Detection device and detection method for multicellular clusters in circulation

Abstract

The invention provides a detection method and a detection device for multicellular clusters in circulation. The detection method specifically comprises the following steps: exciting and receiving fluorescent pulse signals when fluorescent cells flow through a detection area; the exciting light is focused on an interested blood vessel through a microscope objective; when the fluorescently-labeled cells flow through the laser detection part, fluorescence is released; converting into an electric signal through a photoelectric conversion device; the fluorescence pulse signal subsequently enters a signal conversion device; signal conversion is carried out through a comparator; and when the fluorescence trigger signal is transmitted to the imaging device, the lighting equipment is turned on, and the imaging device records an image sequence of the cells flowing through the detection area after receiving the fluorescence trigger signal. According to the detection device and the detection method for the multicellular clusters in circulation, the physiological environment of the human body can be efficiently, quickly and accurately detected, in-vitro operation such as blood sampling and centrifugation is not needed, a detection target does not leave the living body environment, and a detection result can better reflect the real situation in the physiological environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical detection, in particular to a device and method for detecting multiple cell clusters in circulation. BACKGROUND

[0002] Circulating tumor cells (CTC) are tumor cells that fall off from primary or metastatic lesions into the blood circulation system during tumor occurrence and development. The content of CTC in blood is extremely low, usually only a few to tens per milliliter of blood, but its detection is of great significance for early diagnosis, disease monitoring, treatment effect evaluation and prognosis of tumor patients.

[0003] Traditional CTC detection methods usually require blood sampling, consuming a lot of time and manpower. For example, the method of realizing CTC detection through microfluidic chip technology, which utilizes the microchannel structure and special surface modification of microfluidic chip to realize the detection of CTC. Specifically, the microfluidic method introduces blood samples into the channel of the microfluidic chip, uses the microfluidic channel with surface modification to capture and enrich CTC, and then analyzes the captured CTC, which consumes a lot of manpower and material resources. Therefore, developing an efficient CTC detection method has been an urgent demand of clinical and scientific researchers. SUMMARY

[0004] The purpose of the present application is to provide a device and method for detecting multiple cell clusters in circulation, which solves the technical problem of how to efficiently, quickly and accurately detect the physiological environment of the human body. The fluorescence signal of the circulating cell is used as the trigger source of the imaging system capture signal, and the corresponding image sequence of the cell flowing through the detection area is recorded by a high-sensitivity high-speed camera. Then the system analyzes the morphology of the cell in the image sequence to detect the multiple cell clusters in circulation. The present application does not require blood sampling, centrifugation and other in vitro operations, saving manpower and material resources, and the detection target does not leave the biological environment, so the detection result can better reflect the true situation in the physiological environment.

[0005] A method for detecting multiple cell clusters in circulation, specifically comprising the following steps:

[0006] Step S1: exciting and receiving the fluorescence pulse signal of the fluorescent cell flowing through the detection area;

[0007] Step S2: the excitation light emitted by the laser source is focused on the blood vessel of interest by the microscope objective;

[0008] When the fluorescently labeled cell flows through the laser detection site, it will release fluorescence;

[0009] Step S3: the fluorescence is collected by the microscope objective and converted into an electrical signal by the photoelectric conversion device;

[0010] Step S4: the fluorescent pulse signal subsequently enters the signal conversion device to generate a trigger signal for the imaging part;

[0011] Step S5: signal conversion is performed by a comparator, and when the intensity of the received fluorescent signal pulse is greater than a certain threshold value, the signal conversion device generates a rising edge pulse signal for starting the subsequent imaging device;

[0012] Step S6: when the fluorescent trigger signal is transmitted to the imaging device, the illumination device is turned on, and the imaging device records an image sequence of cells flowing through the detection area after receiving the fluorescent trigger signal.

[0013] In the step S1, the laser irradiation and fluorescence receiving device is a living body flow cytometer or a living cell fluorescence detection device based on an optical fiber type.

[0014] In the step S6, the illumination duration T of the illumination device is estimated and adjusted according to the blood vessel flow rate V and the detection area length L.

[0015] The estimation formula of the illumination duration is T = L / V.

[0016] A detection device for multiple cell clusters in circulation, comprising a laser irradiation and fluorescence receiving device for exciting and receiving a fluorescent pulse signal when fluorescent cells flow through a detection area;

[0017] A signal conversion device for converting the fluorescent pulse signal into an effective trigger signal for the imaging part;

[0018] A high-sensitivity high-speed imaging device for recording an image sequence of cells flowing through the detection area after receiving the fluorescent trigger signal;

[0019] The laser irradiation and fluorescence receiving device, the signal conversion device, and the high-sensitivity high-speed imaging device are sequentially connected.

[0020] The laser irradiation and fluorescence receiving device is a living body flow cytometer or a living cell fluorescence detection device based on an optical fiber type.

