CN109358026A - Fluorescence drop detection method, device and server - Google Patents

Fluorescence drop detection method, device and server Download PDF

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Publication number
CN109358026A
CN109358026A CN201811070596.7A CN201811070596A CN109358026A CN 109358026 A CN109358026 A CN 109358026A CN 201811070596 A CN201811070596 A CN 201811070596A CN 109358026 A CN109358026 A CN 109358026A
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fluorescence
droplet
degree
membership
droplets
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CN109358026B (en
Inventor
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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Suzhou Institute of Biomedical Engineering and Technology of CAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/10—Segmentation; Edge detection
    • G06T7/11—Region-based segmentation
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/10—Segmentation; Edge detection
    • G06T7/136—Segmentation; Edge detection involving thresholding
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/10—Segmentation; Edge detection
    • G06T7/187—Segmentation; Edge detection involving region growing; involving region merging; involving connected component labelling
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/10—Image acquisition modality
    • G06T2207/10004—Still image; Photographic image

Abstract

The invention discloses a kind of fluorescence drop detection method, device and servers, wherein the detection method includes: the fluorescence Liquid particle image for obtaining sample to be tested, is split to fluorescence Liquid particle image to extract multiple first fluorescence droplets in fluorescence Liquid particle image；According to the area and degree of membership of multiple first fluorescence droplets, judge in multiple first fluorescence droplets whether adhesion；When, there are when adhesion, being split in multiple first fluorescence droplets the connected domain of adhesion droplet formation to generate the second fluorescence droplet；According to the size and degree of membership of the second fluorescence droplet, judge whether the second fluorescence droplet is effective droplet.Fluorescence drop detection method, device and server provided in an embodiment of the present invention, the detection combined by area with degree of membership, improve the reliability to connected domain detection, and due to the hardware device for not increasing fluorescence drop detection, testing cost is advantageously reduced, and is easy to be integrated with existing hardware detection device.

Description

Fluorescence drop detection method, device and server
Technical field
The present invention relates to digital polymerase chain reaction technical fields, and in particular to a kind of fluorescence drop detection method, dress It sets and server.
Background technique
Digital pcr (Polymerase Chain Reaction polymerase chain reaction, abbreviation PCR) is to develop in recent years Very rapid Measurement for Biotechnique.Digital pcr is divided into droplet type and micro chamber two major classes, wherein droplet type digital pcr is city Leading products in, the signal detecting method of droplet type digital pcr include flow cytometer detection method and based on digital droplet image Planar detector method.
Flow cytometer detection method has many advantages, such as that background fluorescence intensity is low, and recognizer is simple, but this method optical path it is complicated, at This is high, and is difficult to be integrated with the droplet generation module of instrument front end, PCR amplification module, digital pcr it is integrated and Seem subsequent inability under cost effective development trend.
Planar detector method based on digital droplet image has the following problems: since the intensity of excitation fluorescence is weaker, usually Need the longer time for exposure that could obtain the image that can be used to identify, nonetheless, the brightness and comparison of droplet fluorescent image Degree is still obvious relatively low compared with bright field image, meanwhile, the noise of droplet fluorescent image is relatively low.
For the above problem existing for flow cytometer detection method and plane probe method, it would be highly desirable to study the glimmering of the new high reliability of one kind Light drop detection method improves its integrated level to reduce the cost of droplet type digital pcr.
Summary of the invention
In view of this, the embodiment of the invention provides a kind of fluorescence drop detection method, device and server, it is existing to solve Reliability present in some droplet type digital pcr technologies is lower, higher cost and it is not easy of integration the problems such as.
According in a first aspect, the embodiment of the invention provides a kind of fluorescence drop detection methods, comprising: obtain sample to be tested Fluorescence Liquid particle image, the fluorescence Liquid particle image is split multiple first glimmering in the fluorescence Liquid particle image to extract Light droplet；According to the area and degree of membership of the multiple first fluorescence droplet, judge in the multiple first fluorescence droplet whether Adhesion；When there are when adhesion, being split in the multiple first fluorescence droplet the connected domain of adhesion droplet formation to generate Second fluorescence droplet；According to the size and degree of membership of the second fluorescence droplet, judge whether the second fluorescence droplet is to have Imitate droplet.
