WO2021169822A1 - 一种适于生物分子多重检测的成像系统 - Google Patents

一种适于生物分子多重检测的成像系统 Download PDF

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Publication number
WO2021169822A1
WO2021169822A1 PCT/CN2021/076515 CN2021076515W WO2021169822A1 WO 2021169822 A1 WO2021169822 A1 WO 2021169822A1 CN 2021076515 W CN2021076515 W CN 2021076515W WO 2021169822 A1 WO2021169822 A1 WO 2021169822A1
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light source
excitation light
excitation
fluorescence
image
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PCT/CN2021/076515
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English (en)
French (fr)
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郭庆生
徐宏
古宏晨
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上海交通大学
上海迈景纳米科技有限公司
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Priority claimed from CN202020226457.5U external-priority patent/CN211927701U/zh
Priority claimed from CN202010129135.3A external-priority patent/CN113376127A/zh
Application filed by 上海交通大学, 上海迈景纳米科技有限公司 filed Critical 上海交通大学
Priority to EP21761813.1A priority Critical patent/EP4134660A4/en
Priority to US17/802,512 priority patent/US20230296518A1/en
Publication of WO2021169822A1 publication Critical patent/WO2021169822A1/zh

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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
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1425—Optical investigation techniques, e.g. flow cytometry using an analyser being characterised by its control arrangement
    • G01N15/1427—Optical investigation techniques, e.g. flow cytometry using an analyser being characterised by its control arrangement with the synchronisation of components, a time gate for operation of components, or suppression of particle coincidences
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N2015/1438—Using two lasers in succession
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N2015/144—Imaging characterised by its optical setup
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1486—Counting the particles
    • 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
    • G01N2021/6463—Optics
    • G01N2021/6471—Special filters, filter wheel
    • 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
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence

Definitions

  • the patent of the present invention relates to the field of biological detection, in particular to an imaging system suitable for multiple detection of biomolecules.
  • the purpose of the present invention is to provide an imaging system suitable for multiple detection of biomolecules.
  • the first aspect of the present invention provides an imaging system suitable for multiple detection of biomolecules, which can realize fluorescence and photo-induced chemiluminescence imaging at the same time.
  • the second aspect of the present invention provides an imaging system suitable for multiple detection of biomolecules.
  • the imaging system includes at least a first optical path and a second optical path; the first optical path is from an excitation light source to an objective lens, and the excitation light source A filter group is provided between the lens and the objective lens, and the filter group is configured and arranged to guide the excitation light emitted from the excitation light source to the objective lens; the second optical path is from the An objective lens to an image acquisition device, and a reflector is provided between the objective lens and the image acquisition device, and the reflector is configured and arranged to project the fluorescence received by the objective lens to the image acquisition device;
  • the excitation light source includes at least a first excitation light source and a second excitation light source. The first excitation light source is used for excitation of fluorescent signals, and the second excitation light source is used for excitation of photo-excited chemiluminescence signals.
  • the third aspect of the present invention provides the aforementioned imaging system suitable for multiple detection of biomolecules, and its application in the field of multiple detection of biomolecules.
  • the fourth aspect of the present invention provides an imaging method for multiple detection of biomolecules, which adopts the aforementioned imaging system suitable for multiple detection of biomolecules, and at least includes the following steps:
  • the fifth aspect of the present invention provides an image analysis method for multiple detection of biomolecules, which includes the following steps:
  • S1 matches the fluorescence image of the detection liquid with the photo-excited chemiluminescence image according to the spot position to obtain the fluorescence signal and the photo-excited chemiluminescence signal corresponding to each spot.
  • the fluorescence image and the photo-excited chemiluminescence image of the detection liquid adopt the aforementioned biological molecules Obtained by multiple detection imaging methods;
  • S2 clusters and classifies the fluorescent signals, and obtains the average photoinduced chemiluminescence intensity corresponding to each fluorescent signal
  • S3 determines the content of each molecule to be tested in the detection solution according to the classification of the fluorescent signal and the average photo-induced chemiluminescence intensity.
  • a sixth aspect of the present invention provides a computer-readable storage medium on which a computer program is stored, and when the program is executed by a processor, the steps of the aforementioned image analysis method for multiple detection of biomolecules are realized.
  • the imaging system suitable for multiple detection of biomolecules of the present invention has the following beneficial effects:
  • the invention combines fluorescence imaging and photo-induced chemiluminescence imaging for the first time, can simultaneously detect fluorescence signals and photo-induced chemiluminescence signals, and is suitable for multiple photo-induced chemiluminescence detection.
  • Fig. 1 is a schematic diagram of the imaging system suitable for multiple detection of biomolecules of the present invention, and the arrows in the figure indicate the propagation direction of light.
  • Figure 2 shows the imaging system suitable for multiple detection of biomolecules of the present invention used for multiple photoinduced chemiluminescence to obtain images.
  • Fig. 3 shows a flow chart of the imaging system suitable for multiple detection of biomolecules for multiple photo-induced chemiluminescence image analysis of the present invention.
  • Figure 4 shows the cluster analysis results of the imaging system image analysis software for multiple detection of biomolecules used in multiple photo-induced chemiluminescence images of the present invention.
  • the results show the fluorescence classification and corresponding photo-induced chemistry of the four detection particles. light intensity.
  • Figure 5 shows a communication diagram of the imaging system of the present invention.
  • An imaging system suitable for multiple detection of biomolecules can realize fluorescence and photo-induced chemiluminescence imaging at the same time.
  • an imaging system suitable for multiple detection of biomolecules in an embodiment of the present invention includes at least a first optical path and a second optical path;
  • the first optical path is from Excitation light source 1 to objective lens 15, and a filter group 13 is provided between the excitation light source and the objective lens, and the filter group 13 is configured and arranged to guide the excitation light emitted from the excitation light source to The objective lens;
  • the second optical path is from the objective lens 15 to the image acquisition device 2, and a reflector 16 is provided between the objective lens 15 and the image acquisition device 2, and the reflector 16 is configured and Arranged to project the fluorescence received by the objective lens 15 to the image acquisition device 2;
  • the excitation light source 1 includes at least a first excitation light source 11 and a second excitation light source 12, and the first excitation light source 11 is used for fluorescence signals
  • the second excitation light source 12 is used for the excitation of photo-induced chemiluminescence signals.
  • the imaging system can simultaneously realize fluorescence and photo-induced chemiluminescence imaging.
  • the imaging system further includes a sample stage 14 which is arranged above the objective lens and is used to place the object to be observed.
  • the imaging system further includes a workstation 3 and a light source switching switch 17.
  • the workstation is in communication with the image acquisition device 2, and the light source switching switch 17 is connected to the excitation light source 1 and the workstation 3. Communication connection.
  • the communication connection refers to the formation of communication between connected devices through the transmission and interaction of signals.
