An imaging system suitable for multiplex detection of biomolecules

Abstract

The present invention provides an imaging system suitable for multiplex detection of biomolecules, the imaging system comprising 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 a filter group is provided between the excitation light source and the objective lens, 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 objective lens to an image acquisition device, and a reflector is provided between the objective lens and the image acquisition device, the reflector is configured and arranged to project the fluorescence received by the objective lens to the image acquisition device; the excitation light source comprises at least a first excitation light source and a second excitation light source, the first excitation light source is used for excitation of a fluorescence signal, and the second excitation light source is used for excitation of a photochemiluminescence signal. The imaging system can simultaneously realize fluorescence and photochemiluminescence imaging.

Description

Technical Field

[0001] The invention relates to the field of biological detection, and in particular to an imaging system suitable for multiple detection of biological molecules. Background Art

[0002] At present, photoluminescence detection instruments, such as Siemens Dimension EXL analyzer, PerkinElmer Both Multimode Plate Reader and Beijing Kemei's LiCA500 fully automatic chemiluminescence analyzer can only detect the unified signal in the entire homogeneous solution, and cannot detect the signal of a single detection particle, so they cannot realize the function of multiple detection. Summary of the invention

[0003] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide an imaging system suitable for multiplex detection of biomolecules.

[0004] A first aspect of the present invention provides an imaging system suitable for multiplex detection of biomolecules, wherein the imaging system can simultaneously realize fluorescence and photoinduced chemiluminescence imaging.

[0005] A second aspect of the present invention provides an imaging system suitable for multiplex detection of biological molecules, the imaging system comprising 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 a filter group is provided between the excitation light source and the objective lens, 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 objective lens to an image acquisition device, and a reflector is provided between the objective lens and the image acquisition device, the reflector is configured and arranged to project the fluorescence received by the objective lens to the image acquisition device; the excitation light source comprises at least a first excitation light source and a second excitation light source, the first excitation light source is used for excitation of a fluorescence signal, and the second excitation light source is used for excitation of a photoinduced chemiluminescence signal.

[0006] The third aspect of the present invention provides the aforementioned imaging system suitable for multiplex detection of biomolecules, and its application in the field of multiplex detection of biomolecules.

[0007] A fourth aspect of the present invention provides an imaging method for multiplex detection of biomolecules, which is performed using the aforementioned imaging system suitable for multiplex detection of biomolecules, and comprises at least the following steps:

[0008] 1) turning on the first excitation light source of the imaging system, so that the light emitted by the excitation light source passes through the filter set and then reaches the sample through the objective lens and forms an image, thereby obtaining a fluorescent image of the sample;

[0009] 2) Switch to the second excitation light source, so that the light emitted by the excitation light source passes through the filter set and then reaches the sample through the objective lens and forms an image, thereby obtaining a photochemiluminescence image of the sample.

[0010] A fifth aspect of the present invention provides an image analysis method for multiplex detection of biomolecules, comprising the following steps:

[0011] S1 matches the fluorescence image and the photochemiluminescence image of the detection liquid according to the position of the light spot to obtain the fluorescence signal and the photochemiluminescence signal corresponding to each light spot, wherein the fluorescence image and the photochemiluminescence image of the detection liquid are obtained by the aforementioned imaging method for multiple detection of biomolecules;

[0012] S2 clusters and classifies the fluorescence signals and obtains the average photoluminescence intensity corresponding to each fluorescence signal;

[0013] S3 determines the content of each molecule to be tested in the test solution according to the different classifications of the fluorescence signals and the average photochemiluminescence intensity.

[0014] A sixth aspect of the present invention provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the steps of the aforementioned image analysis method for multiplex detection of biological molecules.

[0015] As described above, the imaging system suitable for multiplex detection of biomolecules of the present invention has the following beneficial effects:

[0016] The present invention combines fluorescence imaging and photochemiluminescence imaging for the first time, and can detect fluorescence signals and photochemiluminescence signals at the same time, and is suitable for multiple photochemiluminescence detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of an imaging system suitable for multiplex detection of biomolecules according to the present invention, wherein arrows indicate the propagation direction of light.

[0018] Figure 2 The imaging system suitable for multiplex detection of biomolecules according to the present invention is shown to be used for multiplex photochemiluminescence to obtain images.

