CN109358026B - Fluorescent liquid drop detection method and device and server - Google Patents

Fluorescent liquid drop detection method and device and server Download PDF

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CN109358026B
CN109358026B CN201811070596.7A CN201811070596A CN109358026B CN 109358026 B CN109358026 B CN 109358026B CN 201811070596 A CN201811070596 A CN 201811070596A CN 109358026 B CN109358026 B CN 109358026B
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刘聪
董文飞
黎海文
蒋克明
张涛
周武平
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Suzhou Institute of Biomedical Engineering and Technology of CAS
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Abstract

The invention discloses a fluorescent liquid drop detection method, a fluorescent liquid drop detection device and a server, wherein the fluorescent liquid drop detection method comprises the following steps: acquiring a fluorescent liquid drop image of a sample to be detected, and segmenting the fluorescent liquid drop image to extract a plurality of first fluorescent droplets in the fluorescent liquid drop image; judging whether the first fluorescent droplets are adhered or not according to the areas and membership degrees of the first fluorescent droplets; when there is adhesion in the plurality of first fluorescent droplets, dividing a connected domain formed by the adhered droplets to generate second fluorescent droplets; and judging whether the second fluorescent droplet is a valid droplet according to the size and membership grade of the second fluorescent droplet. The fluorescent liquid drop detection method, the fluorescent liquid drop detection device and the server provided by the embodiment of the invention improve the reliability of connected domain detection by combining the area and the membership degree, are favorable for reducing the detection cost because a hardware device for fluorescent liquid drop detection is not added, and are easy to integrate with the conventional hardware detection device.

Description

Fluorescent liquid drop detection method and device and server
Technical Field
The invention relates to the technical field of digital polymerase chain reaction, in particular to a fluorescent liquid drop detection method, a fluorescent liquid drop detection device and a server.
Background
Digital PCR (Polymerase Chain Reaction, PCR for short) is a very rapidly developed biological detection technology in recent years. The digital PCR is divided into two major types, namely a droplet type and a microcavity type, wherein the droplet type digital PCR is the dominant product in the market, and the signal detection method of the droplet type digital PCR comprises a flow detection method and a plane detection method based on a digital droplet image.
The flow detection method has the advantages of low background fluorescence intensity, simple identification algorithm and the like, but the method has complex optical path and high cost, is difficult to integrate with a microdroplet generation module and a PCR amplification module at the front end of an instrument, and appears to be subsequently powerless under the development trend of digital PCR integration and low cost.
The planar detection method based on digital droplet images has the following problems: because of the weak intensity of the excited fluorescence, it usually requires a long exposure time to obtain an image that can be used for identification, and even then, the brightness and contrast of the droplet fluorescence image are still significantly lower compared to the bright field image, and at the same time, the signal-to-noise ratio of the droplet fluorescence image is lower.
In view of the above problems of the flow detection method and the planar detection method, a new high-reliability fluorescent droplet detection method is urgently needed to be researched to reduce the cost of the droplet-type digital PCR and improve the integration level thereof.
Disclosure of Invention
In view of this, embodiments of the present invention provide a fluorescent droplet detection method, apparatus, and server, so as to solve the problems of low reliability, high cost, and difficult integration in the existing droplet-type digital PCR technology.
According to a first aspect, embodiments of the present invention provide a fluorescent droplet detection method, including: acquiring a fluorescent liquid drop image of a sample to be detected, and segmenting the fluorescent liquid drop image to extract a plurality of first fluorescent droplets in the fluorescent liquid drop image; judging whether the first fluorescent droplets are adhered or not according to the areas and membership degrees of the first fluorescent droplets; when there is a conglutination in the plurality of first fluorescent droplets, segmenting a connected domain formed by the conglutination droplets to generate second fluorescent droplets; and judging whether the second fluorescent droplet is a valid droplet or not according to the size and membership degree of the second fluorescent droplet.
