Single-particle fluorescence spectrum and label-free imaging multi-parameter measurement flow type device and method

Abstract

The invention provides a single-particle fluorescence spectrum and label-free imaging multi-parameter measurement flow type device and method, and the device comprises a liquid flow system which comprises a sheath flow device, the sheath flow device comprises sheath fluid and sample fluid, and the sample fluid comprises a cell sample; the optical system comprises an excitation end and a receiving end, the excitation end comprises a first light source and a second light source, and the first light source and the second light source jointly irradiate the cell sample to excite the particles to generate fluorescence spectrum information, a light scattering image and a bright field image; and the signal acquisition system is used for simultaneously acquiring a fluorescence spectrum signal, a bright field image and a light scattering image.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical detection, and in particular relates to a single particle fluorescence spectrum and label-free imaging multi-parameter measurement flow device and method. Background Art

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

[0003] Traditional flow cytometry is a fluorescent labeling detection technology that converts the forward light scatter signal (FSC), side light scatter signal (SSC) and multi-band fluorescence signal generated by laser-irradiated cells labeled with fluorescent dye into electrical signals through a photodetector (such as PMT). By analyzing the electrical signals, cell-related characteristics such as cell size, cell morphology, DNA / RNA content, cell surface / internal markers, etc. are obtained, and cell analysis is performed.

[0004] Flow cytometry has been widely used in many fields such as cell biology, immunology, hematology, stem cell research, and oncology. In cell biology research, by analyzing the cell DNA content distribution curve and distinguishing G0 / G1, S, and G2 / M phase cells, the percentage of each cell in the cell cycle can be determined; in immunology research, combined with immunofluorescence methods, by fluorescently labeling different surface-specific antigens, T and B lymphocyte subsets can be distinguished, the number of antigens can be determined, etc., which plays an important role in judging immunodeficiency, autoimmune diseases, leukemia, and lymphoma phenotypes; in genetics research, flow cytometry can determine the chromosomal DNA content, perform flow chromosome karyotype analysis, quickly analyze the karyotype and sort out different chromosomes, which are used for human genome research, genetic diseases and cancer diagnosis.

[0005] Specifically, traditional flow cytometry focuses the stained or labeled cell suspension to be tested into a single column through sheath fluid. The cells pass through the detection area one by one and are excited by the laser to produce FSC, SSC and multi-color fluorescence signals. The scattered light signal and fluorescence signal are received by the photodetector and converted into electrical signals. The electrical signals are amplified and digitized and collected and stored by the computer. The collected data are displayed in the form of scatter plots or histograms. By setting gates on the scatter plots or histograms, the cells of interest can be well distinguished from other cells. Traditional flow cytometry has the advantage of high-throughput detection, and the detection speed can reach more than 10,000 cells per second.

[0006] Although traditional flow cytometry has so many advantages, it still has some shortcomings: because traditional flow cytometry only receives a part of the signal of the maximum peak of each fluorescent dye, it cannot distinguish similar fluorescent dyes and the detection parameters are limited; traditional flow cytometry cannot provide morphological information of cells. Currently, with the development of biomedicine, cell morphology detection plays an important role in cell cycle research and cell signal transduction. Complex biological problems require more comprehensive and detailed multi-parameter research on cells.

[0007] In order to solve these shortcomings, traditional flow cytometry is developing towards imaging flow cytometry or full-spectrum flow cytometry. Imaging flow cytometry can reflect the morphological information of cells, but it has limitations such as low throughput, complex technology, and low image contrast. Full-spectrum flow cytometry greatly improves the spectral detection capability, but it also has problems such as high cost, difficulty in data analysis, high sample preparation requirements, and complex experiments. At the same time, it cannot provide cell morphological information.

