Bioluminescence imaging flow cytometry method and device

Abstract

The present invention provides a bioluminescence imaging flow cytometry method and apparatus, comprising: a free reflection unit configured to obtain a bioluminescence signal by irradiating a sample suspension, and to re-reflect and converge the collected bioluminescence signal to enhance the detected bioluminescence signal; the sample control unit is configured to comprise a multi-channel flow type assembly, a fixing mechanism and a three-axis displacement table; the multi-channel flow type assembly is used for fixing and bearing a sample suspension, the fixing mechanism is used for fixing the multi-channel flow type assembly, and the three-axis displacement table drives the fixing mechanism to perform three-axis movement so as to adjust an imaging area of the sample suspension fixedly borne by the multi-channel flow type assembly; and an optical imaging unit configured to capture a bioluminescent cell image at a low signal level in space.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell analysis, and particularly relates to a bioluminescence imaging flow cytometer and method. BACKGROUND

[0002] The statements in this section merely provide background information related to the present application and do not necessarily constitute prior art.

[0003] Traditional flow cytometry can quickly analyze the basic characteristics of cells, such as size and granularity, but cannot obtain morphological details of cells. Imaging flow cytometry is an innovative tool that combines flow cytometry and microscopic imaging technology, which can provide multi-dimensional data and high-resolution images in cell analysis. Specifically, it can not only measure the physical characteristics of cells, but also provide cell morphology, structure and subcellular information, thereby achieving more comprehensive analysis.

[0004] With the continuous advancement of technology, imaging flow cytometry has continuously improved its multi-channel detection capability, integrated multiple lasers and fluorescence channels, and made multiple labeling and complex analysis possible. Today, imaging flow cytometry is widely used in cell biology, drug screening and clinical diagnosis, and provides strong support for scientific research and clinical diagnosis.

[0005] The single-cell detection method of the imaging flow cytometer mainly relies on fluorescence imaging. Fluorescence imaging uses fluorescent proteins, fluorescent dyes, quantum dot labels, etc. to label the genes, proteins, etc. of cells, and through laser excitation of the labels to produce fluorescence, thereby achieving the purpose of single-cell signal detection and collection. Labeling can cause damage to the structure and function of cells, and long-term excitation can also cause photobleaching of the labels, leading to fluorescence failure, which is not conducive to long-term observation of living cells; at the same time, the excitation process can cause autofluorescence of the cells, resulting in low signal-to-noise ratio.

[0006] The inventors found in their research that bioluminescence imaging technology is an optical imaging technology that can be used to visualize molecules and cells in health and disease. This technology uses luciferase-based gene reporters to track and image cells with high sensitivity. The high sensitivity of this technology is derived from the effective photons produced by the chemical reaction of luciferase and substrate luciferin. Compared with traditional fluorescence imaging technology, it has the advantages of no excitation light, no photobleaching, high sensitivity, dynamic tracking, etc., and is the direction of future optical imaging assisted diagnosis.

[0007] However, the current bioluminescence imaging technology detection equipment is less, mainly focusing on large-scale static imaging at the mouse level, and single-cell static imaging at the cell or tissue level. Therefore, the current detection equipment cannot realize high-throughput non-destructive single-cell bioluminescence imaging, especially flow accurate detection. SUMMARY

[0008] In order to overcome the above-mentioned deficiencies of the prior art, the present application provides a bioluminescence imaging flow cytometer, which is based on bioluminescence and microfluidic technologies, and realizes a single-cell optical imaging flow cytometer without excitation light source.

[0009] In order to achieve the above-mentioned purpose, one or more embodiments of the present application provide the following technical solutions: In a first aspect, a bioluminescence imaging flow cytometer device is disclosed, comprising: A free reflection unit is configured to obtain a bioluminescence signal by irradiating a sample suspension, and to re-reflect and converge the collected bioluminescence signal to enhance the detected bioluminescence signal; A sample control unit is configured to include a multi-channel flow assembly, a fixing mechanism, and a three-axis displacement table; The multi-channel flow assembly is used to fix the sample suspension, the fixing mechanism is used to fix the multi-channel flow assembly, and the three-axis displacement table drives the fixing mechanism to move in three axes to adjust the imaging area of the sample suspension fixed by the multi-channel flow assembly; An optical imaging unit is configured to capture a low signal level bioluminescence cell image in space based on the enhanced bioluminescence signal.

