI region can be obtained by counting the cumulative result of the pixel values ​​in the ROI region at the well units at different positions corresponding to the original fluorescence image. Fig.14 The d graph and Fig.15 As shown in Figure d, for fluorescence detection, multiple fluorescence images can be obtained at different times or different cycle numbers, and then the method of the present application is applied multiple times to obtain the pixel accumulation results in the ROI area of ​​multiple well units at different times. The accumulated results can be used to depict the actual amplification curves in different well units.

[0056] A computer-readable medium storing the processing method may be solidified in mobile storage, solid-state storage, or various modules containing storage, which are not limited here.

[0057] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that fall within the scope defined by the claims of the present invention fall within the protection scope of the present invention.

Claims

1. A method for correcting a fluorescence image to obtain an amplified fluorescence result, characterized in that: The array optical detector obtains multiple fluorescence images at different times during the amplification process, and the multiple fluorescence images are all original images without gain; directly obtains the first threshold value from the key area corresponding to the fluorescence amplification area in the well unit of the biological detection consumables in the image, or directly uses the fixed position of the image or an adaptive method to obtain the first threshold value corresponding to the multiple images; the first threshold value is a non-fixed value that changes adaptively according to the multiple fluorescence images corresponding to different times; Acquire an associated image of a saturation / null value binary state from at least one of the fluorescence images based on a first threshold value, calculate at least one of the fluorescence images and the associated image using a first calculation method to obtain a corrected amplification reference image, and divide the amplification reference image into a plurality of ROI regions using a segmentation algorithm; calculate all pixel values ​​of each ROI region of the amplification reference image using a second calculation method, and use all pixel values ​​of each ROI region as amplification fluorescence result reference values ​​of at least one of the fluorescence images at a corresponding moment; Among them, the first operation method is a multiplication operation, and at least one of the fluorescence images and the associated image is multiplied to obtain a corrected amplified reference image; the second operation method is one of the RGB method, the HSL method, the grayscale method and the cumulative calculation method.

2. The method for obtaining amplified fluorescence results by correcting fluorescence images according to claim 1, characterized in that: The segmentation algorithm is the Hough circle method, which divides the amplified reference image into a plurality of circular ROI areas.

3. The method for obtaining amplified fluorescence results by correcting fluorescence images according to claim 1, characterized in that: The segmentation algorithm is an area equalization method, which divides the amplified reference image into a plurality of quadrilateral ROI regions with equal areas.

4. An amplification detection system, using the method for obtaining amplification fluorescence results by correcting fluorescence images according to claim 1, characterized in that: The invention relates to a biological detection consumable comprising an emission source capable of generating an excitation source of at least one wavelength and a plurality of well units; each well unit in the biological detection consumable comprises a fluorescent agent, a quencher and a fragment to be detected; the biological detection consumable can be subjected to a constant temperature or a variable temperature amplification cycle, and cooperates with an array-type detector to obtain a plurality of fluorescence images corresponding to different moments; and further comprises a processing module, which obtains the amplified fluorescence result by correcting the fluorescence image.

5. The amplification detection system according to claim 4, characterized in that: A microlens unit is also arranged above each well unit.

6. A computer-readable medium, characterized in that The method for correcting a fluorescence image and obtaining an amplified fluorescence result as described in claim 1 is performed.

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