  • the communication connection includes a wired connection and a wireless connection, for example, an electric signal transmission connection, a digital signal transmission connection, and the like.
  • the light source switching switch 17 can switch the excitation light source 1.
  • the workstation includes a control center 31 and a trigger control box 32 that are communicatively connected to each other.
  • the control center 31 is in communication connection with the image acquisition device 2.
  • the control center 31 may be a computer, and the computer may be used to carry image processing programs for matching and signal analysis between the image obtained after excitation of the microcarrier signal with the coding function and the image obtained after the detection of the particle signal excitation .
  • the trigger control box can also be equipped with a trigger control box control program for controlling the trigger control box.
  • the control center is used to control the trigger control box 32, and the trigger control box is used to control the light source switch 17, the opening and closing of the excitation light source 1 and the image acquisition device 2.
  • the control center 31 can directly control the opening and closing of the excitation light source 1 through the trigger control box 32, or can control the image acquisition device through the trigger control box 32, so as to realize the opening and closing of the excitation light source.
  • the image acquisition device 2 can be activated to make the image acquisition device generate a TTL (transistor-transistor logic integrated circuit) signal and send it to the trigger control box.
  • TTL transistor-transistor logic integrated circuit
  • the trigger control box After the trigger control box receives the signal, it immediately sends the TTL control signal to the excitation light source 1, and then The activation of the excitation light source 1 can be controlled.
  • the control center can switch the first excitation light source 11 and the second excitation light source 12 by controlling the opening and closing of the light source switching switch.
  • the imaging system includes a normal fluorescence imaging mode and a time-resolved fluorescence mode.
  • the normal fluorescence imaging mode means that the light of the excitation light source is synchronized with the exposure of the image acquisition device;
  • the time-resolved fluorescence mode means that the excitation The light source and luminescence are not synchronized with the exposure of the image capture device.
  • control center can control the trigger control box to switch between the two fluorescence modes.
  • the normal fluorescence imaging mode is that the excitation light source is turned on when the image acquisition device 2 starts to expose.
  • the excitation time of the excitation light source can be set to be the same as the camera exposure time, and the image acquisition device 2 is started to make the image acquisition device generate TTL (transistor -Transistor logic integrated circuit) signal and send it to the trigger control box.
  • the trigger control box receives the signal, it immediately sends a TTL control signal to the excitation light source 1, and starts the excitation light source 1.
  • the camera starts to synchronize the exposure to realize the light emission and image acquisition device Exposure synchronization.
  • the time-resolved fluorescence imaging mode is that the control center controls the excitation light source to turn on through the trigger control box, and the image acquisition device 2 is exposed to turn off at this time. After a period of time, the excitation light source is turned off, and at this time, the image acquisition device 2 is exposed and then turned on. At this time, start the image acquisition device 2 so that the image acquisition device generates a TTL (transistor-transistor logic integrated circuit) signal and sends it to the trigger control box.
  • TTL transistor-transistor logic integrated circuit
  • the trigger control box After the trigger control box receives the signal, it immediately sends the TTL control signal to the excitation light source 1, and starts Excitation light source 1, at this time the camera has not yet started exposure; when the set time is reached, the excitation light source is turned off, at this time the camera starts exposure again to obtain an image. This can further reduce the background noise generated by the excitation light.
  • the time-resolved fluorescence function can be controlled by the program on the control center 31 to trigger the control box, and then the time-resolved function can be turned on or off.
  • the trigger control box may be a conventional trigger control box, such as FluoCa's VV-TRGBOX trigger control box.
  • the first excitation light source 11 may be one or more of a laser, a xenon lamp, a mercury lamp, a halogen lamp, or a light emitting diode.
  • the second excitation light source 12 may be one or more of a laser, a xenon lamp, a mercury lamp, a halogen lamp, or a light emitting diode.
  • the image acquisition device 2 may be a camera.
  • the image acquisition device 2 is selected from one or more of CCD, EMCCD, CMOS or sCMOS.
  • the filter group (13) includes at least a first filter group, a second filter group, and a third filter group.
  • Each filter group includes an excitation filter, an emission filter, and two To the color mirror.
  • the filter set 13 is detachable. It can be replaced according to fluorescent substances with different excitation and emission wavelengths, or according to the types of photosensitizers and chemiluminescent agents filled in different photo-excited chemiluminescent particles.
  • the number of the filter groups can be adjusted according to the types and quantities of fluorescent substances.
  • the first filter set is: excitation filter 488/15nm, dichroic mirror 495nm long pass, and emission filter 535/23nm.
  • the second filter set is: excitation filter 488/15nm, dichroic mirror 495nm long pass, and emission filter 600/40nm.
  • the third filter set is: excitation filter 680/13nm, dichroic mirror 653nm short pass, and emission filter 615/20nm.
  • an excitation filter In the filter group, an excitation filter, a dichroic mirror, and an emission filter are arranged in sequence.
  • the angle between the dichroic mirror and the propagation direction of the excitation light generated by the excitation light source is 45°.
  • the excitation light generated by the excitation light source in the filter group first passes through the excitation filter, and then reaches the surface of the dichroic mirror.
  • the dichroic mirror According to the nature of the dichroic mirror, light with a wavelength below 495nm is reflected, and light above 495nm is reflected.
  • the light is transmitted, so the incident excitation light reaches the dichroic mirror, is reflected to the objective lens, and then propagates to the sample.
  • the light signal excited by the sample returns to the dichroic mirror through the objective lens, passes through the dichroic mirror, and then passes through the emission filter, reaches the mirror, and is reflected by the mirror to the image acquisition device.
  • the imaging system can realize fluorescence imaging function and photoinduced chemiluminescence imaging function.
  • 1Fluorescence imaging function pass the first filter group (excitation filter 488/15nm, dichroic mirror 495nm long pass, emission filter 535/23nm) or second filter group (excitation Filter 488/15nm, dichroic mirror 495nm long pass, emission filter 600/40nm) and objective lens to excite the sample, the fluorescence signal generated by the sample passes through the objective lens and the first filter group or the second filter group again , And finally collected by the camera to produce two fluorescence images.
  • Photo-induced chemiluminescence imaging function the sample is excited by the photo-induced chemiluminescence light source through the third filter group (excitation filter 680/13nm, dichroic mirror 653nm short pass, emission filter 615/20nm) and objective lens , The generated photo-induced chemiluminescence signal passes through the objective lens and the third filter group again, and is finally collected by the camera to generate a photo-induced chemiluminescence image.
  • the third filter group excitation filter 680/13nm, dichroic mirror 653nm short pass, emission filter 615/20nm
  • the control center of the workbench of the present invention can install different image processing programs and trigger control box control programs according to needs.
  • the control program of the trigger control box can be easily implemented by those skilled in the art as long as they know the two modes of the present invention.
  • the imaging system can switch any one of the three filter groups into the imaging light path through the filter wheel.
  • the imaging system has two imaging modes.