According to the fluorescent droplet detection method provided by the embodiment of the invention, whether each first fluorescent droplet belongs to a connected domain formed by adhesion of a plurality of droplets is judged by respectively calculating the area and membership degree of each first fluorescent droplet. Since the area of the connected domain formed by the adhered droplets is generally larger, the area can be used as one of the criteria for judging whether the first fluorescent droplet is the connected domain. The connected domain detection only by the area may cause misjudgment on the non-connected domain with larger area, so that the fluorescent droplet detection method provided by the embodiment of the invention designs a membership function aiming at fluorescent droplet detection, and the possibility of droplet adhesion occurs through membership reaction. Through the detection of combining the area and the membership degree, the reliability of the detection of the connected domain is improved, and the misjudgment in the detection of the connected domain can be reduced. In addition, the fluorescent liquid drop detection method provided by the embodiment of the invention realizes the detection of the fluorescent liquid drop through image processing methods such as segmentation and the like and calculation methods such as calculation area and membership function, does not increase a hardware device for fluorescent liquid drop detection, is favorable for reducing the detection cost, and is easy to integrate with the existing hardware detection device.
With reference to the first aspect, in a first embodiment of the first aspect, the determining whether the first fluorescent droplets are stuck according to the areas and membership degrees of the first fluorescent droplets includes: judging whether the area of the first fluorescent droplet is larger than a preset area threshold value or not and whether the membership degree of the first fluorescent droplet is larger than a preset first membership degree threshold value or not; and when the area of the first fluorescent droplet is larger than a preset area threshold value and the membership degree of the first fluorescent droplet is larger than a preset first membership degree threshold value, judging that the first fluorescent droplet is adhered.
According to the fluorescent liquid drop detection method provided by the embodiment of the invention, the detection of the connected domain by combining the area and the membership degree is realized by setting the area threshold and the first membership degree threshold. The threshold detection algorithm is low in complexity and easy to realize, so that the detection cost can be reduced while the detection effect is ensured.
With reference to the first embodiment of the first aspect, in a second embodiment of the first aspect, the degree of membership of the first fluorescent droplet is calculated according to the following formula:
Figure BDA0001799438870000031
wherein f isiRepresenting membership of the ith first fluorescent droplet; a. theiDenotes the area of the ith first fluorescent droplet, PiDenotes the perimeter, h, of the ith first fluorescent dropletiRepresents the length of the ith first fluorescent droplet; w is aiRepresents the width of the ith first fluorescent droplet; r is0Represents a predetermined effective droplet radius; α, β, and k are constants respectively representing preset coefficients.
According to the fluorescent droplet detection method provided by the embodiment of the invention, the membership degree of the first fluorescent droplet, namely the possibility that the first fluorescent droplet has the adhered droplet, is represented as a real numerical value between [0 and 1] according to the length and the width of the first fluorescent droplet, so that the membership degree numerical value of the first fluorescent droplet is obtained through calculation, and the judgment basis for judging whether the first fluorescent droplet has the adhered droplet is taken as the result that the membership degree of the first fluorescent droplet is smaller than the corresponding threshold. The closer the membership is to 1, the more likely the corresponding first fluorescent droplet is to be a connected domain with a sticky droplet; closer membership to 0 indicates that the corresponding first fluorescent droplet is less likely to be a connected domain with an adherent droplet present.
With reference to the first aspect, in a third embodiment of the first aspect, the dividing the connected domain formed by the adherent droplets to generate second fluorescent droplets includes: generating a Euclidean distance topographic map of the connected domain; respectively determining the central position of each droplet in the connected domain according to the Euclidean distance topographic map of the connected domain; segmenting the connected domain according to the central position to generate a second fluorescent droplet corresponding to the central position.
According to the fluorescent droplet detection method provided by the embodiment of the invention, after the connected domains containing the sticky droplets are detected, each connected domain is divided to eliminate the sticky droplets, so that the accuracy of fluorescent droplet detection is improved.
With reference to the third embodiment of the first aspect, in the fourth embodiment of the first aspect, the determining whether the second fluorescent droplet is an effective droplet according to the size and membership of the second fluorescent droplet includes: judging whether the height of the second fluorescent droplet is within a preset height range or not, the width of the second fluorescent droplet is within a preset width range or not, and the membership degree of the second fluorescent droplet is smaller than a preset second membership degree threshold or not; and when the height of the second fluorescent droplet is in a preset height range, the width of the second fluorescent droplet is in a preset width range, and the membership degree of the second fluorescent droplet is smaller than a preset second membership degree threshold value, judging the second fluorescent droplet as an effective droplet.
According to the fluorescent droplet detection method provided by the embodiment of the invention, after the second fluorescent droplets in the connected domain are segmented, the effective droplets in the second fluorescent droplets are identified by judging whether the height, the width and the membership degree of the second fluorescent droplets meet the requirements of corresponding threshold values, so that the identification precision is higher.