[0010] As a further technical solution, the free reflection unit includes an auxiliary light source, a mirror, and a plano-convex lens. The auxiliary light source is used to emit a light beam, the mirror is used to adjust the incident direction of the emitted light beam, and the plano-convex lens is used to modulate and converge the adjusted light beam into a point-shaped illumination light beam. When the auxiliary light source is turned off and the mirror is rotated by 45°, the mirror and the plano-convex lens will form an enhancement structure to realize the enhanced detection of the single-cell bioluminescence signal without excitation light source excitation.

[0011] As a further technical solution, the auxiliary light source is a sleeve light source that can freely adjust the light intensity and has full-spectrum emission.

[0012] As a further technical solution, the mirror is a coated mirror with an effective reflection area of 400 nm to 700 nm.

[0013] As a further technical solution, the plano-convex lens is a plano-convex lens with a focal length of 25 mm and one flat and one convex side.

[0014] As a further technical solution, the multi-channel flow assembly is formed by parallel arrangement of a plurality of quartz glass tubes, and the quartz glass tubes are fixed on a glass slide.

[0015] As a further technical solution, the optical imaging unit comprises a collection objective, a focusing objective and a high-sensitivity detector. The collection objective is used for efficiently collecting bioluminescence signals, and the focusing objective is used for converging the acquired bioluminescence signals onto the high-sensitivity detector, on which the images of bioluminescence cells at a low signal level are captured.

[0016] As a further technical solution, the collection objective and the focusing objective are both microscopic objectives. Preferably, the collection objective is a high-numerical-aperture objective with a magnification of 20 times, and the focusing objective is a short-focal-length objective with a magnification of 4 times.

[0017] In a second aspect, an imaging method of a bioluminescence imaging flow cytometer is disclosed, comprising: The white light emitted by the auxiliary light source is reflected by the mirror to the plano-convex lens and then converged onto the multi-channel flow assembly, so as to illuminate the imaging area of the internal structure of the multi-channel flow assembly; Adjusting the imaging area of the sample to be measured: adjusting the position of the multi-channel flow assembly so that the sample to be measured moves to the central imaging area of the collection objective, aligning all the optical components so that the signal source is at the central position of the detector chip, and fine-tuning the three-axis displacement table so that the multi-channel flow assembly is at the focal point position under the irradiation of the auxiliary light source; After the focal point is determined, the auxiliary light source is turned off, the mirror is rotated by 45 The bioluminescence signals collected by the plano-convex lens are reflected again to enhance the weak bioluminescence signals of single cells in the flow assembly; The enhanced signals of the sample to be measured are collected and then converged onto the high-sensitivity detector, so as to generate the images of bioluminescence cells.

[0018] As a further technical solution, the sample to be measured is in a suspended state.

[0019] In a third aspect, a high-throughput single-cell bioluminescence stripe imaging detection method is disclosed, which is realized based on a bioluminescence imaging flow cytometer and comprises: Under the flow cytometry environment, the single-cell bioluminescence signals in the sample solution are acquired by the free reflection unit, and the bioluminescence signals are imaged by the optical imaging unit; In the imaging process of the bioluminescence signals by the optical imaging unit, the frame rate of the detector in the optical imaging unit is set; The sample flow rate is controlled by the injection pump, and the single-cell bioluminescence signals under different flow rates are obtained; The bioluminescence signals under different flow rates are analyzed, and the flow rate meeting the requirements is selected as the optimal flow rate for forming the cell stripe according to the analysis result, under which the highest cell throughput can be obtained under the condition of allowing single-cell bioluminescence detection.

[0020] In a fourth aspect, the imaging method of the bioluminescence imaging flow cytometer can be used for bioluminescence detection of different ovarian cancer cell lines and for detection of the development of mouse ovarian cancer cells.