  • the first is the common fluorescence mode for photo-induced chemiluminescence signal excitation, which specifically includes the following workflows:
  • Step 1 Place the sample on the sample stage and focus on the object to be tested in the sample.
  • Step 2 Switch the first filter group into the imaging light path, and switch to the fluorescence excitation light source through the light source switching module. Turn off the time-resolved fluorescence function, set the camera exposure time to 200ms to take pictures, and get fluorescence image 1, as shown in Figure 2.
  • Step 3 Switch the second filter set to enter the imaging light path, and other conditions are the same as in Step 2, and take a photo to obtain a fluorescent image 2, as shown in FIG. 2.
  • Step 4 Switch the third filter group into the imaging light path, switch to the photo-induced chemiluminescence excitation light source through the light source switching module, turn off the time-resolved fluorescence function, set the light source excitation time to 1000ms, and the camera exposure time to 1000ms.
  • the light source switch is triggered by the camera control to turn on, and the camera starts to expose synchronously at this time. After 1000ms, the exposure ends and the light source is turned off to obtain the photo-induced chemiluminescence image, as shown in Figure 2.
  • the second type is photo-induced chemiluminescence signal excitation using time-resolved fluorescence mode, which specifically includes the following workflow:
  • Step 1 Place the sample on the sample stage, and focus on the object under test in the sample through the focusing knob that adjusts the distance between the objective lens and the sample stage.
  • Step 2 Switch the first filter group into the imaging light path, and switch to the fluorescence excitation light source through the light source switching module. Turn off the time-resolved fluorescence function, set the camera exposure time to 200ms to take pictures, and get the fluorescence image 1.
  • Step 3 Switch the second filter set to enter the imaging light path, other conditions are the same as in Step 2, and take a photo to obtain a fluorescent image 2.
  • Step 4 Switch the third filter group into the imaging light path, switch to the photo-induced chemiluminescence excitation light source through the light source switching module, turn on the time-resolved fluorescence function, set the light source excitation time to 500ms, and the camera exposure time to 1000ms.
  • the light source switch is triggered by the camera control to turn on, and the camera exposure is turned off at this time; the light source is turned off after 500ms, and the camera starts to expose at this time, and the exposure ends after 1000ms, and the photo-induced chemiluminescence image is obtained.
  • the imaging method for multiple detection of biomolecules is performed using the aforementioned imaging system suitable for multiple detection of biomolecules, and at least includes the following steps:
  • the imaging method further includes the following steps: changing the parameters of the filter set, repeating step 1), and obtaining various fluorescence images of the sample.
  • the fluorescence image and the photo-induced chemiluminescence image of the sample are collected by an image collecting device.
  • the imaging method may adopt a normal fluorescence mode or a time-resolved fluorescence mode, wherein when the time-resolved fluorescence mode is adopted, in step 2), the imaging system is switched to the photo-induced chemiluminescence excitation light source and then turned on Time-resolved fluorescence mode to obtain the photo-induced chemiluminescence image of the sample.
  • the imaging system is switched to the photo-induced chemiluminescence excitation light source and then turned on Time-resolved fluorescence mode to obtain the photo-induced chemiluminescence image of the sample.
  • the image obtained by the above method can be analyzed and processed in the control center 31 of the imaging system of the present invention.
  • S1 matches the fluorescence image of the detection liquid with the photo-excited chemiluminescence image according to the spot position to obtain the fluorescence signal and the photo-excited chemiluminescence signal corresponding to each spot.
  • the fluorescence image and the photo-excited chemiluminescence image of the detection liquid adopt the aforementioned biological molecules Obtained by multiple detection imaging methods;
  • S2 clusters and classifies the fluorescent signals, and obtains the average photoinduced chemiluminescence intensity corresponding to each fluorescent signal
  • S3 determines the content of each molecule to be tested in the detection solution according to the classification of the fluorescent signal and the average photo-induced chemiluminescence intensity.
  • the detection solution suitable for the above analysis method is a multiple photo-excited chemical detection reaction solution.
  • the reaction solution for multiple photo-excited chemical detection is a homogeneous reaction solution, and its reactants generally include detection particles suitable for multiple detection of biomolecules, test molecules, and matching particles.
  • the molecules to be tested can form complexes with corresponding detection particles and matching particles, and the complexes can emit light signals under laser irradiation.
  • the multiple photo-excited chemical detection reaction solution can simultaneously detect multiple test molecules in a single homogeneous reaction solution.
  • the molecule to be tested can be a protein, a small molecule antigen or a nucleic acid.
  • Detection particles suitable for multiple detection of biomolecules include microcarriers with coding functions, and detection particles are connected to the microcarriers.
  • the microcarrier with encoding function can be selected from one or more of fluorescent encoding microcarriers, Raman signal encoding microcarriers, photonic crystal encoding microcarriers or pattern encoding microcarriers.
  • the detection particles should be suitable for photo-induced chemiluminescence detection.
  • the detection particles should include donor particles (DB) or acceptor particles (AB) suitable for photo-induced chemiluminescence detection.
  • the acceptor particles can emit fluorescence, and the donor particles are used to excite the acceptor particles to emit light.
  • DB is doped with photosensitizer, which can generate singlet oxygen after being excited by light.
  • the AB is doped with chemiluminescent agent and fluorescent agent.
  • the chemiluminescent agent converts the energy of singlet oxygen into 360nm emission light and excites the fluorescent agent to produce fluorescence.
  • the chemiluminescent agent is selected from one or more of dioxane or dimethylthiophene.
  • the detection particles should generally also include specific biological capture materials, which include, but are not limited to, one or more of biological capture probes, antigens, antibodies, ProtinA, ProtinG, or streptavidin.
  • the biocapture material can be coupled with donor particles or acceptor particles.
  • the biological capture material can specifically bind to the molecule to be tested.
  • the matching particles are photo-excited chemiluminescent particles that match the detection particles in the detection particles.
  • the matching particles should include acceptor particles; when the detection particles include acceptor particles, the matching particles should include donor particles.
  • Matching particles should generally also include detection substances, which include, but are not limited to, one or more of probes, antigens, antibodies, ProtinA, ProtinG, or streptavidin.
  • the detection substance can be coupled to donor particles or acceptor particles.
  • the detection substance should not directly bind to the detection particle, but can form a complex with the detection particle through the molecule to be detected. Therefore, the detection substance should be able to specifically bind to the test molecule, or the detection substance can specifically bind to the test molecule via an intermediate binding substance.
  • the test molecule When the test molecule exists, the test molecule forms a complex with the corresponding detection particles and matching particles. After being irradiated by the laser, the DB in the complex can activate the oxygen in the surrounding environment to convert into singlet oxygen, and its survival time is only It is 4 microseconds. The short survival time determines that the propagation diameter of ionic oxygen is very small (about 200nm). Because the distance between AB and DB in the composite is less than 200nm, AB receives the singlet oxygen generated by DB and is excited to emit light signals. When there is no molecule to be tested, the distance between AB and DB is too far to excite the light signal of AB.