Fluorescence drop detection method provided in an embodiment of the present invention, by the area for calculating separately each first fluorescence droplet And degree of membership, judge whether each first fluorescence droplet belongs to by the connected domain of multiple droplet Adhesion formations.Due to adhesion drop The general area of the connected domain of formation can be bigger so that area can be used as judge the first fluorescence droplet whether be connected domain mark One of standard.Only according to area carry out connected domain detection may non-interconnected domain biggish to area cause to judge by accident, therefore, the present invention is real The fluorescence drop detection method for applying example offer devises the subordinating degree function detected for fluorescence droplet, is reflected by degree of membership A possibility that existing droplet adhesion.The detection combined by area with degree of membership improves the reliability to connected domain detection, energy Enough reduce the erroneous judgement in connected domain detection.In addition, fluorescence drop detection method provided in an embodiment of the present invention, passes through the figure such as segmentation As processing method and the calculation methods such as reference area and subordinating degree function, the detection to fluorescence drop is realized, is not increased The hardware device of fluorescence drop detection advantageously reduces testing cost, and is easy to be integrated with existing hardware detection device.
With reference to first aspect, described according to the multiple first fluorescence droplet in first aspect first embodiment Area and degree of membership, judge in the multiple first fluorescence droplet whether adhesion, comprising: judge the face of the first fluorescence droplet Whether product is greater than preset area threshold, and whether the degree of membership of the first fluorescence droplet is greater than preset first degree of membership threshold Value；When the area of the first fluorescence droplet is greater than preset area threshold, and the degree of membership of the first fluorescence droplet is greater than When preset first degree of membership threshold value, determine that there are adhesions in the first fluorescence droplet.
Fluorescence drop detection method provided in an embodiment of the present invention, by setting area threshold value and the first degree of membership threshold value, Realize detection that area is combined with degree of membership, for connected domain.Since the algorithm complexity of threshold test is lower, it is easy It realizes, testing cost can be reduced while guaranteeing detection effect.
First embodiment with reference to first aspect calculates institute in first aspect second embodiment according to the following formula State the degree of membership of the first fluorescence droplet:
Wherein, fiIndicate the degree of membership of i-th of first fluorescence droplets；AiIndicate the area of i-th of first fluorescence droplets, Pi Indicate the perimeter of i-th of first fluorescence droplets, hiIndicate the length of i-th of first fluorescence droplets；wiIt indicates i-th first The width of fluorescence droplet；r0Indicate preset effective droplet radius；α, β and k are constant, respectively indicate preset coefficient.
Fluorescence drop detection method provided in an embodiment of the present invention, according to the length and width of the first fluorescence droplet, by The degree of membership of one fluorescence droplet, i.e. there are a possibility that adhesion droplet in the first fluorescence droplet to be expressed as the real number value between [0,1], To which the degree of membership numerical value of the first fluorescence droplet be calculated, and then the degree of membership of the first fluorescence droplet is less than corresponding threshold value and is made To judge to whether there is the judgment basis of adhesion drop in the first fluorescence droplet.Degree of membership indicates that corresponding first is glimmering closer to 1 Light droplet more may be in the presence of the connected domain for being adhered droplet；Degree of membership indicates that corresponding first fluorescence droplet more can not closer to 0 It can be in the presence of the connected domain for being adhered droplet.
With reference to first aspect, in first aspect third embodiment, the connected domain to adhesion droplet formation is carried out Segmentation is to generate the second fluorescence droplet, comprising: generates the Euclidean distance topographic map of the connected domain；According to the Europe of the connected domain Family name determines the center of each droplet in the connected domain apart from topographic map respectively；According to the center to the connection Domain is split, to generate the second fluorescence droplet corresponding with the center.
Fluorescence drop detection method provided in an embodiment of the present invention detecting comprising after being adhered the connected domain of droplet, The accuracy for being conducive to improve the detection of fluorescence droplet to eliminate being adhered between droplet is split to each connected domain.