[0021] The above one or more technical solutions have the following beneficial effects: The technical scheme of the present application realizes the irradiation of the sample suspension to obtain the bioluminescence signal, and the collected bioluminescence signal is reflected again to enhance the detected bioluminescence signal; the multi-channel flow component in the sample control unit is used to fix the sample suspension, the fixing mechanism is used to fix the multi-channel flow component, and the three-axis displacement table drives the fixing mechanism to move in three axes to adjust the imaging area of the sample suspension fixed and carried by the multi-channel flow component; and then the optical imaging unit is used to capture the bioluminescence cell image with low signal level in the space, so as to realize the single-cell bioluminescence weak light imaging. The technical scheme of the present application develops a sheath-free flow single-cell bioluminescence high-throughput imaging technology based on the bioluminescence characteristics, which can be expanded to multiple channels; at the same time, the strip imaging bioluminescence high-throughput imaging technology is proposed for the weak light property of bioluminescence; and the present application realizes the application of the bioluminescence imaging flow cytometer technology in cell detection and classification, drug effect research and mouse tumor monitoring.

[0022] The traditional flow detection is related to light scattering and fluorescence emission, and needs excitation of a light source. The technical scheme of the present application creates a precedent of sheath-free flow detection, and combines the characteristics that bioluminescence can self-illuminate without excitation light to realize the single-cell bioluminescence flow imaging detection without using an excitation light source.

[0023] When preparing a biological sample smear based on the traditional microscopy, drying, fixing and freezing are often needed, and it is not suitable for live cell detection. The present application proposes a sheath-free flow bioluminescence high-throughput flow detection technology, and uses a quartz glass tube, a glass slide and other tools to build a sample carrying table for single-cell flow and with good optical properties. The cells are in a suspension state to ensure their cell activity, and the sheath-free flow can realize the high-throughput flow detection of the bioluminescence single-cell signal.

[0024] The unique design of the plano-convex lens-mirror and double-objective coupling developed by the present application realizes the high-sensitivity detection of low-level bioluminescence signals. The plano-convex lens-mirror structure uses a plano-convex lens to collect the bioluminescence signals from the sample, and the collected signals are reflected to the objective lens through the mirror, thereby increasing the light collection amount of the objective lens; the double-objective coupling structure uses two back-to-back objective lenses to have a large numerical aperture while keeping a low magnification, so that more light can be converged on each pixel of the camera.

[0025] The application realizes further improvement of the flux by using the strip imaging bioluminescence high flux imaging method, and controls the cell tailing phenomenon in the optimal analysis range by balancing the flow rate of the bioluminescence sample and the exposure time of the detector, so that the detection efficiency of the sample is greatly improved.

[0026] Advantages of the additional aspects of the application will be in part apparent from the following description, will in part be apparent from the corresponding drawings, and will in part result from the practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0027] The accompanying drawings, which form a part of the specification, are included to provide a further understanding of the application and are incorporated herein by reference. The illustrations are shown schematically and are not intended to limit the application.

[0028] Figure 1 It is a structural schematic diagram of the bioluminescence imaging flow cytometry detection device in the embodiment one of the application. Figure 2 It is a light path schematic diagram after the rotating mirror 45 Figure 3 It is a bioluminescence image strip detection simulation in the embodiment two of the application. Figure 4 It is a schematic diagram of the cell strip image under different flow rates in the embodiment two of the application. Figure 5 It is a high flux single cell bioluminescence image of the sheath-free flow cell classification in the embodiment three of the application. Figure 6 It is a high flux single cell bioluminescence image of the sheath-free flow drug effect in the embodiment three of the application. Wherein, 1, auxiliary light source, 2, mirror, 3, plano-convex lens, 4, multi-channel flow component, 5, acquisition objective lens, 6, focusing objective lens, 7, high-sensitivity detector, 8, detection and analysis system, 9, micro-channel detailed structure. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs.

[0030] It should be noted that the terms used herein are only for the purpose of describing the specific embodiments and are not intended to limit the exemplary embodiments according to the application.

[0031] The embodiments in the application and the features in the embodiments can be combined with each other without conflict.

[0032] Embodiment one Referring to the drawings​Figure 1 The embodiment disclosed in the present embodiment is a bioluminescence imaging flow cytometer device, which converges bioluminescence signals into a high-sensitivity detector through a unique optical path structure design. The cytometer structure comprises: a free reflection unit configured to obtain bioluminescence signals by irradiating a sample suspension, and to re-reflect and converge the collected bioluminescence signals to enhance the detected bioluminescence signals; a sample control unit configured to comprise a multi-channel flow assembly, a fixing mechanism, and a three-axis displacement table; The multi-channel flow assembly is used to fix the sample suspension, the fixing mechanism is used to fix the multi-channel flow assembly, and the three-axis displacement table drives the fixing mechanism to move in three axes to adjust the imaging area of the sample suspension fixed by the multi-channel flow assembly; an optical imaging unit configured to capture a low-signal-level bioluminescence cell image in space based on the enhanced bioluminescence signals.