  • the multiple photochemical detection reaction solution should include a variety of detection particles suitable for multiple detection of biomolecules. Based on the different codes of the detection particles, they can emit different fluorescence to distinguish the detection results of different molecules to be tested. And/or, the detection results of different molecules to be detected can also be distinguished based on the difference of the photoinduced chemiluminescence of the complex formed by the molecule to be detected and the corresponding detection particles and matching particles.
  • the light spot of the fluorescent image comes from the coding substance of the detection particle and/or the detection particle reaction complex, and the light spot of the photo-induced chemiluminescence image comes from the detection particle reaction complex.
  • the fluorescence image can be matched with the light spot at the same position in the photo-induced chemiluminescence image, so as to obtain the fluorescence signal and photo-induced chemiluminescence signal corresponding to the detection particle or the detection particle reaction complex at the position. Furthermore, by clustering and classifying the fluorescence signals, the average photoinduced chemiluminescence intensity corresponding to various types of fluorescence signals is obtained.
  • the average photo-induced chemiluminescence intensity corresponding to each molecule to be tested can be obtained according to the molecule to be tested, the type of fluorescent signal, and the average photo-induced chemiluminescence intensity corresponding to the fluorescent signal.
  • the corresponding relationship between the intensity of excited chemiluminescence and the concentration can obtain the content of each molecule to be tested.
  • step S1 the position of the light spot in the image can be identified by global thresholding, and the fluorescence image of the detection liquid and the photo-induced chemiluminescence image can be matched according to the position of the light spot.
  • Various optical signal parameters can be collected to characterize fluorescence signals and photo-induced chemiluminescence signals, such as the gray value or RGB value of the light spot.
  • noise reduction is performed before the fluorescence image and the photo-induced chemiluminescence image are matched to distinguish the fluorescence signal or the photo-induced chemiluminescence signal from the background signal.
  • step S2 the fluorescent signal can be clustered and classified according to the difference in the characteristic value of the optical signal.
  • the average photoinduced chemiluminescence intensity corresponding to each fluorescence signal can be calculated by using conventional statistical methods.
  • step S3 the specific steps may include the following steps:
  • the concentration of each molecule to be tested is obtained according to the standard curve of photo-induced chemiluminescence of each molecule to be tested and the average photo-induced chemiluminescence intensity.
  • an embodiment of the present invention also provides a computer-readable storage medium on which a computer program is stored, and when the program is executed by a processor, the steps of the foregoing method are implemented.
  • the aforementioned computer program can be stored in a computer-readable storage medium.
  • the computer-readable storage medium may include, but is not limited to, a floppy disk, an optical disk, a CD-ROM (read-only optical disk memory), a magneto-optical disk, and a ROM (only Read memory), RAM (random access memory), EPROM (erasable programmable read-only memory), EEPROM (electrically erasable programmable read-only memory), magnetic or optical card, flash memory, or suitable for storage Other types of media/machine-readable media that execute instructions.
  • the computer-readable storage medium may be a product that has not been connected to a computer device, or a component that has been connected to a computer device for use.
  • control center 31 in the embodiment of the present invention may be equipped with image analysis software to implement the above analysis method.
  • the software image analysis process includes four steps: image preprocessing, co-localization, grayscale analysis, and cluster analysis.
  • Image preprocessing denoise the obtained fluorescence image and photo-induced chemiluminescence image, distinguish the fluorescence signal or photo-induced chemiluminescence signal from the background signal, and identify the position of the signal in the image through global thresholding.
  • 2Co-localization unify the position of the fluorescence signal and the photo-induced chemiluminescence signal in the image.
  • 3Grayscale analysis read the grayscale values of the fluorescence signal and the photo-induced chemiluminescence signal respectively.
  • 4Clustering analysis cluster the fluorescent signals at different positions in the image according to the intensity of the gray value, and obtain different classifications, which are used as the types of different molecules to be tested. Then, the gray value of the photo-induced chemiluminescence signal of different fluorescence classifications is counted, so as to obtain the content (concentration) level of the different types of molecules to be tested.
  • Step 1 Image input.
  • the software reads the fluorescence image and photo-induced chemiluminescence image obtained in the foregoing.
  • Step 2 Image preprocessing. The noise is reduced, the fluorescence signal or photo-induced chemiluminescence signal is distinguished from the background signal, and the position of the signal in the image is identified through global thresholding.
  • Step 3 Co-localization: unify the positions of the fluorescence signal and the photo-induced chemiluminescence signal in the image.
  • Step 4 Grayscale analysis: Read the grayscale values of the fluorescence signal and the photoinduced chemiluminescence signal respectively.
  • Step 5 Cluster analysis: cluster the fluorescence signals at different positions in the image according to the intensity of the gray value to obtain different classifications. Then, the gray values of the photo-induced chemiluminescence signals of different fluorescence classifications are counted, and the average photo-induced chemiluminescence intensity is obtained.
  • Step 6 Data output: One-to-one correspondence between the different fluorescence classifications obtained in Step 5 and the types of different analytes. Then, the average photo-induced chemiluminescence intensity of the different fluorescence classifications is substituted into the standard curve of the corresponding analyte to obtain the actual content (concentration) of the analyte.
  • the cluster analysis result of the image analysis software used for multiple photo-induced chemiluminescence images is shown in Figure 4.
  • the result shows the fluorescence classification and corresponding photo-induced chemiluminescence intensity of the four detection particles.
  • the aforementioned system suitable for multiple detection of biomolecules can be used for applications in the field of multiple detection of biomolecules.