[0019] Figure 3 The flowchart is shown of the imaging system suitable for multiplex detection of biomolecules of the present invention for multiplex photochemiluminescence image analysis.

[0020] Figure 4 Shown is the cluster analysis result of multiple photochemiluminescence images using the image analysis software of the imaging system suitable for multiplex detection of biomolecules of the present invention, which shows the fluorescence classification of four detection particles and the corresponding photochemiluminescence intensity.

[0021] Figure 5 Shown is a communication relationship diagram of the imaging system of the present invention.

[0022] Component number description

[0023] 1 Excitation light source

[0024] 11. First excitation light source

[0025] 12. Second excitation light source

[0026] 13 filter sets

[0027] 14 Sample table

[0028] 15 Objective lens

[0029] 16 Reflector

[0030] 17 Light source switch

[0031] 2 Image acquisition device

[0032] 3 Workstations

[0033] 31 Control Center

[0034] 32 Trigger control box DETAILED DESCRIPTION

[0035] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0036] See also Figures 1 to 5 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0037] An imaging system suitable for multiplex detection of biological molecules according to an embodiment of the present invention can simultaneously realize fluorescence and photoinduced chemiluminescence imaging.

[0038] like Figure 1As shown, in one embodiment of the present invention, an imaging system suitable for multiplex detection of biological molecules, the imaging system comprises at least a first optical path and a second optical path; the first optical path is from an excitation light source 1 to an 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 an 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 comprises at least a first excitation light source 11 and a second excitation light source 12, the first excitation light source 11 is used for excitation of fluorescence signals, and the second excitation light source 12 is used for excitation of photoinduced chemiluminescence signals.

[0039] The imaging system can realize fluorescence and photoinduced chemiluminescence imaging simultaneously.

[0040] The imaging system further comprises a sample stage 14 which is arranged above the objective lens and is used for placing an observed object.

[0041] like Figure 5 As shown, the imaging system further includes a workstation 3 and a light source switching switch 17 . The workstation is communicatively connected to the image acquisition device 2 , and the light source switching switch 17 is communicatively connected to the excitation light source 1 and the workstation 3 .

[0042] The communication connection refers to the communication between connected devices through the transmission interaction of signals. The communication connection includes wired connection and wireless connection, for example, it can be a telecommunication signal transmission connection, a digital signal transmission connection, etc.

[0043] The light source switching switch 17 can switch the excitation light source 1 .

[0044] like Figure 5 As shown, the workstation includes a control center 31 and a trigger control box 32 which are communicatively connected to each other. The trigger control box 32 is communicatively connected to the light source switching switch 17, the excitation light source 1 and the image acquisition device 2, and the control center 31 is communicatively connected to the image acquisition device 2.

[0045] The control center 31 can be a computer, which can be used to carry an image processing program to match and analyze the image obtained after the microcarrier signal with the coding function is excited with the image obtained after the detection particle signal is excited. The trigger control box can also carry a trigger control box control program for controlling the trigger control box.

[0046] The control center is used to control the trigger control box 32 , and the trigger control box is used to control the light source switching switch 17 , the opening and closing of the excitation light source 1 , and the image acquisition device 2 .

[0047] The control center 31 can directly control the opening and closing of the excitation light source 1 through the trigger control box 32, and can also control the image acquisition device through the trigger control box 32, so as to realize the opening and closing of the excitation light source. Specifically, the image acquisition device 2 can be started to generate a TTL (transistor-transistor logic integrated circuit) signal and send it to the trigger control box. After receiving the signal, the trigger control box immediately sends a TTL control signal to the excitation light source 1, thereby being able to control the opening of the excitation light source 1. The control center can realize the switching of 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.

[0048] Optionally, the imaging system includes a common fluorescence imaging mode and a time-resolved fluorescence mode. The common fluorescence imaging mode means that the emission of the excitation light source is synchronized with the exposure of the image acquisition device; the time-resolved fluorescence mode means that the emission of the excitation light source is not synchronized with the exposure of the image acquisition device.

[0049] In one embodiment, the control center can control the trigger control box to switch between the two fluorescence modes.