Third embodiment with reference to first aspect, it is described glimmering according to described second in the 4th embodiment of first aspect The size and degree of membership of light droplet judge whether the second fluorescence droplet is effective droplet, comprising: judge second fluorescence Whether the height of droplet is in preset altitude range, and whether the width of the second fluorescence droplet is in preset width range It is interior, and whether the degree of membership of the second fluorescence droplet is less than preset second degree of membership threshold value；When the second fluorescence droplet Height in preset altitude range, the width of the second fluorescence droplet is in preset width range, and described second When the degree of membership of fluorescence droplet is less than preset second degree of membership threshold value, determine that the second fluorescence droplet is effective droplet.
Fluorescence drop detection method provided in an embodiment of the present invention, after the second fluorescence droplet being partitioned into connected domain, By judging whether height, width and the degree of membership of the second fluorescence droplet meet the requirement of corresponding threshold value, identify therein effective Droplet, accuracy of identification with higher.
4th embodiment with reference to first aspect calculates institute in the 5th embodiment of first aspect according to the following formula State the degree of membership of the second fluorescence droplet:
Wherein, fijIndicate the degree of membership for j-th of second fluorescence droplets being partitioned into from i-th of connected domain；AijIndicate from The area for j-th of second fluorescence droplets being partitioned into i-th of connected domain, PijIt indicates to be partitioned into from i-th of connected domain J-th of second fluorescence droplets perimeter, hijIndicate the length for j-th of second fluorescence droplets being partitioned into from i-th of connected domain Degree；wijIndicate the width for j-th of second fluorescence droplets being partitioned into from i-th of connected domain；r0Indicate preset effective droplet Radius；α, β and k are constant, respectively indicate preset coefficient.
Fluorescence drop detection method provided in an embodiment of the present invention, according to the length and width of the second fluorescence droplet, by The degree of membership of two fluorescence droplets, i.e. there are a possibility that adhesion droplet in the second fluorescence droplet to be expressed as the real number value between [0,1], To which the degree of membership numerical value of the second fluorescence droplet be calculated, and then the degree of membership of the second fluorescence droplet is less than corresponding threshold value and is made To judge the whether effective judgment basis of the second fluorescence droplet.
With reference to first aspect or any embodiment in the first to the 5th embodiment of first aspect, in first aspect In six embodiments, the fluorescence Liquid particle image for obtaining sample to be tested, comprising: the Raw fluorescence for obtaining the sample to be tested is micro- Drip image and corresponding darkfield image；The Raw fluorescence droplet image is corrected according to the darkfield image, generates school Fluorescence droplet image after just.
Fluorescence drop detection method provided in an embodiment of the present invention utilizes the Raw fluorescence droplet image pair with sample to be tested The darkfield image answered corrects Raw fluorescence droplet image, is conducive to the brightness and comparison that improve Raw fluorescence droplet image Degree improves the accuracy and reliability of the detection of fluorescence droplet.
With reference to first aspect or any embodiment of the first aspect first into sixth embodiment, in first aspect It is described the fluorescence Liquid particle image to be split to extract multiple first in the fluorescence Liquid particle image in seven embodiments Fluorescence droplet, comprising: global threshold segmentation is carried out to the fluorescence Liquid particle image, to filter out the back in the fluorescence Liquid particle image Scape；Local auto-adaptive Threshold segmentation is carried out to the fluorescence Liquid particle image after wiping out background, it is micro- to extract the multiple first fluorescence Drop.
Fluorescence drop detection method provided in an embodiment of the present invention is utilized respectively global threshold segmentation and local adaptive thresholding Value segmentation handles fluorescence Liquid particle image, to obtain multiple first fluorescence droplets, subsequent step is directed to and is mentioned The the first fluorescence droplet taken out carries out droplet adhesion detection and segmentation, to avoid directly carrying out validity to the first fluorescence droplet It causes to judge by accident since the size of adhesion droplet is not inconsistent when identification, is conducive to the accuracy for improving the detection of fluorescence droplet and reliable Property.
According to second aspect, the embodiment of the invention provides a kind of fluorescence drop detecting devices, comprising: the first segmentation is single Member is split to extract the fluorescent liquid fluorescence Liquid particle image for obtaining the fluorescence Liquid particle image of sample to be tested Drip multiple first fluorescence droplets in image；First judging unit, for according to the area of the multiple first fluorescence droplet and Degree of membership, judge in the multiple first fluorescence droplet whether adhesion；Second cutting unit, when the multiple first fluorescence droplet In there are when adhesion, second cutting unit is for being split the connected domain of adhesion droplet formation to generate the second fluorescence Droplet；Second judgment unit judges the second fluorescence droplet for the size and degree of membership according to the second fluorescence droplet It whether is effective droplet.