[0033] It should be noted that the free-rotating mirror in the free reflection unit is combined with the converging of the plano-convex lens to irradiate the sample suspension, which is used to control the auxiliary light source to determine the sample position, and the mirror is rotated by 45 The mirror combined with the plano-convex lens after the mirror is used to re-reflect and converge the collected bioluminescence signals in space to enhance the detected bioluminescence signals, so as to realize the enhancement detection of the bioluminescence signals without excitation. The optical path diagram is shown in Figure 2 .

[0034] In a specific embodiment, the free reflection unit comprises an auxiliary light source 1, a mirror 2, and a plano-convex lens 4. The auxiliary light source 1 is used to emit a white light beam for illuminating the imaging area to determine the sample shape. The mirror 2 is used to adjust the incidence of the white light source, wherein the mirror 2 can be freely rotated to control the incidence of the light source and the reflection from the sample signal. The plano-convex lens 4 is used to modulate and converge the white light source into a point-shaped illumination light beam, which is used for focusing of the auxiliary light source or collection of the bioluminescence signals in space, and cooperates with the mirror to re-focus on the bioluminescence sample to enhance the bioluminescence signals. When the auxiliary light source 1 is turned off, the mirror 2 is rotated by 45 , so that it can completely reflect the bioluminescence signals from the sample. The mirror 2 and the plano-convex lens 3 form an enhancement structure to realize the enhancement detection of the bioluminescence signals without excitation.

[0035] In the present embodiment, the sample is a cell that can express a new type of NanoLuc, and the exogenous substrate is Furimazine. When the NanoLuc produced in the cell specifically binds to the substrate, a biochemical reaction can be carried out and a bioluminescence signal can be emitted, which can be collected and analyzed by the device of the present embodiment.

[0036] In this embodiment, the auxiliary light source 1 is a sleeve light source with adjustable light intensity and full-spectrum emission. The reflector 2 is a coated reflector with an effective reflection area of ​​400 nm to 700 nm. Selecting a reflector in this wavelength range can completely reflect the bioluminescent signal from the sample. The plano-convex lens 3 is a plano-convex lens with one flat and one convex side and a focal length of 25 mm. It can fully collect signals from the sample that were not collected by the objective lens, thereby enhancing the signal.

[0037] In this embodiment, the sample control unit includes: a multi-channel flow cytometry component 4 for fixing the sample suspension, a fixing mechanism for fixing the sheath flowmeter component, and a three-axis displacement stage for driving the fixing mechanism to move in three axes. The sample control unit can control the three-dimensional spatial movement of the sample to be tested in the vertical and horizontal planes. By adjusting the imaging area of ​​the sample suspension through the above structure, high-throughput flow cytometry detection can be achieved without the sample forming a sheath flow.

[0038] The detailed structure of the multi-channel flow cytometer module 4 consists of three quartz glass tubes; for details, please refer again. Figure 1 As shown, the detailed structure 9 of the microchannel consists of three channels with an inner diameter of 100 mm. The wall thickness is 200. The high-optical-value quartz glass tubes are arranged in parallel and fixed to a glass slide using silica gel. The system can be expanded from a single channel to a multi-channel design. The fixing structure consists of orthogonal rods that clamp the sheath flow unit and are fixed to a triaxial displacement stage to control the sample's movement along the x, y, and z axes. Existing structures can be used for all of the above, and will not be described in detail here.

[0039] This embodiment's optical imaging unit utilizes a unique dual-objective coupling design to capture low-signal-level bioluminescent cell images on a high-sensitivity detector. Specifically, it includes an acquisition objective 5, a focusing objective 6, and a high-sensitivity detector 7. The acquisition objective efficiently acquires the bioluminescent signal, while the focusing objective converges the acquired bioluminescent signal onto the high-sensitivity detector.

[0040] The acquisition and focusing objectives are microscope objectives. The acquisition objective has a high numerical aperture (NA=0.8) and a magnification of 20x, while the focusing objective has a short focal length (45 mm) and a magnification of 4x. The distance between the acquisition and focusing objectives is minimized without affecting operation to reduce the loss of low-level bioluminescent signals in the transmission path.