[0050] Specifically, in the ordinary fluorescence imaging mode, the excitation light source is turned on while the image acquisition device 2 starts to expose. At this time, the excitation time of the excitation light source and the camera exposure time can be set to be the same, and the image acquisition device 2 is started, so that the image acquisition device generates a TTL (transistor-transistor logic integrated circuit) signal and sends it to the trigger control box. After receiving the signal, the trigger control box immediately sends a TTL control signal to the excitation light source 1 to start the excitation light source 1. At this time, the camera starts to expose synchronously, so as to achieve synchronization between the light source emission and the exposure of the image acquisition device.

[0051] 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 exposure of the image acquisition device 2 is turned off. After a period of time, the excitation light source is turned off, and the exposure of the image acquisition device 2 is turned on again. At this time, the image acquisition device 2 is started, so that the image acquisition device generates a TTL (transistor-transistor logic integrated circuit) signal and sends it to the trigger control box. After receiving the signal, the trigger control box immediately sends a TTL control signal to the excitation light source 1 to start the excitation light source 1. At this time, the camera has not started to expose; when the set time is reached, the excitation light source is turned off, and the camera starts to expose again to obtain an image. This can further reduce the background noise generated by the excitation light. The time-resolved fluorescence function can be turned on or off by controlling the trigger control box through the program carried by the control center 31.

[0052] The trigger control box may be a conventional trigger control box, such as the VV-TRGBOX trigger control box of FluoCa.

[0053] 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.

[0054] 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.

[0055] The image acquisition device 2 may be a camera.

[0056] The image acquisition device 2 is selected from one or more of CCD, EMCCD, CMOS or sCMOS.

[0057] The filter group (13) comprises at least a first filter group, a second filter group and a third filter group, and each filter group comprises an excitation filter, an emission filter and a dichroic mirror.

[0058] The filter set 13 is detachable and can be replaced according to the fluorescent substances with different excitation and emission wavelengths, or according to the types of photosensitizers and chemiluminescent agents filled in different photoinduced chemiluminescent particles.

[0059] The number of the filter sets can be adjusted according to the number of types of fluorescent substances.

[0060] In one embodiment, the first filter set is: excitation filter 488 / 15nm, dichroic mirror 495nm long pass, emission filter 535 / 23nm.

[0061] In one embodiment, the second filter set is: excitation filter 488 / 15nm, dichroic mirror 495nm long pass, emission filter 600 / 40nm.

[0062] In one embodiment, the third filter set is: excitation filter 680 / 13nm, dichroic mirror 653nm short pass, emission filter 615 / 20nm.

[0063] In the filter set, the excitation filter, the dichroic mirror and the emission filter are arranged in sequence.

[0064] In one embodiment, the angle between the dichroic mirror and the propagation direction of the excitation light generated by the excitation light source is 45°.

[0065] The excitation light generated by the excitation light source in the filter set first passes through the excitation filter and then reaches the surface of the dichroic mirror. According to the properties of the dichroic mirror itself, light with a wavelength below 495nm is reflected and light above 495nm is transmitted. Therefore, the incident excitation light reaches the dichroic mirror and is reflected to the objective lens, and then propagates to the sample. The light signal excited by the sample returns to the dichroic mirror after passing through the objective lens, passes through the dichroic mirror, and then passes through the emission filter to reach the reflector, which is reflected by the reflector to the image acquisition device.

[0066] The imaging system can realize fluorescence imaging function and photochemiluminescence imaging function. ① Fluorescence imaging function: The sample is excited by the fluorescent illumination light source through the first filter group (excitation filter 488 / 15nm, dichroic mirror 495nm long pass, emission filter 535 / 23nm) or the second filter group (excitation filter 488 / 15nm, dichroic mirror 495nm long pass, emission filter 600 / 40nm) and the objective lens, and the fluorescence signal generated by the sample passes through the objective lens and the first filter group or the second filter group again, and is finally collected by the camera to generate two fluorescence images. ② Photochemiluminescence imaging function: The sample is excited by the photochemiluminescence light source through the third filter group (excitation filter 680 / 13nm, dichroic mirror 653nm short pass, emission filter 615 / 20nm) and the objective lens, and the generated photochemiluminescence signal passes through the objective lens and the third filter group again, and is finally collected by the camera to generate a photochemiluminescence image.

[0067] The control center of the workbench of the present invention can be installed with different image processing programs and trigger control box control programs as needed. 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.