According to the third aspect, the embodiment of the invention provides a kind of servers, comprising: memory and processor, it is described to deposit Connection is communicated with each other between reservoir and the processor, computer instruction is stored in the memory, and the processor passes through The computer instruction is executed, thereby executing glimmering as described in any one of first aspect or first aspect embodiment Light drop detection method.
Detailed description of the invention
The features and advantages of the present invention will be more clearly understood by referring to the accompanying drawings, and attached drawing is schematically without that should manage Solution is carries out any restrictions to the present invention, in the accompanying drawings:
Fig. 1 shows the application scenarios schematic diagram of the embodiment of the present invention；
Fig. 2 shows the flow charts of a specific example of one of embodiment of the present invention fluorescence droplet detection method；
Fig. 3 shows the process of a specific example of another fluorescence droplet detection method in the embodiment of the present invention Figure；
Fig. 4 shows the structural representation of a specific example of one of embodiment of the present invention fluorescence droplet detection device Figure；
Fig. 5 shows the structural schematic diagram of a specific example of one of embodiment of the present invention server.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is A part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, those skilled in the art are not having Every other embodiment obtained under the premise of creative work is made, shall fall within the protection scope of the present invention.
As shown in Figure 1, being the application scenarios schematic diagram of the embodiment of the present invention, wherein the direction of propagation of arrow expression optical path. Excitation light source 1 is the higher source luminance with specific wavelength, such as laser, highlighted LED.Excitation light source 1, which issues exciting light, to be passed through After plus lens 2 and optical filter 3, irradiating and detecting chip 4 simultaneously excites drop to be measured to generate fluorescence.The fluorescence that drop issues is through filtering After mating plate 6 filters out the exciting light of scattering, into ccd sensor 7, ccd sensor 7 acquires image and by image transmitting to computer It is handled in 8, to realize the detection for being directed to fluorescence droplet.
In some embodiments, as shown in Fig. 2, computer 8 can be realized by executing following steps to fluorescence droplet Detection:
Step S101: the fluorescence Liquid particle image of sample to be tested is obtained.In practical applications, the Raw fluorescence of sample to be tested is micro- Drop image L generally has lower brightness and contrast, and noise is relatively low.It, can in order to improve the quality of fluorescence Liquid particle image Carried out to Raw fluorescence droplet image L using the darkfield image D with Raw fluorescence droplet image L with the identical time of integration Correction, the fluorescence droplet image I after generating correction.Since darkfield image D is by identical as acquisition Raw fluorescence droplet image L The image that is acquired under the identical time of integration of acquisition equipment so that including in darkfield image D and Raw fluorescence droplet image L In similar random noise, by the way that Raw fluorescence droplet image L and darkfield image D is made the difference, can substantially Removing Random No, To improve the signal-to-noise ratio that house of correction obtains fluorescence droplet image I.It in a specific embodiment, can also be to the fluorescence after correction Droplet image I carries out histogram equalization processing, to improve its brightness and contrast；It when necessary, can also be to fluorescence droplet figure As I progress gaussian filtering, further to eliminate noise, to improve its signal-to-noise ratio.Further, it is also possible to by several fluorescence droplet figures As I is spliced, so that subsequent step can simultaneously be handled several fluorescence droplet images I, realize that fluorescence droplet is parallel Detection, to improve detection efficiency.
Step S102: fluorescence Liquid particle image is split micro- to extract multiple first fluorescence in fluorescence Liquid particle image Drop.In a specific embodiment, global threshold segmentation first can be carried out to fluorescence droplet image I, to filter out fluorescence drop figure As the background in I；Local auto-adaptive Threshold segmentation then is carried out to the fluorescence Liquid particle image I after wiping out background, to extract wherein Multiple first fluorescence droplets.