With reference to the fourth embodiment of the first aspect, in the fifth embodiment of the first aspect, the degree of membership of the second fluorescent droplet is calculated according to the following formula:
Figure BDA0001799438870000041
wherein f isijRepresenting membership of a jth second fluorescent droplet segmented from an ith connected domain; a. theijDenotes the area of the jth second fluorescent droplet divided from the ith communication domain, PijThe expression represents the perimeter h of the jth second fluorescent droplet divided from the ith communication domainijIndicating the length of a jth second fluorescent droplet segmented from an ith connected domain; w is aijIndicating the width of a jth second fluorescent droplet segmented from an ith connected domain; r is0Represents a predetermined effective droplet radius; α, β, and k are constants respectively representing preset coefficients.
According to the fluorescent droplet detection method provided by the embodiment of the invention, the membership degree of the second fluorescent droplet, namely the possibility that the second fluorescent droplet has adhesive droplets, is represented as a real numerical value between [0 and 1] according to the length and the width of the second fluorescent droplet, so that the membership degree numerical value of the second fluorescent droplet is obtained through calculation, and the judgment basis for judging whether the second fluorescent droplet is effective or not is taken as the judgment basis that the membership degree of the second fluorescent droplet is smaller than the corresponding threshold.
With reference to the first aspect or any one of the first to fifth embodiments of the first aspect, in a sixth embodiment of the first aspect, the acquiring a fluorescent droplet image of a sample to be tested includes: acquiring an original fluorescent droplet image and a corresponding dark field image of the sample to be detected; and correcting the original fluorescent droplet image according to the dark field image to generate a corrected fluorescent droplet image.
According to the fluorescent droplet detection method provided by the embodiment of the invention, the original fluorescent droplet image is corrected by utilizing the dark field image corresponding to the original fluorescent droplet image of the sample to be detected, so that the brightness and the contrast of the original fluorescent droplet image are improved, and the accuracy and the reliability of fluorescent droplet detection are improved.
With reference to the first aspect or any one of the first to sixth embodiments of the first aspect, in a seventh embodiment of the first aspect, the segmenting the fluorescent drop image to extract a plurality of first fluorescent droplets in the fluorescent drop image includes: performing global threshold segmentation on the fluorescent liquid drop image to filter out a background in the fluorescent liquid drop image; locally adaptive threshold segmentation is performed on the background-filtered fluorescent droplet image to extract the plurality of first fluorescent droplets.
According to the fluorescent droplet detection method provided by the embodiment of the invention, the fluorescent droplet image is processed by utilizing global threshold segmentation and local self-adaptive threshold segmentation respectively, so that a plurality of first fluorescent droplets are obtained, droplet adhesion detection and segmentation can be carried out on the extracted first fluorescent droplets in subsequent steps, misjudgment caused by inconsistent sizes of the adhesion droplets when the first fluorescent droplets are directly identified in effectiveness is avoided, and the accuracy and reliability of fluorescent droplet detection are improved.
According to a second aspect, embodiments of the present invention provide a fluorescent droplet detection apparatus, comprising: the first segmentation unit is used for acquiring a fluorescent liquid drop image of a sample to be detected, and segmenting the fluorescent liquid drop image to extract a plurality of first fluorescent droplets in the fluorescent liquid drop image; the first judging unit is used for judging whether the first fluorescent droplets are adhered or not according to the areas and membership degrees of the first fluorescent droplets; a second dividing unit configured to divide a connected domain formed by the adhered droplets to generate second fluorescent droplets when there is adhesion among the plurality of first fluorescent droplets; and the second judging unit is used for judging whether the second fluorescent droplet is a valid droplet or not according to the size and membership degree of the second fluorescent droplet.
According to a third aspect, an embodiment of the present invention provides a server, including: a memory and a processor, the memory and the processor being communicatively connected to each other, the memory having stored therein computer instructions, and the processor executing the computer instructions to perform the fluorescent droplet detection method according to the first aspect or any one of the embodiments of the first aspect.
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The features and advantages of the present invention will be more clearly understood by reference to the accompanying drawings, which are illustrative and not to be construed as limiting the invention in any way, and in which:
FIG. 1 shows a schematic diagram of an application scenario of an embodiment of the present invention;
FIG. 2 shows a flow diagram of a specific example of a fluorescent droplet detection method in an embodiment of the invention;
FIG. 3 shows a flow diagram of one particular example of another fluorescent droplet detection method in an embodiment of the invention;
FIG. 4 shows a schematic structural diagram of a specific example of a fluorescent droplet detection device in an embodiment of the invention;
fig. 5 is a schematic structural diagram showing a specific example of a server in the embodiment of the present invention.