[0008] In order to solve the problem of lack of information related to cell morphology, flow cytometry is currently developing in the direction of multimodal imaging flow cytometry, for example, it can simultaneously combine fluorescence imaging, bright field imaging and light scattering imaging. Among them, light scattering imaging is a label-free, highly sensitive technology that uses the internal information of the medium carried by scattered light, without the need for fluorescent labeling, to convert scattered light signals into image signals through a specific optical system and detection device. Light scattering imaging has a wide range of uses. For example, light scattering imaging can distinguish between normal cells and diseased cells, which can bring a new cell analysis method to scientific research and clinical diagnosis. Although light scattering images can provide rich information on the internal structure of cells, they cannot currently reflect the cell morphology very well, while bright field imaging can achieve better cell morphology detection.

[0009] Although imaging flow cytometry solves the defects in cell morphology, it cannot make good use of the high-speed characteristics of flow cytometry, and the sensitivity of fluorescence imaging is low, and it cannot detect weak fluorescence signals.

[0010] Specifically, the inventors discovered in their research that existing measuring devices use a labeling method to measure fluorescent single-cell signals when measuring parameters of single-particle cells. However, the existing devices use a labeled method to achieve detection, and can only obtain a one-dimensional voltage signal, but cannot obtain a cell image. In addition, the components used for fluorescence detection result in a complex structure, which is difficult to implement and cannot obtain comprehensive detection parameters of single-particle cells.

[0011] In addition, in the inventor's previous related research, there was a label-free method for detecting single particle cell parameters. The existing scheme cannot perform fluorescence detection. Similarly, the above method also has the problem of obtaining a single single particle cell parameter.

[0012] In summary, when faced with complex sample detection requirements, a single flow detection and measurement device is difficult to adapt, and often requires the cooperation of multiple flow devices to meet complex analysis requirements, which not only complicates the detection and analysis process, but also greatly increases the cost of the device. It is of great significance to develop flow devices and methods that can simultaneously perform multi-parameter measurements of single particle / single cell labeled fluorescence spectra and label-free light scattering and bright field imaging. Summary of the invention

[0013] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a single-particle fluorescence spectroscopy and label-free imaging multi-parameter measurement flow device, which can simultaneously collect fluorescence spectral information, bright field images, and light scattering images, and realize the combination of fluorescence spectrum and image to perform single particle classification, particle size identification and specific fluorescence detection.

[0014] To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions:

[0015] In a first aspect, a single particle fluorescence spectroscopy and label-free imaging multi-parameter measurement flow device is disclosed, comprising:

[0016] A liquid flow system, wherein the liquid flow system comprises a sheath flow device, wherein the sheath flow device contains a sheath fluid and a sample fluid, wherein the sample fluid contains a cell sample;

[0017] An optical system, wherein the optical system comprises an excitation end and a receiving end, wherein the excitation end comprises a first light source and a second light source, wherein the first light source and the second light source jointly illuminate the cell sample to excite particles to generate fluorescence spectrum information and a light scattering image and a bright field image;

[0018] The signal acquisition system is used to simultaneously acquire fluorescence spectrum signals, bright field images and light scattering images.

[0019] As a further technical solution, the liquid flow system also includes a sample injection pump and a sheath flow injection pump. The upper end of the sheath flow device is provided with an injection port, and the injection port is connected to the syringe clamped in the sample injection pump through a pipeline. The left and right ends of the sheath flow device are respectively provided with a first sheath flow port and a second sheath flow port, and the first sheath flow port and the second sheath flow port are respectively connected to the syringe clamped in the sheath flow injection pump through pipelines.

[0020] As a further preferred technical solution, the sheath flow device is a rectangular glass channel, and the speeds of the sample liquid and the sheath liquid are changed by the sample injection pump and the sheath flow injection pump, thereby controlling the position and flow speed of the cell sample in the observation area of ​​the sheath flow device.

[0021] As a further preferred technical solution, the first light source is a laser light source, and the second light source is a mercury lamp light source.