Step S103: according to the area and degree of membership of multiple first fluorescence droplets, judge be in multiple first fluorescence droplets No adhesion.When there are when adhesion drop, executing step S104 in multiple first fluorescence droplets；When in multiple first fluorescence droplets not There are when adhesion drop, according to the size and degree of membership of the first fluorescence droplet, judge whether the first fluorescence droplet is effective droplet, And retain effective first fluorescence droplet, delete the first invalid fluorescence droplet.In a specific embodiment, as shown in figure 3, The process of step S103 can be realized by following steps:
Step S1031: judge whether the area of the first fluorescence droplet is greater than preset area threshold.When the first fluorescence droplet Area be greater than preset area threshold when, execute step S1032；When the area of the first fluorescence droplet is not more than preset area When threshold value, it can be determined that the first fluorescence droplet is not that multiple drops are adhered the connected domain to be formed, and then can according to this first The size and degree of membership of fluorescence droplet judge whether the first fluorescence droplet is effective droplet.
Step S1032: judge whether the degree of membership of the first fluorescence droplet is greater than preset first degree of membership threshold value.When first When the degree of membership of fluorescence droplet is greater than preset first degree of membership threshold value, step S1033 is executed；When being subordinate to for the first fluorescence droplet When degree is no more than preset first degree of membership threshold value, it can be determined that the first fluorescence droplet is not the company of multiple drop Adhesion formations Logical domain, and then can judge whether the first fluorescence droplet is effectively micro- according to the size and degree of membership of the first fluorescence droplet Drop.
Step S1033: determining the first fluorescence droplet, there are adhesion drops, and then execute step S104 to adhesion droplet formation Connected domain be split so that the drop being adhered in connected domain separate.Due to general comprising multiple connected domains for being adhered drop With the area for being greater than individual droplets, fluorescence drop detection method provided in an embodiment of the present invention is provided with area threshold, thus Realize the area detecting to the first drop.In a specific embodiment, area threshold can beWherein r0It is default Effective droplet radius.There is missing inspection when detecting connected domain in order to avoid simple usable floor area, it is provided in an embodiment of the present invention glimmering Light drop detection method devises subordinating degree function, to detect a possibility that being adhered in the first fluorescence droplet.Specifically, Can according to the following formula (1) calculate the first fluorescence droplet degree of membership:
Wherein, fiIndicate the degree of membership of i-th of first fluorescence droplets；AiIndicate the area of i-th of first fluorescence droplets, Pi Indicate the perimeter of i-th of first fluorescence droplets, hiIndicate the length of i-th of first fluorescence droplets；wiIt indicates i-th first The width of fluorescence droplet；r0Indicate preset effective droplet radius；α, β and k are constant, respectively indicate preset coefficient.
Formula (1) is according to the length and width of the first fluorescence droplet, by the degree of membership of the first fluorescence droplet, i.e. the first fluorescence There are a possibility that adhesion droplet in droplet to be expressed as the real number value between [0,1], so that the person in servitude of the first fluorescence droplet be calculated Belong to degree value, and then the degree of membership of the first fluorescence droplet is less than corresponding threshold value as judging to whether there is in the first fluorescence droplet The judgment basis of adhesion drop.Degree of membership indicates that corresponding first fluorescence droplet more may be adhered droplet to exist closer to 1 Connected domain；Degree of membership indicates that corresponding first fluorescence droplet more can not be in the presence of the connected domain for being adhered droplet closer to 0.One In specific embodiment, preset first degree of membership threshold value can be 0.5.
Step S104: connected domain is split to generate the second fluorescence droplet.Exist to the purpose that connected domain is split In interrupting the droplet being adhered in connected domain, them is made to be changed into multiple second fluorescence droplets independent by being adhered state.? In one specific embodiment, as shown in figure 3, can realize that step S104 is split connected domain by following sub-step To generate the process of the second fluorescence droplet:
Step S1041: the Euclidean distance topographic map of connected domain is generated.Can be used the prior art to connected domain at Reason, to obtain its corresponding Euclidean distance topographic map, the embodiment of the present application is without limitation.
Step S1042: the centre bit of each droplet in connected domain is determined respectively according to the Euclidean distance topographic map of connected domain It sets.In the corresponding Euclidean distance topographic map of each connected domain, local minimum, which is likely to be, is adhered a drop in drop Center, therefore, can using the local minimum in Euclidean distance topographic map as prospect mark.