[0068] The imaging system can switch any one of the three filter groups through a filter wheel to enter the imaging light path.

[0069] The imaging system has two imaging modes. The first one is a normal fluorescence mode for photoinduced chemiluminescence signal excitation, which specifically includes the following workflow:

[0070] Step 1: Place the sample on the sample stage and focus on the object to be tested in the sample.

[0071] Step 2: Switch the first filter set 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 obtain the fluorescence image 1, as shown in Figure 2 shown.

[0072] Step 3: Switch the second filter set to enter the imaging light path. Other conditions are the same as step 2. Take a photo to obtain the fluorescent image 2, such as Figure 2shown.

[0073] Step 4: Switch the third filter set to enter the imaging light path, switch to the photochemiluminescence 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 camera control triggers the light source switch 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 photochemiluminescence image, such as Figure 2 shown.

[0074] The second method is to use time-resolved fluorescence mode for photoinduced chemiluminescence signal excitation, which specifically includes the following workflow:

[0075] Step 1: Place the sample on the sample stage and focus on the object to be tested in the sample by adjusting the focusing knob to adjust the distance between the objective lens and the sample stage.

[0076] Step 2: Switch the first filter set to enter 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 obtain fluorescence image 1.

[0077] Step 3: Switch the second filter set to enter the imaging light path. Other conditions are the same as in step 2. Take a photo to obtain fluorescent image 2.

[0078] Step 4: Switch the third filter set to enter the imaging light path, switch to the photochemiluminescence 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 turned on by the camera control trigger, and the camera exposure is turned off at this time; after 500ms, the light source is turned off, and the camera exposure starts at this time, and the exposure ends after 1000ms to obtain the photochemiluminescence image.

[0079] An imaging method for multiplex detection of biomolecules according to an embodiment of the present invention is performed using the aforementioned imaging system suitable for multiplex detection of biomolecules, and comprises at least the following steps:

[0080] 1) turning on the first excitation light source of the imaging system, so that the light emitted by the excitation light source passes through the filter set and then reaches the sample through the objective lens and forms an image, thereby obtaining a fluorescent image of the sample;

[0081] 2) Switch to the second excitation light source, so that the light emitted by the excitation light source passes through the filter set and then reaches the sample through the objective lens and forms an image, thereby obtaining a photochemiluminescence image of the sample.

[0082] Furthermore, the imaging method further comprises the following steps: changing the parameters of the filter set, repeating step 1), and obtaining a plurality of fluorescence images of the sample.

[0083] The fluorescence image and the photoluminescence image of the sample are collected by an image collection device.

[0084] In one embodiment, 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), after the imaging system is connected to the photochemiluminescence excitation light source, the time-resolved fluorescence mode is turned on to obtain a photochemiluminescence image of the sample. Specifically, the workflow of the two modes mentioned above may be referred to.

[0085] The images obtained by the above method can be analyzed and processed in the control center 31 of the imaging system of the present invention.

[0086] An image analysis method for multiplex detection of biomolecules according to an embodiment of the present invention comprises the following steps:

[0087] S1 matches the fluorescence image and the photochemiluminescence image of the detection liquid according to the position of the light spot to obtain the fluorescence signal and the photochemiluminescence signal corresponding to each light spot, wherein the fluorescence image and the photochemiluminescence image of the detection liquid are obtained by the aforementioned imaging method for multiple detection of biomolecules;

[0088] S2 clusters and classifies the fluorescence signals and obtains the average photoluminescence intensity corresponding to each fluorescence signal;

[0089] S3 determines the content of each molecule to be tested in the test solution according to the different classifications of the fluorescence signals and the average photochemiluminescence intensity.

[0090] The detection solution suitable for the above analysis method is a multiplex photochemical detection reaction solution.

[0091] The multiple photochemical detection reaction solution is a homogeneous reaction solution, and its reactants generally include detection particles suitable for multiple detection of biomolecules, molecules to be detected, and matching particles. The molecules to be detected can form a complex with the corresponding detection particles and matching particles, and the complex can emit a light signal under laser irradiation.

[0092] Multiple photochemical detection reaction solutions can simultaneously detect multiple molecules to be detected in a single homogeneous reaction solution.