[0021] The laser irradiation and fluorescence receiving device comprises, in order according to the direction of light propagation, a laser source, a mirror, a cylindrical lens, a mechanical slit, an achromatic lens, a beam splitter, a fluorescence filter, an achromatic lens, a mechanical slit, and a photomultiplier tube.

[0022] The photomultiplier tube is connected with a fluorescence signal recording and viewing device.

[0023] The beam splitter is connected with the objective lens.

[0024] The fluorescence signal recording and viewing device can be an oscilloscope or a PC display. When the laser irradiation and fluorescence receiving device share an objective lens with the high-speed imaging device, the objective lens can be marked as a microscope objective.

[0025] The high-sensitivity high-speed imaging device comprises an illumination device and a microscopic imaging device, the imaging unit of the microscopic imaging device adopts a photomultiplier CCD or an EMCCD, and the illumination device is a laser source or an LED illumination device.

[0026] The illumination device comprises a light source irradiating on a light splitting filter at an included angle, a microscope objective arranged on one side of the light splitting filter, and a light source filter through which the light of the light source passes, and a fluorescence filter and a fluorescence imaging unit arranged on the other side of the light splitting filter.

[0027] The present application achieves the following remarkable effects:

[0028] The present application discloses a device and method for detecting multiple cell clusters in circulation. When the fluorescently labeled cells flow through the laser detection site focused on the blood vessel, corresponding fluorescence signal pulses are generated. The fluorescence signal pulses are used as the trigger source of the imaging part to start the high-sensitivity high-speed camera to record the image sequence when the cells flow through the detection position. The system detects and identifies the multiple cell clusters in circulation by analyzing the cell morphology in the image sequence. The method has the ability of non-invasive dynamic detection of multiple cell clusters in circulation without blood sampling, avoids cumbersome operations such as blood sampling, reduces the burden of manpower and material resources, and reduces the detection cost. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a schematic diagram of the device for detecting multiple cell clusters in circulation in the present application.

[0030] Figure 2 It is an image of a typical multiple cell cluster in the present application.

[0031] Figure 3 It is a schematic diagram of the structure of the laser irradiation and fluorescence receiving device in the present application.

[0032] Figure 4 It is a schematic diagram of the structure of the high-sensitivity high-speed imaging device in the present application.

[0033] Wherein, the reference signs are: 1, laser irradiation and fluorescence receiving device; 2, signal conversion device; 3, high-speed imaging device; 4, blood vessel; 5, laser source; 6, reflecting mirror; 7, cylindrical mirror; 8, mechanical slit one; 9, achromatic lens one; 10, beam splitter; 101, objective lens; 11, fluorescence filter one; 12, achromatic lens two; 13, mechanical slit two; 14, photomultiplier tube; 141, fluorescence signal recording and viewing device; 15, fluorescence filter two; 16, fluorescence imaging unit; 17, light source filter; 18, light source; 19, microscope objective; 20, light splitting filter. DETAILED DESCRIPTION

[0034] In order to more clearly illustrate the technical features of the present scheme, the present scheme will be described below through specific embodiments.

[0035] A method for detecting multiple cell clusters in circulation, specifically comprising the following steps:

[0036] Step S1: exciting and receiving the fluorescence pulse signal when the fluorescent cells flow through the detection area;

[0037] Step S2: the excitation light emitted by the laser source 5 is focused on the blood vessel 4 of interest through the microscope objective 19;

[0038] When the fluorescently labeled cells flow through the laser detection site, fluorescence will be released;

[0039] Step S3: after the fluorescence is collected by the microscope objective 19, it is converted into an electrical signal by the photoelectric conversion device;

[0040] Step S4: the fluorescence pulse signal subsequently enters the signal conversion device 2, generating a trigger signal for the imaging part;

[0041] Step S5: signal conversion is performed by the comparator, and when the received fluorescence signal pulse intensity is greater than a certain threshold value, the signal conversion device 2 generates a rising edge pulse signal for starting the subsequent imaging device;

[0042] Step S6: when the fluorescence trigger signal is transmitted to the imaging device, the illumination device is turned on, and the imaging device records the image sequence of the cells flowing through the detection area after receiving the fluorescence trigger signal.

[0043] In step S1, the fluorescence pulse signal is excited and received by a live flow cytometer or a live cell fluorescence detection device based on an optical fiber type.

[0044] In step S6, the illumination duration T of the illumination device is estimated and adjusted according to the blood vessel 4 flow rate V and the detection area length L;

[0045] The estimation formula of the illumination duration is T=L / V.