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Abstract

本发明专利提供一种适于生物分子多重检测的成像系统，所述成像系统至少包括第一光学路径和第二光学路径；所述第一光学路径为从激发光源至物镜，且所述激发光源和所述物镜之间设置有滤光片组，所述滤光片组被配置和布置为将从所述激发光源发出的激发光引导至所述物镜；所述第二光学路径为从所述物镜至图像采集装置，且所述物镜和所述图像采集装置之间设置有反光镜，所述反光镜被配置和布置为将由所述物镜接收到的荧光投射到所述图像采集装置；所述激发光源至少包括第一激发光源和第二激发光源，所述第一激发光源用于荧光信号的激发，所述第二激发光源用于光激化学发光信号的激发。所述成像系统能够同时实现荧光和光激化学发光成像。

Description

一种适于生物分子多重检测的成像系统 技术领域
本发明专利涉及生物检测领域，特别是涉及一种适于生物分子多重检测的成像系统。
背景技术
目前光激化学发光的检测仪器，例如西门子的Dimension EXL analyzer、PerkinElmer的
Figure PCTCN2021076515-appb-000001
Multimode Plate Reader和北京科美的LiCA500全自动化学发光分析仪都只能检测整个均相溶液中的统一信号，无法对单个检测颗粒的信号进行检测，因此无法实现多重检测的功能。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种适于生物分子多重检测的成像系统。
本发明第一方面提供适于生物分子多重检测的成像系统，所述成像系统能够同时实现荧光和光激化学发光成像。
本发明第二方面提供适于生物分子多重检测的成像系统，所述成像系统至少包括第一光学路径和第二光学路径；所述第一光学路径为从激发光源至物镜，且所述激发光源和所述物镜之间设置有滤光片组，所述滤光片组被配置和布置为将从所述激发光源发出的激发光引导至所述物镜；所述第二光学路径为从所述物镜至图像采集装置，且所述物镜和所述图像采集装置之间设置有反光镜，所述反光镜被配置和布置为将由所述物镜接收到的荧光投射到所述图像采集装置；所述激发光源至少包括第一激发光源和第二激发光源，所述第一激发光源用于荧光信号的激发，所述第二激发光源用于光激化学发光信号的激发。
本发明第三方面提供前述适于生物分子多重检测的成像系统，在生物分子多重检测领域的应用。
本发明第四方面提供一种生物分子多重检测的成像方法，采用前述的适于生物分子多重检测的成像系统进行，至少包括如下步骤：
1)打开所述成像系统的第一激发光源，使激发光源的发出的光线通过滤光片组后，通过物镜到达样本并成像，获得样本的荧光图像；
2)切换至第二激发光源，使激发光源的发出的光线通过滤光片组后，通过物镜到达样本并成像，获得样本的光激化学发光图像。
本发明第五方面提供一种生物分子多重检测的图像分析方法，包括如下步骤：
S1根据光斑位置将检测液的荧光图像和光激化学发光图像进行匹配，以获得各光斑对应的荧光信号及光激化学发光信号，所述检测液的荧光图像和光激化学发光图像采用前述的生物分子多重检测的成像方法获得；
S2对荧光信号进行聚类分类，并获得各荧光信号对应的平均光激化学发光强度；
S3根据荧光信号的分类不同和平均光激化学发光强度，确定检测液中各待测分子的含量。
本发明第六方面提供一种计算机可读存储介质，其上存储有计算机程序，该程序被处理器执行时实现前述的生物分子多重检测的图像分析方法的步骤。
如上所述，本发明的适于生物分子多重检测的成像系统，具有以下有益效果：
本发明首次将荧光成像和光激化学发光成像结合，可同时对荧光信号和光激化学发光信号进行检测，适用于多重光激化学发光检测。
附图说明
图1显示为本发明的适于生物分子多重检测的成像系统的示意图，图中箭头表示光线的传播方向。
图2显示为本发明的适于生物分子多重检测的成像系统用于多重光激化学发光得到图像。
图3显示为本发明的适于生物分子多重检测的成像系统用于多重光激化学发光图像分析的流程图。
图4显示为本发明的适于生物分子多重检测的成像系统图像分析软件用于多重光激化学发光图像的聚类分析结果，该结果显示了四种检测颗粒的荧光分类和相应的光激化学发光强度。
图5显示为本发明的成像系统的通讯关系图。
元件标号说明
1                      激发光源
11                     第一激发光源
12                     第二激发光源
13                     滤光片组
14                     样本台
15                     物镜
16                     反光镜
17                     光源切换开关
2                      图像采集装置
3                      工作站
31                     控制中心
32                     触发控制盒
具体实施方式
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效。
请参阅图1至图5。须知，本说明书所附图式所绘示的结构、比例、大小等，均仅用以配合说明书所揭示的内容，以供熟悉此技术的人士了解与阅读，并非用以限定本发明可实施的限定条件，故不具技术上的实质意义，任何结构的修饰、比例关系的改变或大小的调整，在不影响本发明所能产生的功效及所能达成的目的下，均应仍落在本发明所揭示的技术内容得能涵盖的范围内。同时，本说明书中所引用的如“上”、“下”、“左”、“右”、“中间”及“一”等的用语，亦仅为便于叙述的明了，而非用以限定本发明可实施的范围，其相对关系的改变或调整，在无实质变更技术内容下，当亦视为本发明可实施的范畴。
本发明一实施例的适于生物分子多重检测的成像系统，所述成像系统能够同时实现荧光和光激化学发光成像。
如图1所示，本发明的一实施例中的一种适于生物分子多重检测的成像系统，所述成像系统至少包括第一光学路径和第二光学路径；所述第一光学路径为从激发光源1至物镜15，且所述激发光源和所述物镜之间设置有滤光片组13，所述滤光片组13被配置和布置为将从所述激发光源发出的激发光引导至所述物镜；所述第二光学路径为从所述物镜15至图像采集装置2，且所述物镜15和所述图像采集装置2之间设置有反光镜16，所述反光镜16被配置和布置为将由所述物镜15接收到的荧光投射到所述图像采集装置2；所述激发光源1至少包括第一激发光源11和第二激发光源12，所述第一激发光源11用于荧光信号的激发，所述第二激发光源12用于光激化学发光信号的激发。
所述成像系统能够同时实现荧光和光激化学发光成像。