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 and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, 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.
Fig. 1 is a schematic diagram of an application scenario of the embodiment of the present invention, in which arrows indicate propagation directions of optical paths. The excitation light source 1 is a high-brightness light source having a specific wavelength, such as a laser, a high-brightness LED, or the like. The excitation light source 1 emits excitation light which passes through the converging lens 2 and the optical filter 3, and then irradiates the detection chip 4 and excites the liquid drop to be detected to generate fluorescence. The fluorescence emitted by the liquid drop enters the CCD sensor 7 after the scattered exciting light is filtered by the optical filter 6, and the CCD sensor 7 collects images and transmits the images to the computer 8 for processing so as to realize the detection of the fluorescent microdroplets.
In some embodiments, as shown in fig. 2, computer 8 may effect detection of the fluorescent droplet by performing the following steps:
step S101: and acquiring a fluorescent liquid drop image of the sample to be detected. In practical applications, the original fluorescent droplet image L of the sample to be measured generally has low brightness and contrast, and low signal-to-noise ratio. To improve the quality of the fluorescent droplet image, the original fluorescent droplet image L can be corrected using a dark field image D having the same integration time as the original fluorescent droplet image L, resulting in a corrected fluorescent droplet image I. The dark field image D is an image acquired by the same acquisition equipment for acquiring the original fluorescent droplet image L in the same integration time, so that the dark field image D contains random noise similar to that in the original fluorescent droplet image L, and the difference between the original fluorescent droplet image L and the dark field image D can greatly eliminate the random noise, thereby improving the signal-to-noise ratio of the corrected fluorescent droplet image I. In one embodiment, histogram equalization may be performed on the corrected fluorescent droplet image I to improve brightness and contrast thereof; if necessary, the fluorescent droplet image I may also be Gaussian filtered to further remove noise and thereby increase its signal-to-noise ratio. In addition, the multiple fluorescent droplet images I can be spliced, so that the multiple fluorescent droplet images I can be processed simultaneously in the subsequent step, parallel detection of fluorescent droplets is realized, and the detection efficiency is improved.
Step S102: the fluorescent drop image is segmented to extract a plurality of first fluorescent droplets in the fluorescent drop image. In one embodiment, the fluorescent droplet image I may be subjected to global threshold segmentation to filter out the background in the fluorescent droplet image I; and then carrying out local adaptive threshold segmentation on the fluorescence droplet image I after the background is filtered out so as to extract a plurality of first fluorescence droplets in the fluorescence droplet image I.
Step S103: and judging whether the first fluorescent droplets are adhered or not according to the areas and membership degrees of the first fluorescent droplets. When a sticky droplet exists in the plurality of first fluorescent droplets, performing step S104; when no adhesive droplet exists in the plurality of first fluorescent droplets, judging whether the first fluorescent droplets are valid droplets according to the size and membership grade of the first fluorescent droplets, reserving the valid first fluorescent droplets, and deleting the invalid first fluorescent droplets. In a specific embodiment, as shown in fig. 3, the process of step S103 can be implemented by the following steps:
step S1031: and judging whether the area of the first fluorescent droplet is larger than a preset area threshold value. Performing step S1032 when the area of the first fluorescent droplet is larger than the preset area threshold; when the area of the first fluorescent droplet is not greater than the preset area threshold, it can be determined that the first fluorescent droplet is not a connected domain formed by the adhesion of a plurality of droplets, and then it can be determined whether the first fluorescent droplet is an effective droplet according to the size and membership degree of the first fluorescent droplet.
Step S1032: and judging whether the membership degree of the first fluorescent droplet is larger than a preset first membership degree threshold value. When the membership degree of the first fluorescent droplet is greater than a preset first membership degree threshold, performing step S1033; when the membership degree of the first fluorescent droplet is not greater than a preset first membership degree threshold, the first fluorescent droplet is judged not to be a connected domain formed by the adhesion of a plurality of droplets, and whether the first fluorescent droplet is an effective droplet or not can be further judged according to the size and the membership degree of the first fluorescent droplet.