[0046] A detection device for multiple cell clusters in circulation, comprising a laser irradiation and fluorescence receiving device 1 for exciting and receiving fluorescence pulse signals of the fluorescent cells flowing through the detection area;

[0047] A signal conversion device 2 for converting the fluorescence pulse signals into effective trigger signals of the imaging part;

[0048] A high-sensitivity high-speed imaging device 3 for recording the image sequence of the cells flowing through the detection area after receiving the fluorescence trigger signals;

[0049] The laser irradiation and fluorescence receiving device 1, the signal conversion device 2 and the high-sensitivity high-speed imaging device 3 are sequentially connected.

[0050] The laser irradiation and fluorescence receiving device 1 is a living flow cytometer or a living cell fluorescence detection device based on an optical fiber type.

[0051] The laser irradiation and fluorescence receiving device 1 comprises a laser source 5, a mirror 6, a cylindrical lens 7, a mechanical slit 8, an achromatic lens 9, a light splitting mirror 10, a fluorescence filter 11, an achromatic lens 12, a mechanical slit 13 and a photomultiplier tube 14 arranged in sequence according to the light path propagation direction.

[0052] The photomultiplier tube 14 is connected with a fluorescence signal recording and viewing device 141.

[0053] The light splitting mirror 10 is connected with an objective lens 101.

[0054] The fluorescence signal recording and viewing device 141 can be an oscilloscope or a PC display. When the laser irradiation and fluorescence receiving device 1 shares the objective lens with the high-speed imaging device 3, the objective lens can be marked as a microscope objective 19.

[0055] The high-sensitivity high-speed imaging device 3 comprises an illumination device and a microscopic imaging device, the imaging unit of the microscopic imaging device adopts a photomultiplier CCD or an EMCCD, and the illumination device is a laser source 5 or an LED illumination device.

[0056] The illumination device comprises a light source 18 irradiating on a light splitting filter 20 at an angle, the microscope objective 19 arranged on one side of the light splitting filter 20, the light of the light source 18 passing through a light source filter 17, and the other side of the light splitting filter 20 being provided with a fluorescence filter 15 and a fluorescence imaging unit 16.

[0057] The specific working process of the present application is as follows:

[0058] The detection device for multiple cell clusters in circulation proposed by the present application is composed of three parts, which are the laser irradiation and fluorescence receiving device 1, the signal conversion device 2 and the high-sensitivity high-speed imaging device 3.

[0059] The main function of the laser irradiation and fluorescence receiving device 1 is to excite and receive the fluorescence pulse signal when the fluorescent cells flow through the detection area. It can be a traditional in vivo flow cytometer or an in vivo cell fluorescence detection device based on an optical fiber type. The excitation light emitted by the laser source 5 is focused on the blood vessel 4 of interest through the microscope objective 19. When the fluorescently labeled cells flow through the laser detection site, they will emit fluorescence. After the fluorescence is collected by the microscope objective 19, it is converted into an electrical signal by a photoelectric conversion device (such as a PMT). The fluorescence electrical signal generated when the cells flow through the laser detection site is similar to a pulse-like signal. This fluorescence pulse signal subsequently enters the signal conversion device 2, generating a trigger signal for the imaging part.

[0060] Specifically, the laser beam emitted by the laser source 5 serves as excitation light, which is reflected to the cylindrical lens 7 by the mirror 6. The laser beam passes through the cylindrical lens 7, the mechanical slit 8, the achromatic lens 9, and reaches the beam splitter 10, which directs the laser beam to the objective 101 Figure 3 The laser beam emitted from the objective 101 is in the form of a line, which illuminates the blood vessel 4 of interest. When fluorescently labeled cells flow through the laser beam irradiation position, they will emit fluorescence. After the fluorescence is collected by the objective 101, it passes through the fluorescence filter 11, the achromatic lens 12, the mechanical slit 13, and the photomultiplier tube 14 in sequence. The photoelectrically converted fluorescence signal is recorded and visualized by the fluorescence signal recording and viewing device 141 Figure 3 The uppermost structure).

[0061] The main function of the signal conversion device 2 is to convert the fluorescence pulse signal into an effective trigger signal for the imaging part. For example, signal conversion is performed by a comparator. When the intensity of the received fluorescence signal pulse is greater than a certain threshold, the signal conversion device 2 generates a rising edge pulse signal to start the subsequent imaging device.

[0062] The high-sensitivity high-speed imaging device 3 functions to record the image sequence of cells flowing through the detection area after receiving the fluorescence trigger signal. This part is composed of an illumination device and a microscopic imaging device. The imaging unit of the microscopic imaging device uses a high-sensitivity high-speed imaging device 3, such as an electron multiplying CCD (EMCCD). The illumination device can be a laser source 5 or an LED illumination device. When the fluorescence trigger signal is transmitted to the imaging device, the illumination device is turned on.