[0093] The molecule to be detected can be a protein, a small molecule antigen or a nucleic acid.

[0094] The detection particles suitable for multiple detection of biological molecules include microcarriers with coding function, and the microcarriers are connected with detection microparticles.

[0095] The microcarrier with coding function can be selected from one or more of fluorescence coding microcarrier, Raman signal coding microcarrier, photonic crystal coding microcarrier or pattern coding microcarrier.

[0096] 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. Among them, the acceptor particles can emit fluorescence, and the donor particles are used to excite the acceptor particles to emit light. DB is doped with a photosensitizer, which can produce singlet oxygen after being excited by light. AB is doped with a chemiluminescent agent and a fluorescent agent, and the chemiluminescent agent converts the energy of singlet oxygen into 360nm emission light, which excites the fluorescent agent to produce fluorescence. The chemiluminescent agent is selected from one or more of dioxetane or dimethylthiophene.

[0097] The detection particles should generally also include specific biological capture materials, including but not limited to one or more of biological capture probes, antigens, antibodies, ProtinA, ProtinG or streptavidin. The biological capture material can be coupled to donor particles or receptor particles. The biological capture material can specifically bind to the molecule to be detected.

[0098] The matching particles are photoinduced chemiluminescent particles that match the detection particles in the detection particles. Generally, when the detection particles include donor particles, the matching particles should include acceptor particles; when the detection particles include acceptor particles, the matching particles should include donor particles.

[0099] The matching particles should generally also include a detection substance, which includes but is not limited to one or more of a probe, an antigen, an antibody, ProtinA, ProtinG or streptavidin. The detection substance can be coupled to the donor particle or the acceptor particle. The detection substance should not be directly bound 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 molecule to be detected, or the detection substance can specifically bind to the molecule to be detected through an intermediate binding substance.

[0100] When the molecule to be tested exists, it forms a complex with the corresponding detection particles and matching particles. After being irradiated by laser, DB in the complex can activate oxygen in the surrounding environment to convert it into singlet oxygen, which has a survival time of only 4 microseconds. The short survival time determines that the propagation diameter of ion oxygen is very small (about 200nm). Since the distance between AB and DB in the complex is less than 200nm, AB receives the singlet oxygen generated by DB and is stimulated to emit a light signal. When there is no molecule to be tested, the distance between AB and DB is far and the light signal of AB cannot be stimulated.

[0101] 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, different fluorescence can be emitted to distinguish the detection results of different molecules to be detected. And / or, the photochemical fluorescence difference between the complexes formed by the molecules to be detected and the corresponding detection particles and matching particles can also be used to distinguish the detection results of different molecules to be detected.

[0102] Since the position of each detection particle in the detection liquid is relatively static, the light spot of the fluorescence image comes from the coding material of the detection particle and / or the detection particle reaction complex, and the light spot of the photochemiluminescence image comes from the detection particle reaction complex. Therefore, the fluorescence image can be matched with the light spot at the same position in the photochemiluminescence image to obtain the fluorescence signal and photochemiluminescence signal corresponding to the detection particle or the detection particle reaction complex at that position. Further, by clustering and classifying the fluorescence signals, the average photochemiluminescence intensity corresponding to each type of fluorescence signal is obtained. Since different molecules to be tested correspond to different fluorescence signals, the average photochemiluminescence intensity corresponding to each molecule to be tested can be obtained based on the molecule to be tested, the type of fluorescence signal, and the average photochemiluminescence intensity corresponding to the fluorescence signal, and the content of each molecule to be tested can be further obtained based on the corresponding relationship between the photochemiluminescence intensity and the concentration.

[0103] In step S1, the position of the light spot in the image can be identified by global thresholding, and the fluorescence image and the photochemiluminescence image of the detection liquid can be matched according to the position of the light spot.

[0104] Various optical signal parameters can be collected to characterize fluorescence signals and photochemiluminescence signals, such as the grayscale value or RGB value of the light spot.

[0105] In a preferred embodiment, noise reduction is performed before matching the fluorescence image and the photochemiluminescence image to distinguish the fluorescence signal or the photochemiluminescence signal from the background signal.

[0106] In step S2, the fluorescence signals can be clustered and classified according to the different characterization values ​​of the light signals. The average photoinduced chemiluminescence intensity corresponding to each fluorescence signal can be calculated by conventional statistical methods.