Step S1033: judging that the first fluorescent droplet has the adhered droplet, and executing step S104 to divide the connected domain formed by the adhered droplet so as to separate the adhered droplet in the connected domain. Since the connected domain including the plurality of the adhesive droplets generally has an area larger than that of the independent droplet, the fluorescent droplet detection method provided by the embodiment of the invention sets an area threshold value, thereby realizing area detection of the first droplet. In one embodiment, the area threshold may be
Figure BDA0001799438870000082
Wherein r is0Is a predetermined effective droplet radius. In order to avoid missing detection when the connected domain is detected by using only the area, the fluorescent droplet detection method provided by the embodiment of the invention designs the membership function to detect the possibility of adhesion in the first fluorescent droplet. Specifically, the membership of the first fluorescent droplet can be calculated according to the following formula (1):
Figure BDA0001799438870000081
wherein f isiRepresenting membership of the ith first fluorescent droplet; a. theiDenotes the area of the ith first fluorescent droplet, PiDenotes the perimeter, h, of the ith first fluorescent dropletiRepresents the length of the ith first fluorescent droplet; w is aiRepresents the width of the ith first fluorescent droplet; r is0Represents a predetermined effective droplet radius; α, β, and k are constants respectively representing preset coefficients.
According to the length and the width of the first fluorescent droplet, the membership degree of the first fluorescent droplet, namely the possibility that the first fluorescent droplet has the adhesive droplet, is expressed as a real value between [0 and 1], so that the membership degree value of the first fluorescent droplet is obtained through calculation, and the judgment basis for judging whether the first fluorescent droplet has the adhesive droplet is taken as the membership degree smaller than the corresponding threshold. The closer the membership is to 1, the more likely the corresponding first fluorescent droplet is to be a connected domain with a sticky droplet; closer membership to 0 indicates that the corresponding first fluorescent droplet is less likely to be a connected domain with an adherent droplet present. In one embodiment, the preset first membership threshold may be 0.5.
Step S104: the connected domain is partitioned to generate a second fluorescent droplet. The purpose of segmenting the domains is to break down the droplets that are bound within the domains, causing them to transform from a bound state into a plurality of second fluorescent droplets that are independent of each other. In a specific embodiment, as shown in fig. 3, the process of segmenting the connected domain to generate the second fluorescent droplet in step S104 can be realized by the following sub-steps:
step S1041: and generating a Euclidean distance topographic map of the connected domain. The connected domain may be processed by using the prior art to obtain a corresponding euclidean distance topographic map thereof, which is not limited in the embodiment of the present application.
Step S1042: and respectively determining the central position of each droplet in the connected domain according to the Euclidean distance topographic map of the connected domain. In the euclidean distance topographic map corresponding to each connected domain, the local minimum value is possibly the central position of one of the adhered droplets, so that the local minimum value in the euclidean distance topographic map can be used as a foreground mark.
Step S1043: the connected components are segmented according to the central position to generate a second fluorescent droplet corresponding to the central position. And processing the connected domains by combining the foreground marks marked by local minimum values in the Euclidean distance topographic map and adopting an image segmentation algorithm in the prior art, so that the liquid drops adhered in each connected domain can be separated, and a plurality of second fluorescent droplets are obtained. Preferably, the connected domains can be segmented using a watershed algorithm.
Step S105: and judging whether the second fluorescent droplet is a valid droplet according to the size and membership grade of the second fluorescent droplet. When the second fluorescent droplet is a valid droplet, retaining the second fluorescent droplet; deleting the second fluorescent droplet when the second fluorescent droplet is not a valid droplet. Preferably, the height, width and corresponding membership of the second fluorescent droplet can be used together to determine whether it is a valid droplet. And when the height of the second fluorescent droplet is within a preset height range, the width of the second fluorescent droplet is within a preset width range, and the membership degree of the second fluorescent droplet is smaller than a preset second membership degree threshold value, judging the second fluorescent droplet as an effective droplet. Specifically, the membership of the second fluorescent droplet can be calculated according to the following formula (2):
Figure BDA0001799438870000101
wherein f isijRepresenting membership of a jth second fluorescent droplet segmented from an ith connected domain; a. theijDenotes the area of the jth second fluorescent droplet divided from the ith communication domain, PijThe expression represents the perimeter h of the jth second fluorescent droplet divided from the ith communication domainijIndicating the length of a jth second fluorescent droplet segmented from an ith connected domain; w is aijIndicating the width of a jth second fluorescent droplet segmented from an ith connected domain; r is0Represents a predetermined effective droplet radius; α, β, and k are constants respectively representing preset coefficients. Corresponding to the membership function in equation (2), the second membership threshold for determining whether the second fluorescent droplet is a valid droplet may be 0.4.