[0022] As a further technical solution, the excitation end further includes a cylindrical lens, a fluorescent illuminator, a first bandpass filter, and a first focusing objective lens;

[0023] The laser light emitted by the laser light source is shaped into a light sheet by a cylindrical lens and then used as an excitation light source for light scattering and fluorescence;

[0024] The light emitted by the mercury lamp light source is focused by the fluorescent illuminator, the first bandpass filter and the first focusing objective lens in sequence to serve as a bright field light source.

[0025] As a further technical solution, the receiving end of the optical system includes a second focusing objective lens, a first long-pass dichroic mirror, a first short-pass dichroic mirror, and a plurality of channel fluorescence spectrum information acquisition units, and the fluorescence spectrum information acquisition unit of each channel has the same structure, including a long-pass dichroic mirror, a band-pass filter and a focusing objective lens arranged in sequence;

[0026] The second focusing objective lens collects the fluorescence spectrum information and light scattering image emitted by the cell sample perpendicular to the laser, as well as the bright field image parallel to the mercury lamp light source;

[0027] The information collected by the second focusing objective lens is reflected by the first long-pass dichroic mirror to form a bright field image, and the light scattering image is transmitted through the first short-pass dichroic mirror. The fluorescence spectrum information reflected by the first short-pass dichroic mirror is first separated into multiple fluorescence bands by the long-pass dichroic mirror of the fluorescence spectrum information collection unit of multiple channels. The fluorescence of multiple different bands is respectively transmitted through band-pass filters to transmit the fluorescence of specific bands, and then respectively transmitted after being focused by the objective lens.

[0028] As a further technical solution, a bandpass filter is used behind the focusing objective lens so that the three fluorescence channels can obtain fluorescence with bandwidths of 575-585nm, 630-640nm, and 675-685nm respectively.

[0029] The spatial filters are the second band-pass filter, the third band-pass filter, and the fourth band-pass filter in the implementation example. The objective lens is the third focusing objective lens, the fourth focusing objective lens, and the fifth focusing objective lens in the implementation example.

[0030] As a further technical solution, the signal acquisition system includes a plurality of PMTs, a data acquisition card, a first CMOS camera, a second CMOS camera, and a data analysis system; the number of the PMTs is consistent with the number of channels of the fluorescence spectrum information acquisition unit;

[0031] The bright field image is reflected to the second CMOS camera after passing through the first long-pass dichroic mirror;

[0032] The light scattering image is transmitted to the first CMOS camera through the first short-pass dichroic mirror;

[0033] The fluorescence spectrum information of multiple bands is focused into point shape by multiple focusing lenses and then transmitted to the corresponding light windows of PMT respectively;

[0034] The output lines of the PMT are respectively connected to the analog voltage input channels of the data acquisition card. The data acquisition card converts the analog signal into a digital signal and transmits it to the data analysis system through an interface.

[0035] As a further preferred technical solution, the fluorescence spectrum information acquisition unit has three channels.

[0036] In the second aspect, a single particle fluorescence spectrum and label-free imaging multi-parameter measurement method is disclosed, including:

[0037] Prepare a sample suspension and introduce it into the sheath flow device through a sample syringe, and adjust the flow rate ratio of the sheath liquid and the sample liquid through a syringe pump so that the cell sample forms a stable focused flow in the middle of the sheath flow device;

[0038] Adjust the position of the sheath flow device so that the illumination areas of the two light sources on the sheath flow device overlap, adjust the bright field focus and light scattering defocus, and the two light sources jointly illuminate the cell sample to excite the particles to generate fluorescence spectrum information and light scattering images and bright field images;

[0039] Select the appropriate sampling rate and filter cutoff frequency of the data acquisition card, select the appropriate sampling number of the camera, and then select the file saving path to collect the generated information;

[0040] The collected information is analyzed to perform image recognition and sample classification.

[0041] As a further preferred technical solution, when the information is collected and generated, the three working modes of fluorescence spectrum, light scattering imaging and bright field imaging can work separately or simultaneously, and the fluorescence spectrum, light scattering image and bright field image of the labeled single particle or one or two of the information can be detected simultaneously.