[0107] In step S3, the following steps may be specifically included:

[0108] According to the corresponding relationship between the molecules to be tested and the types of fluorescent signals, the average photoinduced chemiluminescence intensity corresponding to each molecule to be tested is obtained;

[0109] The concentration of each molecule to be tested is obtained according to the photochemiluminescence standard curve of each molecule to be tested and the average photochemiluminescence intensity.

[0110] Furthermore, 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 aforementioned method are implemented.

[0111] Those skilled in the art can understand that all or part of the steps for implementing the above-mentioned method embodiments can be completed by hardware related to the computer program. The aforementioned computer program can be stored in a computer-readable storage medium. When the program is executed, the steps of the above-mentioned method embodiments are executed; 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, a ROM (read-only memory), a RAM (random access memory), an EPROM (erasable programmable read-only memory), an EEPROM (electrically erasable programmable read-only memory), a magnetic card or an optical card, a flash memory, or other types of media / machine-readable media suitable for storing machine-executable instructions. The computer-readable storage medium can be a product that is not connected to a computer device, or it can be a component that is connected to a computer device for use.

[0112] Furthermore, the control center 31 in the embodiment of the present invention may be equipped with image analysis software to implement the above analysis method.

[0113] In a specific embodiment, the process of software analysis of images includes four steps: image preprocessing, colocalization, grayscale analysis, and cluster analysis. ① Image preprocessing: reduce the noise of the obtained fluorescence image and photochemiluminescence image, distinguish the fluorescence signal or photochemiluminescence signal from the background signal, and identify the position of the signal in the image through global thresholding. ② Colocalization: unify the position of the fluorescence signal and the photochemiluminescence signal in the image. ③ Grayscale analysis: read the grayscale values ​​of the fluorescence signal and the photochemiluminescence signal respectively. ④ Cluster analysis: cluster the fluorescence signals at different positions in the image according to the strength of the grayscale value, and obtain different classifications, which are used as the categories of different molecules to be tested. Then, the grayscale values ​​of the photochemiluminescence signals in different fluorescence classifications are statistically analyzed to obtain the content (concentration) levels of different categories of molecules to be tested.

[0114] like Figure 3 As shown in the figure, the process of multiplex photochemiluminescence image analysis is as follows:

[0115] Step 1: Image input: The software reads the fluorescence image and photochemiluminescence image obtained above.

[0116] Step 2: Image preprocessing: De-noise the obtained image, separate the fluorescence signal or photoluminescence signal from the background signal, and identify the location of the signal in the image through global thresholding.

[0117] Step 3: Colocalization: Unify the positions of the fluorescence signal and the photoluminescence signal in the image.

[0118] Step 4: Grayscale analysis: read the grayscale values ​​of the fluorescence signal and the photochemiluminescence signal respectively.

[0119] Step 5: Cluster analysis: Cluster the fluorescence signals at different locations in the image according to the strength of the grayscale value to obtain different classifications. Then, the grayscale values ​​of the photochemiluminescence signals in different fluorescence classifications are counted to obtain the average photochemiluminescence intensity.

[0120] Step 6: Data output: The different fluorescence classifications obtained in step 5 are matched one by one with the types of different analytes. Then the average photoluminescence 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.

[0121] The image analysis software was used for cluster analysis of multiple photochemiluminescence images. Figure 4 As shown, the results show the fluorescence classification of the four detected particles and the corresponding photochemiluminescence intensity.

[0122] The aforementioned system suitable for multiplex detection of biomolecules can be used in applications in the field of multiplex detection of biomolecules.

[0123] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. An imaging system suitable for multiplex detection of biomolecules, characterized in that: The imaging system is capable of simultaneously realizing fluorescence and photoinduced chemiluminescence imaging, and the imaging system comprises at least a first optical path and a second optical path; the first optical path is from an excitation light source (1) to an 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) comprises at least a first excitation light source (11) and a second excitation light source (12), the first excitation light source (11) being used for excitation of the fluorescence signal, and the second excitation light source (12) being used for excitation of the photochemiluminescence signal; the imaging system further comprises a workstation (3), the workstation being communicatively connected to the image acquisition device (2); the workstation comprises a control center (31), the control center (31) being communicatively connected to the image acquisition device (2), the control center (31) being equipped with an image processing program for matching and performing signal analysis between the fluorescence image obtained after the microcarrier signal with the coding function is excited and the photochemiluminescence image obtained after the detection particle signal is excited.