Step S1043: being split connected domain according to center, to generate the second fluorescence corresponding with center Droplet.It is marked in conjunction with the prospect for utilizing local minimum to mark in Euclidean distance topographic map, using image in the prior art point It cuts algorithm to handle connected domain, the drop being adhered in each connected domain can be separated, to obtain multiple second fluorescence Droplet.Preferably, watershed algorithm can be used to be split each connected domain.
Step S105: according to the size and degree of membership of the second fluorescence droplet, judge whether the second fluorescence droplet is effectively micro- Drop.When the second fluorescence droplet is effective droplet, retain the second fluorescence droplet；When the second fluorescence droplet is not effective droplet, Delete the second fluorescence droplet.Preferably, can be sentenced simultaneously using the height of the second fluorescence droplet, width and its corresponding degree of membership Breaking, whether it is effective droplet.When the height of the second fluorescence droplet is in preset altitude range, the width of the second fluorescence droplet In preset width range, and the degree of membership of the second fluorescence droplet be less than preset second degree of membership threshold value when, determine second Fluorescence droplet is effective droplet.Specifically, can according to the following formula (2) calculate the second fluorescence droplet degree of membership:
Wherein, fijIndicate the degree of membership for j-th of second fluorescence droplets being partitioned into from i-th of connected domain；AijIndicate from The area for j-th of second fluorescence droplets being partitioned into i-th of connected domain, PijIt indicates to be partitioned into from i-th of connected domain J-th of second fluorescence droplets perimeter, hijIndicate the length for j-th of second fluorescence droplets being partitioned into from i-th of connected domain Degree；wijIndicate the width for j-th of second fluorescence droplets being partitioned into from i-th of connected domain；r0Indicate preset effective droplet Radius；α, β and k are constant, respectively indicate preset coefficient.It is corresponding with the subordinating degree function in formula (2), for judging It can be 0.4 that whether the second fluorescence droplet, which is the second degree of membership threshold value of effective droplet,.
Fluorescence drop detection method provided in an embodiment of the present invention, by the area for calculating separately each first fluorescence droplet And degree of membership, judge whether each first fluorescence droplet belongs to by the connected domain of multiple droplet Adhesion formations.Due to adhesion drop The general area of the connected domain of formation can be bigger so that area can be used as judge the first fluorescence droplet whether be connected domain mark One of standard.Only according to area carry out connected domain detection may non-interconnected domain biggish to area cause to judge by accident, therefore, the present invention is real The fluorescence drop detection method for applying example offer devises the subordinating degree function detected for fluorescence droplet, is reflected by degree of membership A possibility that existing droplet adhesion.The detection combined by area with degree of membership improves the reliability to connected domain detection, energy Enough reduce the erroneous judgement in connected domain detection.In addition, fluorescence drop detection method provided in an embodiment of the present invention, passes through the figure such as segmentation As processing method and the calculation methods such as reference area and subordinating degree function, the detection to fluorescence drop is realized, is not increased The hardware device of fluorescence drop detection advantageously reduces testing cost, and is easy to be integrated with existing hardware detection device.
The embodiment of the invention also provides a kind of fluorescence drop detecting devices, as shown in figure 4, the fluorescence drop detecting device It may include: the first cutting unit 401, the first judging unit 402, the second cutting unit 403 and second judgment unit 404.
Wherein, the first cutting unit 401 is used to obtain the fluorescence Liquid particle image of sample to be tested, carries out to fluorescence Liquid particle image Segmentation is to extract multiple first fluorescence droplets in fluorescence Liquid particle image；Its specific course of work can refer to above method implementation In example described in step S101 to step S102.
First judging unit 402 is used for area and degree of membership according to multiple first fluorescence droplets, judges that multiple first is glimmering It whether there is adhesion in light droplet；Its specific course of work can refer in above method embodiment described in step S103.
When, there are when adhesion, the second cutting unit 403 is used for the company to adhesion droplet formation in multiple first fluorescence droplets Logical domain is split to generate the second fluorescence droplet；Its specific course of work can refer to step S104 in above method embodiment It is described.
Second judgment unit 404 is used for size and degree of membership according to the second fluorescence droplet, judges that the second fluorescence droplet is No is effective droplet；Its specific course of work can refer in above method embodiment described in step S105.