[0041] The operation process of the device in this embodiment includes: The white light emitted by the auxiliary light source is reflected by a mirror and then focused onto a plano-convex lens to illuminate the imaging area of ​​the sample flow cytometry module to determine that the sample is at the focal point of the objective lens.

[0042] After the focus is determined, the bright field light source is turned off. The reflector is rotated 45° to reflect the bioluminescence signal in the space collected by the plano-convex lens again to enhance the weak bioluminescence signal in the flow cytometry component. The acquisition objective lens collects the enhanced signal of the sample, which is then focused onto the high-sensitivity detector by the converging objective lens. Subsequently, the bioluminescence image data is transmitted to the detection and analysis unit for data processing and analysis.

[0043] This invention employs a unique optical design to achieve excitation-free bioluminescent cell imaging, enabling rapid single-cell bioluminescent imaging in flow cytometry based on hydrodynamic principles. During image acquisition, the sample is suspended, eliminating the damaging procedures such as drying and fixation involved in smear processing during optical microscopy, thus ensuring the accuracy and validity of the measurements.

[0044] In addition, a high-throughput single-cell bioluminescence stripe imaging detection method for low-light bioluminescence properties was also disclosed, including: Cell streaks caused by cell tailing are controlled by adjusting the bioluminescent sample flow rate and exposure time. When imaging moving bioluminescent cells, the brightness of the cell image increases with exposure time until it reaches a point where it remains essentially constant. This transition point is defined as the distance the cell image can move, which is equal to its own pixel length plus the exposure time of one pixel. Figure 3 As shown.

[0045] Next, controlling the cell streaks within the analyzable range greatly improves the efficiency of sample detection and analysis, enabling high-throughput imaging of bioluminescent cells.

[0046] In one implementation example, high-throughput single-cell bioluminescence stripe imaging requires a flow cytometry environment to be formed by a flow cytometry component, an injection pump, and a bioluminescent cell sample solution. A free-reflection unit acquires the single-cell bioluminescence signal from the sample solution, and an optical imaging unit images the bioluminescence signal. For example... Figure 4As shown, first, the frame rate of the detector in the optical imaging unit is fixed at 10 frames. The sample flow rate is controlled by a syringe pump. The sample flow rate is controlled at 50 μL / h, 200 μL / h, 1 ml / h, and 2 ml / h, respectively. The single-cell bioluminescence signals are acquired at the four flow rates, respectively. The bioluminescence signals at the four flow rates are analyzed. The cell bioluminescence signal at the flow rate of 50 μL / h shows a single point, the single-cell bioluminescence signal at the flow rate of 200 μL / h shows a short stripe, the single-cell bioluminescence signal at the flow rate of 1 ml / h shows a proper cell stripe, and the single-cell bioluminescence signal at the flow rate of 2 ml / h shows an excessively long cell stripe, which exceeds the imaging field of view and is not conducive to the analysis of experimental results. Finally, in order to capture the optimal flow rate that does not exceed the imaging field of view and improve the efficiency of analyzing rare cells in a large amount of liquid, the flow rate of 1 ml / h is selected as the optimal flow rate for forming a cell stripe, which can obtain the highest cell flux under the condition of allowing single-cell bioluminescence detection.

[0047] Example Two Example Two of the present application provides a bioluminescence imaging flow cytometer device for ovarian cancer single-cell bioluminescence classification and drug effect analysis, including the following steps: Step 1, generate auxiliary bright field light to irradiate the sample suspension to determine that the sample is in a focused state.

[0048] Step 2, fix the flow assembly carrying the sample suspension and adjust the imaging area of the sample suspension. Specifically, the position of the flow assembly is adjusted by the three-axis displacement table to move the sample suspension to the center imaging area of the collection objective, all optical components are aligned to make the signal source at the center position of the detector chip, and the three-axis displacement table is fine-tuned to make the multi-channel flow assembly in the focal position under the irradiation of the bright field light source.

[0049] Step 3, turn off the auxiliary light source, rotate the mirror 45 and a plano-convex lens to form an enhanced combination to collect the bioluminescence signals of the sample dispersed in space in the reflection flow channel and refocus them on the flow assembly to enhance the detection of bioluminescence signals.

[0050] Step 4, use a high numerical aperture collection objective to collect the bioluminescence signals emitted by the cells, and then converge them to the detector through the focusing objective for recording.