所述成像系统还包括样本台14所述样本台设于物镜上方，用于放置被观察对象。
如图5所示，所述成像系统还包括工作站3和光源切换开关17，所述工作站与所述图像 采集装置2通讯连接，所述光源切换开关17与所述激发光源1和所述工作站3通讯连接。
所述通讯连接是指通过信号的传输交互，在连接的设备之间构成通讯。所述通讯连接包括有线连接和无线连接，例如可以为电讯号传输连接，数字讯号传输连接等。
所述光源切换开关17能够切换激发光源1。
如图5所示，所述工作站包括相互通讯连接的控制中心31和触发控制盒32，所述触发控制盒32与所述光源切换开关17、激发光源1以及图像采集装置2通讯连接，所述控制中心31与所述图像采集装置2通讯连接。
所述控制中心31可以为计算机，所述计算机可用于搭载图像处理的程序，用于将带有编码功能的微载体信号激发后获得的图像与检测微粒信号激发后获得的图像进行匹配和信号分析。所述触发控制盒还可以搭载触发控制盒控制程序，用于控制触发控制盒。
所述控制中心用于控制触发控制盒32，所述触发控制盒用于控制光源切换开关17、激发光源1的开闭以及图像采集装置2。
控制中心31可以通过触发控制盒32直接控制激发光源1的开闭，也可以通过触发控制盒32，控制图像采集装置，从而实现激发光源的开闭。具体的，可以启动图像采集装置2，使图像采集装置产生TTL(晶体管-晶体管逻辑集成电路)信号并发送至触发控制盒，触发控制盒收到信号后立即发送TTL控制信号给激发光源1，进而能够控制激发光源1的开启。所述控制中心可通过控制光源切换开关的开闭实现第一激发光源11和第二激发光源12的切换。
可选的，所述成像系统包括普通荧光成像模式和时间分辨荧光模式，所述普通荧光成像模式是指，激发光源发光与图像采集装置曝光同步；所述时间分辨荧光模式是指，所述激发光源与发光与图像采集装置曝光不同步。
在一种实施方式中，可通过控制中心控制触发控制盒来实现两种荧光模式的切换。
具体的，普通荧光成像模式为图像采集装置2开始曝光的同时激发光源打开，此时，可以设置激发光源的激发时间和相机曝光时间相同，启动图像采集装置2，使图像采集装置产生TTL(晶体管-晶体管逻辑集成电路)信号并发送至触发控制盒，触发控制盒收到信号后立即发送TTL控制信号给激发光源1，启动激发光源1，此时相机开始同步曝光，实现光源发光与图像采集装置曝光同步。
时间分辨荧光成像模式为，控制中心通过触发控制盒控制激发光源打开，此时图像采集装置2曝光关闭。一段时间后激发光源关闭，此时图像采集装置2曝光再打开。此时，启动图像采集装置2，使图像采集装置产生TTL(晶体管-晶体管逻辑集成电路)信号并发送至触发控制盒，触发控制盒收到信号后，立即发送TTL控制信号给激发光源1，启动激发光源1，此 时相机还未开始曝光；当设定的时间到达，激发光源关闭，此时相机再开始曝光，获得图像。这样可以进一步降低由激发光产生的背景噪音。时间分辨荧光功能可通过控制中心31上搭载的程序控制触发控制盒，进而实现时间分辨功能的打开或关闭。
所述触发控制盒可以是常规的触发控制盒，例如FluoCa的VV-TRGBOX触发控制盒。
所述第一激发光源11可以是激光器、氙灯、汞灯、卤素灯或发光二极管中的一种或多种。
所述第二激发光源12可以是激光器、氙灯、汞灯、卤素灯或发光二极管中的一种或多种。
所述图像采集装置2可以为相机。
所述图像采集装置2选自CCD、EMCCD、CMOS或sCMOS中的一种或多种。
所述滤光片组(13)至少包括第一滤光片组、第二滤光片组和第三滤光片组，各滤光片组均包括激发滤光片、发射滤光片和二向色镜。
所述滤光片组13可拆卸。可根据不同激发和发射波长的荧光物质进行更换，或者根据不同光激化学发光微粒中填充的光敏剂种类和化学发光剂种类进行更换。
所述滤光片组的数量可根据荧光物质的种类数量进行调整。
在一种实施方式中，第一滤光片组为：激发滤光片488/15nm，二向色镜495nm长通，发射滤光片535/23nm.。
在一种实施方式中，第二滤光片组为：激发滤光片488/15nm，二向色镜495nm长通，发射滤光片600/40nm。
在一种实施方式中，第三滤光片组为：激发滤光片680/13nm，二向色镜653nm短通，发射滤光片615/20nm。
所述滤光片组中，激发滤光片、二向色镜、发射滤光片依次排列。
在一种实施方式中，所述二象色镜与激发光源产生的激发光的传播方向的夹角为45°。
所述滤光片组中激发光源产生的激发光首先透过激发滤光片，再到达二向色镜表面,根据二向色镜本身的性质，在495nm以下波长的光被反射，495nm以上的光透射，故入射的激发光到达二向色镜后反射至物镜，而后传播至样本。样本被激发出的光信号经物镜后返回二向色镜，并透过二向色镜，而后透过发射滤光片，到达反光镜，被反光镜反射至图像采集装置。
所述成像系统可实现荧光成像功能和光激化学发光成像功能。①荧光成像功能：通过荧光照明光源经过第一滤光片组(激发滤光片488/15nm，二向色镜495nm长通，发射滤光片535/23nm)或第二滤光片组(激发滤光片488/15nm，二向色镜495nm长通，发射滤光片600/40nm)和物镜激发样本，样本产生的荧光信号再次通过物镜和第一滤光片组或第二滤光片组，最终被相机采集，产生两张荧光图像。②光激化学发光成像功能：由光激化学发光光 源经过第三滤光片组(激发滤光片680/13nm，二向色镜653nm短通，发射滤光片615/20nm)和物镜激发样本，产生的光激化学发光信号再次经过物镜和第三滤光片组，最终被相机采集，产生光激化学发光图像。
本发明所述工作台的控制中心可以根据需要安装不同的图像处理程序、触发控制盒控制程序。所述触发控制盒的控制程序只要知晓本发明的两种模式，本领域技术人员很容易即可实现。
所述成像系统可通过的滤光片转盘切换三个滤光片组中的任意一个进入至成像光路。
所述成像系统有两种成像模式，第一种为光激化学发光信号激发采用普通荧光模式，具体包括以下工作流程：
步骤1：将样本置于样本台，对样本中的待测物进行对焦。
步骤2：切换第一滤光片组进入成像光路，通过光源切换模块切换至荧光激发光源。关闭时间分辨荧光功能，设置相机曝光时间为200ms进行拍照，得到荧光图像1，如图2所示。
步骤3：切换第二滤光片组进入成像光路，其他条件与步骤2相同，拍照得到荧光图像2，如图2所示。
步骤4：切换第三滤光片组进入成像光路，通过光源切换模块切换至光激化学发光激发光源，关闭时间分辨荧光功能，设置光源激发时间为1000ms，相机曝光时间为1000ms。通过相机控制触发光源开关打开，此时相机同步开始曝光，1000ms后曝光结束，光源关闭，得到光激化学发光图像，如图2所示。
第二种为光激化学发光信号激发采用时间分辨荧光模式具体包括以下工作流程：
步骤1：将样本置于样本台，通过调节物镜和样本台间的距离的调焦旋钮对样本中的待测物进行对焦。