According to the fluorescent droplet detection method provided by the embodiment of the invention, whether each first fluorescent droplet belongs to a connected domain formed by adhesion of a plurality of droplets is judged by respectively calculating the area and membership degree of each first fluorescent droplet. Since the area of the connected domain formed by the adhered droplets is generally larger, the area can be used as one of the criteria for judging whether the first fluorescent droplet is the connected domain. The connected domain detection only by the area may cause misjudgment on the non-connected domain with larger area, so that the fluorescent droplet detection method provided by the embodiment of the invention designs a membership function aiming at fluorescent droplet detection, and the possibility of droplet adhesion occurs through membership reaction. Through the detection of combining the area and the membership degree, the reliability of the detection of the connected domain is improved, and the misjudgment in the detection of the connected domain can be reduced. In addition, the fluorescent liquid drop detection method provided by the embodiment of the invention realizes the detection of the fluorescent liquid drop through image processing methods such as segmentation and the like and calculation methods such as calculation area and membership function, does not increase a hardware device for fluorescent liquid drop detection, is favorable for reducing the detection cost, and is easy to integrate with the existing hardware detection device.
An embodiment of the present invention further provides a fluorescent droplet detecting apparatus, as shown in fig. 4, the fluorescent droplet detecting apparatus may include: a first segmentation unit 401, a first judgment unit 402, a second segmentation unit 403 and a second judgment unit 404.
The first segmentation unit 401 is configured to acquire a fluorescent droplet image of a sample to be detected, and segment the fluorescent droplet image to extract a plurality of first fluorescent droplets in the fluorescent droplet image; the specific working process can be described with reference to step S101 to step S102 in the above method embodiment.
The first judging unit 402 is configured to judge whether there is adhesion in the plurality of first fluorescent droplets according to the areas and membership degrees of the plurality of first fluorescent droplets; the specific working process may refer to step S103 in the above method embodiment.
When there is adhesion in the plurality of first fluorescent droplets, the second dividing unit 403 is configured to divide the connected domain formed by the adhered droplets to generate second fluorescent droplets; the specific working process may refer to step S104 in the above method embodiment.
The second judging unit 404 is configured to judge whether the second fluorescent droplet is a valid droplet according to the size and membership degree of the second fluorescent droplet; the specific working process can be described with reference to step S105 in the above method embodiment.
An embodiment of the present invention further provides a server, as shown in fig. 5, the server may include a processor 51 and a memory 52, where the processor 51 and the memory 52 may be connected by a bus or in another manner, and fig. 5 takes the connection by the bus as an example.
The processor 51 may be a Central Processing Unit (CPU). The Processor 51 may also be other general purpose processors, Digital Signal Processors (DSPs), Application Specific Integrated Circuits (ASICs), Field Programmable Gate Arrays (FPGAs) or other Programmable logic devices, discrete Gate or transistor logic devices, discrete hardware components, or combinations thereof.
The memory 52, as a non-transitory computer-readable storage medium, can be used to store non-transitory software programs, non-transitory computer-executable programs, and modules, such as program instructions/modules corresponding to the fluorescent droplet detection method in the embodiment of the present invention (for example, the first segmentation unit 401, the first determination unit 402, the second segmentation unit 403, and the second determination unit 404 shown in fig. 4). The processor 51 executes various functional applications and data processing of the processor, i.e. implementing the XX method in the above-described method embodiments, by running non-transitory software programs, instructions and modules stored in the memory 52.
The memory 52 may include a storage program area and a storage data area, wherein the storage program area may store an operating system, an application program required for at least one function; the storage data area may store data created by the processor 51, and the like. Further, the memory 52 may include high speed random access memory, and may also include non-transitory memory, such as at least one magnetic disk storage device, flash memory device, or other non-transitory solid state storage device. In some embodiments, the memory 52 may optionally include memory located remotely from the processor 51, and these remote memories may be connected to the processor 51 via a network. Examples of such networks include, but are not limited to, the internet, intranets, local area networks, mobile communication networks, and combinations thereof.
The one or more modules are stored in the memory 52 and, when executed by the processor 51, perform a fluorescent droplet detection method as in the embodiment of fig. 2-3.
The details of the server may be understood by referring to the corresponding descriptions and effects in the embodiments shown in fig. 2 to fig. 3, and are not described herein again.