[0063] In the microscopic imaging device, the light source 18 emits light, which is filtered by the light source filter 17 to form the illumination light. Under the action of the light splitting filter 20, the illumination light is reflected to the microscopic objective 19. The illumination light emitted from the microscopic objective 19 forms an illumination area around the blood vessel 4 of interest. In the illumination area, the fluorescent cells in the blood vessel are irradiated by the illumination light to release fluorescence, which produces a fluorescent signal in the subsequent image. The blood vessel and the surrounding tissue structure in the illumination area also reflect part of the illumination light or produce autofluorescence, forming background light, which produces a background signal in the subsequent image. The fluorescence produced by the cells and the background light of the blood vessel tissue are collected by the microscopic objective 19, pass through the light splitting filter 20 and the fluorescence filter 15 in turn, reach the fluorescence imaging unit 16, and then produce an image.

[0064] The illumination duration T can be estimated and adjusted according to the blood vessel 4 flow rate V and the detection area length L. The estimation formula of the illumination duration is T = L / V. For example, when the blood vessel 4 flow rate is large (V = 10 mm / s), the cells flow through the detection area with a length of L (140 microns) in a short time, and the illumination device can use a short duration T = 14 milliseconds; when the blood vessel 4 flow rate is small (V = 1 mm / s), the cells flow through the detection area with a length of L (140 microns) in a long time, and the illumination device can use a long duration T = 140 milliseconds. The image of a typical multi-cell cluster is shown in FIG. 6. Figure 2 .

[0065] The technical features not described in the present application can be realized by or using the prior art, which will not be described here. Of course, the above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by ordinary skilled in the art within the scope of the present application should also be within the scope of the present application.

Claims

1. A method for detecting multicellular clusters in circulation, characterized in that: The specific steps include: Step S1: Excite and receive the fluorescence pulse signal when the fluorescent cells flow through the detection area; Step S2: The excitation light emitted by the laser source is focused onto the blood vessel of interest through the microscope objective lens; When fluorescently labeled cells flow through the laser detection site, they release fluorescence; Step S3: The fluorescence is collected by a microscope objective lens and converted into an electrical signal by a photoelectric conversion device; Step S4: The fluorescence pulse signal subsequently enters a signal conversion device to generate a trigger signal for the imaging part; Step S5: performing signal conversion through a comparator. When the intensity of the received fluorescence signal pulse is greater than a certain threshold, the signal conversion device generates a rising edge pulse signal for starting a subsequent imaging device. Step S6: When the fluorescence trigger signal is transmitted to the imaging device, the lighting device is turned on, and the imaging device records an image sequence of cells flowing through the detection area after receiving the fluorescence trigger signal.

2. The method for detecting multicellular clusters in circulation according to claim 1, characterized in that: In step S1, a living cell flow cytometer or an optical fiber-based living cell fluorescence detection device is used to excite and receive a fluorescence pulse signal.

3. The method for detecting multicellular clusters in circulation according to claim 1, characterized in that: In step S6, the illumination duration T of the illumination device is estimated and adjusted according to the blood vessel flow velocity V and the length L of the detection area; The estimation formula for lighting duration is T=L / V.

4. A detection device using the method for detecting multicellular clusters in circulation according to any one of claims 1 to 3, characterized in that: It includes laser irradiation and fluorescence receiving devices, which are used to excite and receive fluorescence pulse signals when fluorescent cells flow through the detection area; A signal conversion device, used for converting the fluorescence pulse signal into an effective trigger signal for the imaging part; A high-sensitivity, high-speed imaging device for recording an image sequence of cells flowing through the detection area after receiving a fluorescence trigger signal; The laser irradiation and fluorescence receiving device, the signal conversion device, and the high-sensitivity high-speed imaging device are connected in sequence.

5. The detection device according to claim 4, characterized in that The laser irradiation and fluorescence receiving device is a living cell flow cytometer or a living cell fluorescence detection device based on an optical fiber type.

6. The detection device according to claim 5, characterized in that The laser irradiation and fluorescence receiving device includes a laser source, a reflector, a cylindrical mirror, a mechanical slit, an achromatic lens, a beam splitter, a fluorescence filter, an achromatic lens, a mechanical slit, and a photomultiplier tube, which are arranged in sequence according to the propagation direction of the light path; The photomultiplier tube is connected to a fluorescence signal recording and viewing device; The beam splitter is connected to the objective lens.

7. The detection device according to claim 4, characterized in that The high-sensitivity and high-speed imaging device includes an illumination device and a microscopic imaging device. The imaging unit of the microscopic imaging device adopts a photomultiplier CCD or an EMCCD, and the illumination device is a laser source or an LED illumination device.

8. The detection device according to claim 7, characterized in that The lighting device includes a light source that illuminates a spectroscopic filter at an angle, a microscope objective lens arranged on one side of the spectroscopic filter, the light from the light source passes through the light source filter, and a fluorescence filter and a fluorescence imaging unit are arranged on the other side of the spectroscopic filter.

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