[0042] One or more of the above technical solutions have the following beneficial effects:

[0043] The flow cytometry adopted by the device of the present invention can perform real-time detection of cell fluorescence signals, and has the advantages of high speed and real-time detection of traditional flow cytometry. At the same time, the three-channel fluorescence signal improves the detection efficiency of the device.

[0044] The present invention not only has the function of fluorescence spectrum detection, but also has the function of label-free imaging, and can simultaneously perform two label-free detection methods: light scattering imaging and bright field imaging. Light scattering imaging has good signal stability, and can reflect the shape and size of the scatterer, the internal structure of the scatterer, and effectively distinguish particles of different sizes or similar sizes. High-resolution bright field imaging improves the imaging performance of the device and can intuitively reflect the morphological information of the particles;

[0045] The flow cytometry device of the present invention has three working modes: fluorescence spectroscopy, light scattering imaging, and bright field imaging. It can work independently or simultaneously. It can simultaneously detect the fluorescence spectrum, light scattering image, and bright field image of labeled single particles, which helps to establish a corresponding relationship between fluorescence specificity and particle morphology. The three modes can correspond well, and single particle label-free detection can also be performed. The particle size is determined by the light scattering image, and the particle structure information is viewed by the bright field image. While making up for the shortcomings of the three modes, it has the function of multi-parameter detection.

[0046] The technical solution of the present invention uses laser and mercury lamp as excitation light sources at the same time. While obtaining the fluorescence spectrum information of the sample, it can also obtain the light scattering image and bright field image of the sample, and can classify fluorescent and non-fluorescent particles of single micron-sized particles and identify particles of different sizes.

[0047] The technical solution of the present invention adopts left and right sheath fluids to focus the middle sample fluid, which has a good sample focusing effect and ensures the accuracy of sample detection.

[0048] The present invention is applicable to specific fluorescence detection, imaging recognition, classification and size identification of single micron-sized particles and has universal applicability.

[0049] Advantages of additional aspects of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0051] Figure 1 This is a schematic diagram of the overall structure of the flow device provided by the present invention;

[0052] Figure 2 It is the collection flow chart of the present invention;

[0053] Figure 3 Bright field images and light scattering images of label-free flow imaging detection of 2 μm non-fluorescent polystyrene beads using the flow device and method provided by one or more embodiments of the present invention.

[0054] Figure 4(a)-Figure 4(b) Fluorescence waveform diagram, bright field image, and light scattering image of a 2μm fluorescent bead flow cytometry experiment using the flow cytometry device provided by one or more embodiments of the present invention.

[0055] Figure 5(a)-Figure 5(b)Fluorescence waveform diagram, bright field image, and light scattering image of a flow cytometry experiment involving 2 μm fluorescent, 3.87 μm non-fluorescent, and 4.19 μm non-fluorescent polystyrene mixed beads conducted using the flow device provided by one or more embodiments of the present invention.

[0056] In the figure: 1. laser, 2. mercury lamp, 3. fluorescent illuminator, 4. first bandpass filter, 5. first focusing objective lens, 6. cylindrical lens, 7. sheath flow device, 8. sample injection pump, 9. sheath flow injection pump, 10. second focusing objective lens, 11. first long-pass dichroic mirror, 12. first short-pass dichroic mirror, 13. second long-pass dichroic mirror, 14. third long-pass dichroic mirror, 15. reflecting mirror, 16. second bandpass filter, 17. third bandpass filter, 18. fourth bandpass filter, 19. third focusing objective lens, 20. fourth focusing objective lens, 21. fifth focusing objective lens, 22. first PMT, 23. second PMT, 24. third PMT, 25. data acquisition card, 26. first CMOS camera, 27. second CMOS camera, 28. data analysis system. DETAILED DESCRIPTION

[0057] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0058] It should be noted that the terms used herein are for describing specific embodiments only and are not intended to be limiting of exemplary embodiments according to the present invention.