步骤2：切换第一滤光片组进入成像光路，通过光源切换模块切换至荧光激发光源。关闭时间分辨荧光功能，设置相机曝光时间为200ms进行拍照，得到荧光图像1。
步骤3：切换第二滤光片组进入成像光路，其他条件与步骤2相同，拍照得到荧光图像2。
步骤4：切换第三滤光片组进入成像光路，通过光源切换模块切换至光激化学发光激发光源，打开时间分辨荧光功能，设置光源激发时间为500ms，相机曝光时间为1000ms。通过相机控制触发光源开关打开，此时相机曝光关闭；500ms后光源关闭，此时相机开始曝光，1000ms后曝光结束，得到光激化学发光图像。
本发明一实施例的生物分子多重检测的成像方法，采用前述的适于生物分子多重检测的成像系统进行，至少包括如下步骤：
1)打开所述成像系统的第一激发光源，使激发光源的发出的光线通过滤光片组后，通过物镜到达样本并成像，获得样本的荧光图像；
2)切换至第二激发光源，使激发光源的发出的光线通过滤光片组后，通过物镜到达样本并成像，获得样本的光激化学发光图像。
进一步的，所述成像方法还包括以下步骤：改变滤光片组的参数，重复步骤1)，获得样本的多种荧光图像。
所述样本的荧光图像和光激化学发光图像通过图像采集装置采集。
在一种实施方式中，所述成像方法可以采用普通荧光模式或时间分辨荧光模式，其中，当采用时间分辨荧光模式时，步骤2)中，将成像系统至光激化学发光激发光源后，打开时间分辨荧光模式，获得样本的光激化学发光图像。具体的，可以参照前述的两种模式的工作流程。
采用以上方法获得的图像可以在本发明所述成像系统的控制中心31进行分析处理。
本发明一实施例的生物分子多重检测的图像分析方法，包括如下步骤：
S1根据光斑位置将检测液的荧光图像和光激化学发光图像进行匹配，以获得各光斑对应的荧光信号及光激化学发光信号，所述检测液的荧光图像和光激化学发光图像采用前述的生物分子多重检测的成像方法获得；
S2对荧光信号进行聚类分类，并获得各荧光信号对应的平均光激化学发光强度；
S3根据荧光信号的分类不同和平均光激化学发光强度，确定检测液中各待测分子的含量。
适用于上述分析方法的检测液为多重光激化学检测反应液。
多重光激化学检测反应液为均相反应液，其反应物一般包括适于生物分子多重检测的检测颗粒、待测分子、和匹配微粒。待测分子与对应的检测颗粒及匹配微粒能形成复合物，复合物在激光照射下可发出光信号。
多重光激化学检测反应液可在单个均相反应液中同时检测多个待测分子。
待测分子可以是蛋白、小分子抗原或核酸。
适于生物分子多重检测的检测颗粒，包括带有编码功能的微载体，微载体连接有检测微粒。
所述带有编码功能的微载体可选自荧光编码微载体、拉曼信号编码微载体、光子晶体编码微载体或图案编码微载体中的一种或多种。
检测微粒应适用于进行光激化学发光检测。检测微粒应当包括适用于光激化学发光检测的供体微粒(DB)或受体微粒(AB)。其中，受体微粒能够发出荧光，供体微粒用于激发受体微粒发光。DB内掺杂光敏剂，受光激发后能产生单线态氧。AB内掺杂化学发光剂和荧光 剂，化学发光剂将单线态氧的能量转换成360nm发射光，激发荧光剂产生荧光。其中化学发光剂选自二氧杂环乙烯或二甲基噻吩中的一种或多种。
检测微粒一般还应包括特异性的生物捕获物质，生物捕获物质包括但不限于生物捕获探针、抗原、抗体、ProtinA、ProtinG或链霉亲和素中的一种或多种。生物捕获物质可与供体微粒或受体微粒偶联。生物捕获物质可特异结合待测分子。
匹配微粒为与检测颗粒中的检测微粒匹配的光激化学发光微粒。一般情况下，当检测微粒包括供体微粒时，匹配微粒应包括受体微粒；当检测微粒包括受体微粒时，匹配微粒应包供体微粒。
匹配微粒一般还应包括检测物质，检测物质包括但不限于探针、抗原、抗体、ProtinA、ProtinG或链霉亲和素中的一种或多种。检测物质可与供体微粒或受体微粒偶联。检测物质不应直接结合检测颗粒，但可经待测分子与检测颗粒形成复合物。因此，检测物质应可特异结合待测分子，或者检测物质可经一中间结合物特异结合待测分子。
当待测分子存在时，待测分子与对应的检测颗粒及匹配微粒形成复合物，在受到激光照射后，复合物中的DB能激活周围环境中的氧转化为单线态氧，其生存时间仅为4微秒，短暂的生存时间决定了离子氧的传播直径很小(约为200nm)，由于复合物中AB与DB间距小于200nm，AB接受DB产生的单线态氧而被激发出光信号。而当没有待测分子存在时，AB与DB间距较远而无法激发出AB的光信号。
多重光激化学检测反应液中应包括多种适于生物分子多重检测的检测颗粒，基于检测颗粒的编码不同，可发出不同荧光以区分不同的待测分子检测结果。和/或，也可基于待测分子与对应的检测颗粒及匹配微粒形成复合物的光激化学荧光差异以区分不同的待测分子检测结果。
由于检测液中，各检测颗粒的位置相对静止，荧光图像的光斑来源于检测颗粒的编码物质和/或检测颗粒反应复合物，光激化学发光图像的光斑则来源于检测颗粒反应复合物，因此可以将荧光图像与光激化学发光图像中相同位置的光斑进行匹配，从而获得该位置检测颗粒或检测颗粒反应复合物对应的荧光信号和光激化学发光信号。进一步地通过对荧光信号聚类分类，获得各类荧光信号对应的平均光激化学发光强度。由于不同待测分子对应不同的荧光信号，因此可依据待测分子、荧光信号类别、与荧光信号对应地平均光激化学发光强度获知各待测分子对应的平均光激化学发光强度，进一步根据光激化学发光强度与浓度的对应关系获得各待测分子的含量。
步骤S1中，可通过全局阈值化识别出光斑在图像中的位置，并根据光斑位置将检测液的 荧光图像和光激化学发光图像进行匹配。
可采集各种光信号参数表征荧光信号及光激化学发光信号，例如光斑的灰度值或者RGB值等。
在一优选的实施方式中，在荧光图像和光激化学发光图像进行匹配前进行降噪，将荧光信号或光激化学发光信号和背景信号区分开。
步骤S2中，可依据光信号表征值的不同对荧光信号进行聚类分类。可采用常规统计方法计算获得各荧光信号对应的平均光激化学发光强度。
步骤S3中，具体的可包括下列步骤：
依据待测分子与荧光信号类别的对应关系获得各待测分子对应的平均光激化学发光强度；
依据各待测分子光激化学发光标准曲线及平均光激化学发光强度获得各待测分子的浓度。
进一步的，本发明实施例还提供一种计算机可读存储介质，其上存储有计算机程序，该程序被处理器执行时实现前述方法的步骤。
本领域普通技术人员可以理解：实现上述各方法实施例的全部或部分步骤可以通过计算机程序相关的硬件来完成。前述的计算机程序可以存储于一计算机可读存储介质中。该程序在执行时，执行包括上述各方法实施例的步骤；所述计算机可读存储介质可包括，但不限于，软盘、光盘、CD-ROM(只读光盘存储器)、磁光盘、ROM(只读存储器)、RAM(随机存取存储器)、EPROM(可擦除可编程只读存储器)、EEPROM(电可擦除可编程只读存储器)、磁卡或光卡、闪存、或适于存储机器可执行指令的其他类型的介质/机器可读介质。所述计算机可读存储介质可以是未接入计算机设备的产品，也可以是已接入计算机设备使用的部件。