The embodiment of the invention also provides a kind of servers, as shown in figure 5, the server may include processor 51 and deposit Reservoir 52, wherein processor 51 can be connected with memory 52 by bus or other modes, to be connected by bus in Fig. 5 For.
Processor 51 can be central processing unit (Central Processing Unit, CPU).Processor 51 can be with For other general processors, digital signal processor (Digital Signal Processor, DSP), specific integrated circuit (Application Specific Integrated Circuit, ASIC), field programmable gate array (Field- Programmable Gate Array, FPGA) either other programmable logic device, discrete gate or transistor logic, The combination of the chips such as discrete hardware components or above-mentioned all kinds of chips.
Memory 52 is used as a kind of non-transient computer readable storage medium, can be used for storing non-transient software program, non- Transient computer executable program and module, as the corresponding program of fluorescence drop detection method in the embodiment of the present invention refers to Order/module is (for example, the first cutting unit 401 shown in Fig. 4, the first judging unit 402, the second cutting unit 403 and second are sentenced Disconnected unit 404).Non-transient software program, instruction and the module that processor 51 is stored in memory 52 by operation, thus Execute the various function application and data processing of processor, i.e. XX method in realization above method embodiment.
Memory 52 may include storing program area and storage data area, wherein storing program area can storage program area, Application program required at least one function；It storage data area can the data etc. that are created of storage processor 51.In addition, storage Device 52 may include high-speed random access memory, can also include non-transient memory, for example, at least a magnetic disk storage Part, flush memory device or other non-transient solid-state memories.In some embodiments, it includes relative to place that memory 52 is optional The remotely located memory of device 51 is managed, these remote memories can pass through network connection to processor 51.The reality of above-mentioned network Example includes but is not limited to internet, intranet, local area network, mobile radio communication and combinations thereof.
One or more of modules are stored in the memory 52, when being executed by the processor 51, are executed Fluorescence drop detection method in embodiment as shown in Figure 2 to Figure 3.
Above-mentioned server detail can correspond to refering to Fig. 2 into embodiment shown in Fig. 3 corresponding associated description and Effect is understood that details are not described herein again.
It is that can lead to it will be understood by those skilled in the art that realizing all or part of the process in above-described embodiment method Computer program is crossed to instruct relevant hardware and complete, the program can be stored in a computer-readable storage medium In, the program is when being executed, it may include such as the process of the embodiment of above-mentioned each method.Wherein, the storage medium can for magnetic disk, CD, read-only memory (Read-Only Memory, ROM), random access memory (Random Access Memory, RAM), flash memory (Flash Memory), hard disk (Hard Disk Drive, abbreviation: HDD) or solid state hard disk (Solid-State Drive, SSD) etc.；The storage medium can also include the combination of the memory of mentioned kind.
Although being described in conjunction with the accompanying the embodiment of the present invention, those skilled in the art can not depart from the present invention Spirit and scope in the case where various modifications and variations can be made, such modifications and variations are each fallen within by appended claims institute Within the scope of restriction.

Claims (10)

1. a kind of fluorescence drop detection method characterized by comprising
The fluorescence Liquid particle image for obtaining sample to be tested, is split the fluorescence Liquid particle image to extract the fluorescence drop figure Multiple first fluorescence droplets as in；
According to the area and degree of membership of the multiple first fluorescence droplet, judge whether glue in the multiple first fluorescence droplet Even；
When there are when adhesion, be split to the connected domain of adhesion droplet formation to generate in the multiple first fluorescence droplet Two fluorescence droplets；
According to the size and degree of membership of the second fluorescence droplet, judge whether the second fluorescence droplet is effective droplet.
2. fluorescence drop detection method according to claim 1, which is characterized in that described according to the multiple first fluorescence The area and degree of membership of droplet, judge in the multiple first fluorescence droplet whether adhesion, comprising:
Judge whether the area of the first fluorescence droplet is greater than preset area threshold, and the first fluorescence droplet is subordinate to Whether degree is greater than preset first degree of membership threshold value；
When the area of the first fluorescence droplet is greater than preset area threshold, and the degree of membership of the first fluorescence droplet is greater than When preset first degree of membership threshold value, determine that there are adhesions for the first fluorescence droplet.