[0059] In the absence of conflict, the embodiments of the present invention and the features of the embodiments may be combined with each other.

[0060] The technical solution of the present invention combines fluorescence signal measurement (one-dimensional voltage signal) and cell imaging. Since cell imaging and fluorescence signal measurement are two different detection technologies and the acquisition systems are two different systems, the acquisition system design of the technical solution of the present invention is more complicated than that of an acquisition system (both are one-dimensional voltage signals or both are images). When fusing them, sufficient hardware design is required, considering the compromise between sampling speeds and how to coordinate analysis of different signals.

[0061] The technical solution of the present invention combines fluorescence signal detection and cell imaging, which not only solves the morphological information, but also has high-sensitivity fluorescence signal detection. It can perform both imaging flow and high-speed fluorescence signal detection, and can perform both label-free detection and labeled detection.

[0062] The overall idea proposed by the present invention is:

[0063] The present invention adopts a dual-light source illumination system, which can simultaneously capture the fluorescence spectrum information, bright field image, and light scattering image of a single particle under hydrodynamic focusing. Fluorescence spectrum information can quickly and accurately classify fluorescent particles from non-fluorescent particles. Bright field images can provide rich morphological information of particles with high resolution; light scattering images can provide scattered light in two angle ranges of polar angle and azimuth angle of particles, which can obtain richer information than one-dimensional light scattering, with higher resolution level, and can realize the correct size identification of particles; the three have complementary advantages, and can perform single particle multi-parameter detection with various detection methods such as label-free or label-free.

[0064] Embodiment 1

[0065] This embodiment discloses a single particle fluorescence spectrum and label-free imaging multi-parameter measurement flow device, such as Figure 1 As shown, it includes: a fluid system, an optical system and a signal acquisition system.

[0066] The liquid flow system uses sheath liquids on both sides to focus the sample liquid, which is used to change the speed of the sheath liquid and the sample liquid, and control the position and flow speed of the sample in the observation area;

[0067] When the sheath liquid on both sides is used to focus the sample liquid, specifically: in a flow channel, the sheath liquid flows from both sides and the sample liquid flows from the middle. Due to the slow flow rate, the sheath liquid and the sample liquid are both laminar flows and will not mix with each other. Due to the fast speed and high pressure of the sheath liquid, the pressure of the sheath flow gradually reduces the width of the sample flow and is finally compressed into a narrow flow (usually a few microns to tens of microns wide). Due to the stability of the laminar flow, the sample flow will not experience turbulence or diffusion during the compression process, thereby maintaining a focused state.

[0068] The liquid flow system is divided into a sheath flow meter for liquid flow and a syringe pump for pressure-driven liquid flow. The liquid flow rate can be changed by adjusting the parameters of the syringe pump.

[0069] When controlling the position and flow speed of the sample in the observation area, the sheath flow needs to be controlled to be narrow and the speed needs to match the speed that the imaging system can collect to prevent image tailing due to excessive speed. The particles need to flow in the center of the flow chamber at an appropriate speed.

[0070] The optical system uses laser and bright field light sources to simultaneously illuminate the cell samples, and through spectroscopic technology, provides fluorescence spectrum information and light scattering images generated by laser irradiation of cells, and bright field images generated by bright field irradiation of cells.

[0071] The signal acquisition system captures the sample's three-channel fluorescence spectrum signal, light scattering image, and bright field image, and transmits them to the computer for different signal acquisition and display.

[0072] In this embodiment, the fluid system includes a sheath flow device 7, a sample injection pump 8 and a sheath flow injection pump 9. The sheath flow device of this embodiment is a rectangular glass channel with a length of 12 mm, a width of 0.5 mm and a thickness of 4 mm, which is used to transport and fluidically focus the introduced cell samples, ensuring that the spatial positions of the cell samples in the sheath flow device do not overlap, and the sample observation area is located at the center of the sheath flow device. There is an inlet at the upper end, which is connected to the syringe clamped on the sample injection pump 8 through a pipeline, and there are sheath flow ports at the left and right ends, which are connected to the syringe clamped on the sheath flow injection pump 9 through a pipeline.