[0051] In bioluminescence single-cell imaging, in order to ensure effective acquisition of signals during imaging, the distance between the collection objective and the focusing objective needs to be shortened as much as possible without affecting the operation to reduce the loss of signals in the transmission process.

[0052] Specific operation steps: (1) Take an appropriate amount of live cell suspension solution transfected with bioluminescent genes, dilute it with culture medium, and use it as the sample liquid; (2) Trigger the high-sensitivity sCMOS detector to record the bioluminescence signal of the cells; (3) Turn on the auxiliary light source, adjust the three-axis displacement table to ensure that the microchannels in the field of view are in focus; (4) Adjust the sample liquid speed to 50 ; (5) Turn off the auxiliary light source, rotate the mirror by 45 to form a plano-convex lens-mirror weak light enhancement combination; (6) Add the prepared substrate solution to the bioluminescent suspension, pass it through the self-assembled multi-channel flow assembly at a constant speed through the injection pump, and then start collecting bioluminescent single cell image signals, wherein different types of ovarian cancer cell line cells transfected with bioluminescent genes are placed in different flow channels; (7) Capture the single cell bioluminescence results of different ovarian cancer cell lines in the multi-channel flow assembly according to the set flow rate and parameters, and collect images as shown in Figure 5 ; (8) Place the same number and concentration of Hey cells transfected with bioluminescent genes in different glass tubes, while keeping glass tube 1 stationary, add anti-ovarian cancer tumor drug 10 to glass tube 2, and add anti-ovarian cancer tumor drug 20 to glass tube 3 to verify the effect of drug analysis, and the drug effect is shown in Figure 6 .

[0053] This example proves that the bioluminescence imaging flow cytometry detection device and method can be used for bioluminescence detection of different ovarian cancer cell lines.

[0054] Of course, the device provided by the present application can also be used for cell analysis in other scenes, and is not limited to the above use scenarios.

[0055] Example Three The mouse tumor monitoring method of the bioluminescence imaging flow cytometry provided by the third embodiment of the present application comprises the following steps: Step 1, according to the operation of Example 2, the mouse ovarian cancer peripheral blood circulating tumor detection is performed.

[0056] Step 2, while performing in vitro detection of mouse ovarian cancer circulating tumor cells, the mouse in vivo imaging system detection is also performed for comparison.

[0057] Specific operation steps: (1) keep the bioluminescence imaging flow cytometry detection system unchanged, i.e. keep the device in Example 1, and keep the basic operation in Example 2 unchanged; (2) improve the flow speed of the bioluminescence sample to perform large-capacity liquid rapid detection, the cell strip detection principle and the flow strip under different speeds are as shown in Figure 3 and Figure 4 (3) perform actual blood simulation detection, i.e. configure bioluminescence single-cell mouse blood samples containing 100, 10 and 1 per milliliter according to different concentrations to perform actual detection; (4) culture the purchased nude mice in the laboratory for adaptability and plant tumors in the mice, and perform mouse extracorporeal circulation tumor cell detection and mouse whole-body in vivo imaging detection on the 12th day, the 18th day and the 24th day, respectively; (5) compare the tumor area statistical results of different mice with the statistical analysis results of mouse peripheral blood ovarian cancer circulating tumor cells detected by the system, and the trends are consistent; This example proves that the bioluminescence imaging flow cytometry detection device and method can be used for the development detection and statistical analysis of mouse ovarian cancer cells.

[0058] Of course, the device provided by the application can also be used for mouse tumor cell analysis in other scenes, and is not limited to the above use scenarios.

[0059] Although the specific embodiments of the application are described above with reference to the drawings, the description is not a limitation on the scope of protection of the application, and those skilled in the art should understand that various modifications or changes made by those skilled in the art on the basis of the technical solutions of the application without creative labor are still within the scope of protection of the application.​

Claims

1. A bioluminescence imaging flow cytometer device, characterized in that, include: The free reflection unit is configured to: obtain single-cell bioluminescence signals by irradiating the sample suspension, and reflect and converge the collected bioluminescence signals again to enhance the detected bioluminescence signals; The sample control unit is configured to include: a multi-channel flow cytometer assembly, a fixing mechanism, and a three-axis displacement stage; The multi-channel flow cytometer is used to fix and support the sample suspension, the fixing mechanism is used to fix the multi-channel flow cytometer, and the three-axis displacement stage drives the fixing mechanism to move in three axes to adjust the imaging area of ​​the sample suspension fixed and supported by the multi-channel flow cytometer. The optical imaging unit is configured to capture images of low-signal-level bioluminescent cells in the space based on enhanced bioluminescent signals.