It will be understood by those skilled in the art that all or part of the processes of the methods of the embodiments described above can be implemented by a computer program, which can be stored in a computer-readable storage medium, and when executed, can include the processes of the embodiments of the methods described above. The storage medium may be a magnetic Disk, an optical Disk, a Read-Only Memory (ROM), a Random Access Memory (RAM), a Flash Memory (Flash Memory), a Hard Disk (Hard Disk Drive, abbreviated as HDD), a Solid State Drive (SSD), or the like; the storage medium may also comprise a combination of memories of the kind described above.
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 (9)

1. A fluorescent droplet detection method, comprising:
acquiring a fluorescent liquid drop image of a sample to be detected, and segmenting the fluorescent liquid drop image to extract a plurality of first fluorescent droplets in the fluorescent liquid drop image;
judging whether the first fluorescent droplets are adhered or not according to the areas and membership degrees of the first fluorescent droplets;
when there is a conglutination in the plurality of first fluorescent droplets, segmenting a connected domain formed by a conglutination droplet to generate a second fluorescent droplet, wherein the segmenting the connected domain formed by the conglutination droplet to generate the second fluorescent droplet comprises: generating a Euclidean distance topographic map of the connected domain; respectively determining the central position of each droplet in the connected domain according to the Euclidean distance topographic map of the connected domain; segmenting the connected domain according to the central position to generate a second fluorescent droplet corresponding to the central position;
and judging whether the second fluorescent droplet is a valid droplet or not according to the size and membership degree of the second fluorescent droplet.
2. The method for detecting fluorescent droplets according to claim 1, wherein the determining whether the first fluorescent droplets are adhered according to the areas and membership degrees of the first fluorescent droplets comprises:
judging whether the area of the first fluorescent droplet is larger than a preset area threshold value or not and whether the membership degree of the first fluorescent droplet is larger than a preset first membership degree threshold value or not;
and when the area of the first fluorescent droplet is larger than a preset area threshold value and the membership degree of the first fluorescent droplet is larger than a preset first membership degree threshold value, judging that the first fluorescent droplet is adhered.
3. The fluorescent droplet detection method of claim 2, wherein the membership of the first fluorescent droplet is calculated according to the following formula:
Figure 109520DEST_PATH_IMAGE001
wherein the content of the first and second substances,f i is shown asiMembership of the first fluorescent droplet;A i is shown asiThe area of the individual first fluorescent droplets,P i is shown asiThe perimeter of the first fluorescent droplet,h i is shown asiA length of the first fluorescent droplet;w i is shown asiA width of the first fluorescent droplet;r 0 represents a predetermined effective droplet radius;α、βandkare all constants and respectively represent preset coefficients.
4. The method of claim 1, wherein the determining whether the second fluorescent droplet is a valid droplet based on the size and membership of the second fluorescent droplet comprises:
judging whether the height of the second fluorescent droplet is within a preset height range or not, the width of the second fluorescent droplet is within a preset width range or not, and the membership degree of the second fluorescent droplet is smaller than a preset second membership degree threshold or not;
and when the height of the second fluorescent droplet is in a preset height range, the width of the second fluorescent droplet is in a preset width range, and the membership degree of the second fluorescent droplet is smaller than a preset second membership degree threshold value, judging the second fluorescent droplet as an effective droplet.
5. The fluorescent droplet detection method of claim 4, wherein the membership of the second fluorescent droplet is calculated according to the following formula:
Figure 951574DEST_PATH_IMAGE002
wherein the content of the first and second substances,f ij represents from the firstiDivided in a connected domainjMembership of the second fluorescent droplet;A ij represents from the firstiDivided in a connected domainjThe area of the second fluorescent droplet,P ij represents from the firstiDivided in a connected domainjThe perimeter of the second fluorescent droplet,h ij represents from the firstiDivided in a connected domainjA length of the second fluorescent droplet;w ij represents from the firstiDivided in a connected domainjA width of the second fluorescent droplet;r 0 represents a predetermined effective droplet radius;α、βandkare all constants and respectively represent preset coefficients.
6. The fluorescent droplet detection method of any one of claims 1-5, wherein the obtaining a fluorescent droplet image of a sample to be tested comprises:
acquiring an original fluorescent droplet image and a corresponding dark field image of the sample to be detected;
and correcting the original fluorescent droplet image according to the dark field image to generate a corrected fluorescent droplet image.