3. fluorescence drop detection method according to claim 2, which is characterized in that calculate described first according to the following formula The degree of membership of fluorescence droplet:
Wherein, fiIndicate the degree of membership of i-th of first fluorescence droplets；AiIndicate the area of i-th of first fluorescence droplets, PiIt indicates Indicate the perimeter of i-th of first fluorescence droplets, hiIndicate the length of i-th of first fluorescence droplets；wiIndicate i-th of first fluorescence The width of droplet；r0Indicate preset effective droplet radius；α, β and k are constant, respectively indicate preset coefficient.
4. fluorescence drop detection method according to claim 1, which is characterized in that the connection to adhesion droplet formation Domain is split to generate the second fluorescence droplet, comprising:
Generate the Euclidean distance topographic map of the connected domain；
Determine the center of each droplet in the connected domain respectively according to the Euclidean distance topographic map of the connected domain；
The connected domain is split according to the center, it is micro- to generate the second fluorescence corresponding with the center Drop.
5. fluorescence drop detection method according to claim 4, which is characterized in that described according to the second fluorescence droplet Size and degree of membership, judge whether the second fluorescence droplet is effective droplet, comprising:
The height of the second fluorescence droplet is judged whether in preset altitude range, and the width of the second fluorescence droplet is It is no in preset width range, and whether the degree of membership of the second fluorescence droplet is less than preset second degree of membership threshold value；
When the height of the second fluorescence droplet is in preset altitude range, the width of the second fluorescence droplet is preset In width range, and the degree of membership of the second fluorescence droplet be less than preset second degree of membership threshold value when, determine described second Fluorescence droplet is effective droplet.
6. fluorescence drop detection method according to claim 5, which is characterized in that calculate described second according to the following formula The degree of membership of fluorescence droplet:
Wherein, fijIndicate the degree of membership for j-th of second fluorescence droplets being partitioned into from i-th of connected domain；AijIt indicates from i-th The area for j-th of second fluorescence droplets being partitioned into connected domain, PijIndicate the jth being partitioned into from i-th of connected domain The perimeter of a second fluorescence droplet, hijIndicate the length for j-th of second fluorescence droplets being partitioned into from i-th of connected domain；wij Indicate the width for j-th of second fluorescence droplets being partitioned into from i-th of connected domain；r0Indicate preset effective droplet radius； α, β and k are constant, respectively indicate preset coefficient.
7. fluorescence drop detection method according to claim 1 to 6, which is characterized in that the acquisition is to test sample This fluorescence Liquid particle image, comprising:
Obtain the Raw fluorescence droplet image and corresponding darkfield image of the sample to be tested；
The Raw fluorescence droplet image is corrected according to the darkfield image, the fluorescence droplet image after generating correction.
8. fluorescence drop detection method described in any one of -7 according to claim 1, which is characterized in that described to the fluorescence Liquid particle image is split to extract multiple first fluorescence droplets in the fluorescence Liquid particle image, comprising:
Global threshold segmentation is carried out to the fluorescence Liquid particle image, to filter out the background in the fluorescence Liquid particle image；
Local auto-adaptive Threshold segmentation is carried out to the fluorescence Liquid particle image after wiping out background, it is micro- to extract the multiple first fluorescence Drop.
9. a kind of fluorescence drop detecting device characterized by comprising
First cutting unit, for obtaining the fluorescence Liquid particle image of sample to be tested, to the fluorescence Liquid particle image be split with Extract multiple first fluorescence droplets in the fluorescence Liquid particle image；
First judging unit judges the multiple first for the area and degree of membership according to the multiple first fluorescence droplet In fluorescence droplet whether adhesion；
Second cutting unit, when, there are when adhesion, second cutting unit is used for viscous in the multiple first fluorescence droplet Even the connected domain of droplet formation is split to generate the second fluorescence droplet；
Second judgment unit judges the second fluorescence droplet for the size and degree of membership according to the second fluorescence droplet It whether is effective droplet.
10. a kind of server characterized by comprising
Memory and processor communicate with each other connection, are stored in the memory between the memory and the processor Computer instruction, the processor is by executing the computer instruction, thereby executing as described in any one of claim 1-8 Fluorescence drop detection method.
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