2. The bioluminescence imaging flow cytometer device as described in claim 1, characterized in that, The free reflection unit includes: an auxiliary light source, a reflector, and a plano-convex lens; The auxiliary light source is used to emit a light beam, the reflector is used to adjust the incident direction of the emitted light beam, and the plano-convex lens is used to modulate and converge the adjusted emitted light beam into a point illumination beam. When the auxiliary light source is turned off, the reflector rotates 45° and the plano-convex lens will form an enhancement structure to achieve enhanced detection of single-cell bioluminescence signals that do not require excitation by an excitation light source.

3. The bioluminescence imaging flow cytometer device as described in claim 2, characterized in that, The auxiliary light source is a sleeve light source with adjustable light intensity and full-spectrum emission.

4. The bioluminescence imaging flow cytometer device as described in claim 2, characterized in that, The reflector is a coated reflector with an effective reflection area of ​​400 nm to 700 nm; Preferably, the plano-convex lens is a plano-convex lens with a focal length of 25 mm and one side flat and the other convex.

5. The bioluminescence imaging flow cytometer device as described in claim 1, characterized in that, The multi-channel flow cytometer is composed of multiple quartz glass tubes arranged in parallel, and the quartz glass tubes are fixed on a glass slide.

6. The bioluminescence imaging flow cytometer device as described in claim 1, characterized in that, The optical imaging unit includes an acquisition objective, a focusing objective, and a high-sensitivity detector; The acquisition objective is used to efficiently acquire bioluminescent signals, and the focusing objective is used to converge the acquired bioluminescent signals onto a high-sensitivity detector to capture images of single-cell bioluminescent cells with low signal levels on the high-sensitivity detector.

7. The bioluminescence imaging flow cytometer device as described in claim 6, characterized in that, Both the acquisition objective and the focusing objective are microscope objectives. Preferably, the acquisition objective is a high numerical aperture objective with a magnification of 20x, and the focusing objective is a short focal length objective with a magnification of 4x.

8. The imaging method of a bioluminescence imaging flow cytometer, characterized by: include: The light emitted by the auxiliary light source is reflected by the plano-convex lens and then focused onto the multi-channel flow cytometry module to illuminate the imaging area of ​​the internal structure of the multi-channel flow cytometry module. Adjusting the imaging area of ​​the sample under test in the multi-channel flow cytometry component specifically includes: adjusting the position of the multi-channel flow cytometry component so that the sample under test is moved to the center imaging area of ​​the acquisition objective lens, aligning all optical components so that the signal source is at the center of the detector chip, and fine-tuning the three-axis displacement stage so that the multi-channel flow cytometry component is at the focal position under the illumination of the light source. After focusing, turn off the auxiliary light source and rotate the reflector 45 degrees. The bioluminescence signal collected in the space by the plano-convex lens is then reflected again to enhance the weak bioluminescence signal of single cells in the flow cytometry component. The enhanced signal of the sample to be tested is collected and then converged onto a high-sensitivity detector to generate a single-cell bioluminescent cell image.

9. A high-throughput single-cell bioluminescent stripe imaging detection method, characterized in that, Based on the bioluminescence imaging flow cytometer device according to any one of claims 1-7, comprising: In a flow cytometry environment, the bioluminescence signal of a single cell in the sample solution is acquired by the free reflection unit, and the bioluminescence signal is imaged by the optical imaging unit; In the process of imaging bioluminescent signals by the optical imaging unit, the frame rate of the detector in the optical imaging unit is set. By controlling the sample flow rate with an injection pump, single-cell bioluminescence signals were obtained at different flow rates. Bioluminescence signals at different flow rates were analyzed, and the optimal flow rate for forming cell stripes was selected based on the analysis results. At this flow rate, the highest cell throughput could be obtained under the condition that single-cell bioluminescence detection is allowed.

10. The imaging method of the bioluminescence imaging flow cytometer as described in claim 8, characterized in that, The method can be used for bioluminescence detection of different ovarian cancer cell lines and for detecting the occurrence and development of mouse ovarian cancer cells, with the sample to be tested in a suspended state.

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