进一步的，本发明实施例中的控制中心31可以搭载图像分析软件以实现上述分析方法。
在一个具体实施方式中，软件分析图像的流程包含图像预处理、共定位、灰度分析、聚类分析四个步骤。①图像预处理：对得到的荧光图像和光激化学发光图像进行降噪，将荧光信号或光激化学发光信号和背景信号区分开，并通过全局阈值化识别出信号在图像中的位置。②共定位：对荧光信号和光激化学发光信号在图像中的位置进行统一。③灰度分析：分别读取荧光信号和光激化学发光信号的灰度值。④聚类分析：对图像中不同位置的荧光信号依据灰度值的强弱进行聚类，得出不同分类，以此作为不同待测分子的类别。然后对不同荧光分类所在的光激化学发光信号的灰度值进行统计，以此得出不同类别待测分子的含量(浓度)水平。
如图3所示，多重光激化学发光图像分析的流程：
步骤1：图像输入。软件读取前述中得到的荧光图像和光激化学发光图像。
步骤2：图像预处理。对得到的进行降噪，将荧光信号或光激化学发光信号和背景信号区分开，并通过全局阈值化识别出信号在图像中的位置。
步骤3：共定位：对荧光信号和光激化学发光信号在图像中的位置进行统一。
步骤4：灰度分析：分别读取荧光信号和光激化学发光信号的灰度值。
步骤5：聚类分析：对图像中不同位置的荧光信号依据灰度值的强弱进行聚类，得出不同分类。然后对不同荧光分类所在的光激化学发光信号的灰度值进行统计，得出平均光激化学发光强度。
步骤6：数据输出：将步骤5中得到的不同荧光分类与不同待测物的种类一一对应。再将不同荧光分类所在的平均光激化学发光强度代入相应待测物的标准曲线中，得到该待测物的实际含量(浓度)。
图像分析软件用于多重光激化学发光图像的聚类分析结果如图4所示，该结果显示了四种检测颗粒的荧光分类和相应的光激化学发光强度。
前述适于生物分子多重检测的系统可以用于在生物分子多重检测领域的应用。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (13)

  1. 一种适于生物分子多重检测的成像系统，其特征在于，所述成像系统能够同时实现荧光和光激化学发光成像。
  2. 一种适于生物分子多重检测的成像系统，其特征在于，所述成像系统至少包括第一光学路径和第二光学路径；所述第一光学路径为从激发光源(1)至物镜(15)，且所述激发光源和所述物镜之间设置有滤光片组(13)，所述滤光片组(13)被配置和布置为将从所述激发光源发出的激发光引导至所述物镜；所述第二光学路径为从所述物镜(15)至图像采集装置(2)，且所述物镜(15)和所述图像采集装置(2)之间设置有反光镜(16)，所述反光镜(16)被配置和布置为将由所述物镜(15)接收到的荧光投射到所述图像采集装置(2)；所述激发光源(1)至少包括第一激发光源(11)和第二激发光源(12)，所述第一激发光源(11)用于荧光信号的激发，所述第二激发光源(12)用于光激化学发光信号的激发。
  3. 如权利要求2所述的适于生物分子多重检测的成像系统，其特征在于，所述成像系统还包括工作站(3)和光源切换开关(17)，所述工作站与所述图像采集装置(2)通讯连接，所述光源切换开关(17)与所述激发光源(1)和所述工作站(3)通讯连接。
  4. 如权利要求3所述的适于生物分子多重检测的成像系统，其特征在于，所述工作站包括控制中心(31)和触发控制盒(32)，所述触发控制盒(32)与所述激发光源(1)、所述光源切换开关(17)以及图像采集装置(2)通讯连接，所述控制中心(31)与所述图像采集装置(2)以及触发控制盒(32)通讯连接。
  5. 如权利要求2或4所述所述的适于生物分子多重检测的成像系统，其特征在于，所述成像系统包括普通荧光成像模式和时间分辨荧光模式，所述普通荧光成像模式是指，激发光源发光与图像采集装置曝光同步；所述时间分辨荧光模式是指，所述激发光源发光与图像采集装置曝光不同步。
  6. 如权利要求2所述的适于生物分子多重检测的成像系统，其特征在于，还包括以下特征的一项或多项：
    a.所述第一激发光源(11)或第二激发光源(12)选自激光器、氙灯、汞灯、卤素灯或发光二极管中的一种或多种；
    b.所述图像采集装置(2)选自CCD、EMCCD、CMOS或sCMOS中的一种或多种；
    c.所述滤光片组(13)至少包括第一滤光片组、第二滤光片组和第三滤光片组，各滤光片组均包括激发滤光片、发射滤光片和二向色镜；
    d.所述滤光片组(13)可拆卸。
  7. 如权利要求6所述的适于生物分子多重检测的成像系统，其特征在于，还包括以下特征中的一项或多项：
    (1)第一滤光片组为：激发滤光片488/15nm，二向色镜495nm长通，发射滤光片535/23nm；
    (2)第二滤光片组为：激发滤光片488/15nm，二向色镜495nm长通，发射滤光片600/40nm；
    (3)第三滤光片组为：激发滤光片680/13nm，二向色镜653nm短通，发射滤光片615/20nm。
  8. 如权利要求1-7任一所述的适于生物分子多重检测的成像系统，在生物分子多重检测领域的应用。
  9. 一种生物分子多重检测的成像方法，采用权利要求1-7任一所述的适于生物分子多重检测的成像系统进行，至少包括如下步骤：
    1)打开所述成像系统的第一激发光源，使激发光源的发出的光线通过滤光片组后，通过物镜到达样本并成像，获得样本的荧光图像；
    2)切换至第二激发光源，使激发光源的发出的光线通过滤光片组后，通过物镜到达样本并成像，获得样本的光激化学发光图像。
  10. 如权利要求9所述的生物分子多重检测的成像方法，其特征在于，所述成像方法还包括以下步骤：改变滤光片组的参数，重复步骤1)，获得样本的多种荧光图像。
  11. 如权利要求9所述的生物分子多重检测的成像方法，其特征在于，步骤2)中，切换至光激化学发光激发光源后，打开时间分辨荧光模式，获得样本的光激化学发光图像。
  12. 一种生物分子多重检测的图像分析方法，包括如下步骤：
    S1根据光斑位置将检测液的荧光图像和光激化学发光图像进行匹配，以获得各光斑对应的荧光信号及光激化学发光信号，所述检测液的荧光图像和光激化学发光图像采用权利要求9-11的生物分子多重检测的成像方法获得；
    S2对荧光信号进行聚类分类，并获得各荧光信号对应的平均光激化学发光强度；
    S3根据荧光信号的分类不同和平均光激化学发光强度，确定检测液中各待测分子的含量。
  13. 一种计算机可读存储介质，其上存储有计算机程序，该程序被处理器执行时实现权利要求12所述的生物分子多重检测的图像分析方法的步骤。
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