7. The fluorescent droplet detection method of any one of claims 1-5, wherein segmenting the fluorescent droplet image to extract a plurality of first fluorescent droplets in the fluorescent droplet image comprises:
performing global threshold segmentation on the fluorescent liquid drop image to filter out a background in the fluorescent liquid drop image;
locally adaptive threshold segmentation is performed on the background-filtered fluorescent droplet image to extract the plurality of first fluorescent droplets.
8. A fluorescent drop detection device, comprising:
the first segmentation unit is used for acquiring a fluorescent liquid drop image of a sample to be detected, and segmenting the fluorescent liquid drop image to extract a plurality of first fluorescent droplets in the fluorescent liquid drop image;
the first judging unit is used for judging whether the first fluorescent droplets are adhered or not according to the areas and membership degrees of the first fluorescent droplets;
a second dividing unit configured to divide a connected domain formed by a stuck droplet to generate a second fluorescent droplet when there is a stick in the plurality of first fluorescent droplets, wherein the dividing of the connected domain formed by the stuck droplet to generate the second fluorescent droplet includes: generating a Euclidean distance topographic map of the connected domain; respectively determining the central position of each droplet in the connected domain according to the Euclidean distance topographic map of the connected domain; segmenting the connected domain according to the central position to generate a second fluorescent droplet corresponding to the central position;
and the second judging unit is used for judging whether the second fluorescent droplet is a valid droplet or not according to the size and membership degree of the second fluorescent droplet.
9. A server, comprising:
a memory and a processor communicatively coupled to each other, the memory having stored therein computer instructions, the processor executing the computer instructions to perform the fluorescent droplet detection method of any of claims 1-7.
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Publication number Priority date Publication date Assignee Title
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005002730A1 (en) * 2003-07-02 2005-01-13 The University Of Manchester Microfluidic method and device
CN103065304A (en) * 2012-12-25 2013-04-24 北京农业信息技术研究中心 Two-dimensional fuzzy entropy based adhesive material partition method
CN104089857A (en) * 2014-07-03 2014-10-08 天津大学 Measuring method of oil drop size
CN104388307A (en) * 2014-11-24 2015-03-04 中国科学院苏州生物医学工程技术研究所 Liquid drop type sample fluorescence detection system and method
CN105861299A (en) * 2016-05-05 2016-08-17 广东顺德工业设计研究院(广东顺德创新设计研究院) Micro-drop digital PCR (polymerase chain reaction) fluorescent detection system and fluorescent detection device
CN106596489A (en) * 2016-12-19 2017-04-26 中国科学院苏州生物医学工程技术研究所 Processing method of fluorescence intensity data in fluorescence droplet detection
CN107084948A (en) * 2017-03-13 2017-08-22 深圳市液芯生物科技有限公司 The data extraction method and system of a kind of fluorescent microsphere
CN107478629A (en) * 2017-09-04 2017-12-15 中国科学院苏州生物医学工程技术研究所 A kind of large area digital pcr droplet fluorescence high pass amount detecting device and method
CN107844683A (en) * 2017-10-17 2018-03-27 华东医药（杭州）基因科技有限公司 A kind of computational methods of digital pcr concentration of liquid drops

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005002730A1 (en) * 2003-07-02 2005-01-13 The University Of Manchester Microfluidic method and device
CN103065304A (en) * 2012-12-25 2013-04-24 北京农业信息技术研究中心 Two-dimensional fuzzy entropy based adhesive material partition method
CN104089857A (en) * 2014-07-03 2014-10-08 天津大学 Measuring method of oil drop size
CN104388307A (en) * 2014-11-24 2015-03-04 中国科学院苏州生物医学工程技术研究所 Liquid drop type sample fluorescence detection system and method
CN105861299A (en) * 2016-05-05 2016-08-17 广东顺德工业设计研究院(广东顺德创新设计研究院) Micro-drop digital PCR (polymerase chain reaction) fluorescent detection system and fluorescent detection device
CN106596489A (en) * 2016-12-19 2017-04-26 中国科学院苏州生物医学工程技术研究所 Processing method of fluorescence intensity data in fluorescence droplet detection
CN107084948A (en) * 2017-03-13 2017-08-22 深圳市液芯生物科技有限公司 The data extraction method and system of a kind of fluorescent microsphere
CN107478629A (en) * 2017-09-04 2017-12-15 中国科学院苏州生物医学工程技术研究所 A kind of large area digital pcr droplet fluorescence high pass amount detecting device and method
CN107844683A (en) * 2017-10-17 2018-03-27 华东医药（杭州）基因科技有限公司 A kind of computational methods of digital pcr concentration of liquid drops

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