2. The imaging system suitable for multiplex detection of biomolecules according to claim 1, characterized in that: The imaging system further comprises a light source switching switch (17), wherein the light source switching switch (17) is communicatively connected to the excitation light source (1) and the workstation (3).

3. The imaging system suitable for multiplex detection of biomolecules according to claim 2, characterized in that: The workstation comprises a trigger control box (32), the trigger control box (32) is communicatively connected to the excitation light source (1), the light source switching switch (17) and the image acquisition device (2), and the control center (31) is communicatively connected to the trigger control box (32).

4. The imaging system suitable for multiplex detection of biomolecules according to claim 1 or 3, characterized in that: The imaging system includes a common fluorescence imaging mode and a time-resolved fluorescence mode. The common fluorescence imaging mode means that the excitation light source emits light synchronously with the image acquisition device; the time-resolved fluorescence mode means that the excitation light source emits light asynchronously with the image acquisition device.

5. The imaging system suitable for multiplex detection of biomolecules according to claim 1, characterized in that: Also includes one or more of the following characteristics: a. the first excitation light source (11) or the second excitation light source (12) is selected from one or more of a laser, a xenon lamp, a mercury lamp, a halogen lamp or a light emitting diode; b. The image acquisition device (2) is selected from one or more of CCD, EMCCD, CMOS or sCMOS; c. The filter set (13) comprises at least a first filter set, a second filter set and a third filter set, each filter set comprising an excitation filter, an emission filter and a dichroic mirror; d. The filter set (13) is detachable.

6. The imaging system suitable for multiplex detection of biomolecules according to claim 5, characterized in that: Also includes one or more of the following characteristics: (1) The first filter set is: excitation filter 488 / 15 nm, dichroic mirror 495 nm long pass, emission filter 535 / 23 nm; (2) The second filter set is: excitation filter 488 / 15 nm, dichroic mirror 495 nm long pass, emission filter 600 / 40 nm; (3) The third filter set is: excitation filter 680 / 13 nm, dichroic mirror 653 nm short pass, emission filter 615 / 20 nm.

7. Use of the imaging system suitable for multiplex detection of biomolecules as claimed in any one of claims 1 to 6 in the field of multiplex detection of biomolecules.

8. An imaging method for multiplex detection of biomolecules, using the imaging system suitable for multiplex detection of biomolecules according to any one of claims 1 to 6, comprising at least the following steps: 1) Turning on the first excitation light source of the imaging system, so that the light emitted by the excitation light source passes through the filter set and then reaches the sample through the objective lens and forms an image, thereby obtaining a fluorescent image of the sample; 2) Switch to the second excitation light source, so that the light emitted by the excitation light source passes through the filter set, reaches the sample through the objective lens and forms an image, thereby obtaining a photochemiluminescence image of the sample.

9. The imaging method for multiplex detection of biomolecules according to claim 8, characterized in that: The imaging method further comprises the following steps: changing the parameters of the filter set, and repeating step 1) to obtain a plurality of fluorescence images of the sample.

10. The imaging method for multiplex detection of biomolecules according to claim 8, characterized in that: In step 2), after switching to the photochemiluminescence excitation light source, the time-resolved fluorescence mode is turned on to obtain the photochemiluminescence image of the sample.

11. An image analysis method for multiplex detection of biomolecules, comprising the following steps: S1 matching the fluorescence image and the photochemiluminescence image of the detection liquid according to the position of the light spot to obtain the fluorescence signal and the photochemiluminescence signal corresponding to each light spot, wherein the fluorescence image and the photochemiluminescence image of the detection liquid are obtained by the imaging method for multiple detection of biomolecules according to any one of claims 8 to 10; S2 clusters and classifies the fluorescence signals and obtains the average photoluminescence intensity corresponding to each fluorescence signal; S3 determines the content of each molecule to be tested in the test solution based on the different classifications of the fluorescence signals and the average photochemiluminescence intensity.

12. A computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the steps of the image analysis method for multiplex detection of biomolecules according to claim 11.

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