[0073] It should be noted that regarding single-ended sheath flow: single-ended sheath flow is usually introduced from one side of the sample flow, which may cause asymmetric compression of the sample flow in the flow chamber, uneven focusing effect, and affect the focusing accuracy. It may cause the sample flow to diffuse in the flow chamber, especially under high flow rate conditions, resulting in unstable position of the sample flow in the detection area, affecting the sensitivity and resolution of the detection signal, causing uneven distribution of the sample flow in the flow chamber, and increasing the risk of clogging.

[0074] Therefore, a double-ended sheath flow is adopted in the sub-technical scheme of this embodiment: the double-ended sheath flow introduces the sheath flow symmetrically from both sides of the sample flow, which can apply uniform pressure to the sample flow, so that it is accurately compressed at the center of the flow chamber, forming a more stable single-cell flow, improving the focusing accuracy, ensuring that cells or particles pass through the detection area one by one, and can effectively inhibit the diffusion of the sample flow, maintain the narrowness and stability of the sample flow, and flexibly control the width of the sample flow to adapt to a wider sample flow range and experimental conditions. The sample flow can be stably confined to the center of the detection area, improving the sensitivity and resolution of the detection signal, and can reduce the risk of blockage of the sample flow in the flow chamber, thereby improving the stability and reliability of the equipment.

[0075] The sample injection pump 8 controls the speed of injecting the cell sample liquid, and the sheath flow injection pump 9 controls the speed of injecting the sheath liquid; the syringe of the sample injection pump 8 is used to continuously introduce the cell sample liquid into the sheath flow device, and the syringe of the sheath flow injection pump 9 is used to continuously introduce the sheath liquid into the sheath flow device.

[0076] The optical system is divided into an excitation end and a receiving end. The excitation end of the optical system includes a laser 1, a mercury lamp 2, a fluorescent illuminator 3, a bandpass filter 4, a first focusing objective lens 5, and a cylindrical lens 6. The laser emitted by the laser 1 is shaped into a light sheet by the cylindrical lens 6 and used as an excitation light source for light scattering and fluorescence. The white light emitted by the mercury lamp 2 is focused by the fluorescent illuminator 3, the bandpass filter 4, and the first focusing objective lens 5 as a bright field light source. In this embodiment, the mercury lamp light source can freely adjust the intensity and has full spectrum emission. The laser light source selected is a helium-neon laser with a wavelength of 532nm. The bandpass filter 4 is 414-60nm, the cylindrical lens focuses at a distance of 50mm, and the focusing objective lens is a 10× objective lens with a numerical aperture of 0.25.

[0077] The sub-optical system of this embodiment uses dual light sources for illumination, in which the optical paths of the laser emitted by the laser and the white light emitted by the mercury lamp need to be designed separately and precisely coupled to ensure that the sheet laser and the point white light overlap in the sample area; complex calibration steps are required. The uniform illumination of the sheet laser avoids local over-excitation of the sample. The local illumination of the point white light reduces the light damage to non-target areas. The sheet laser provides a uniform fluorescence excitation background, and the point white light enhances the contrast of the bright field image. Combining the two can improve the image quality and the accuracy of the analysis.

[0078] The fluorescent illuminator in the sub-optical system of this embodiment is IX3-RFALFE, which adopts a compound eye lens system and can provide uniformly distributed illumination light, which can make the illumination of the entire field of view, including the peripheral area of ​​the field of view, bright and evenly distributed.

[0079] The mercury lamp light source in the sub-optical system of this embodiment includes a mercury lamp and a bandpass filter, which is used to filter the light source generated by the mercury lamp through the bandpass filter to obtain a bright field light source with a wavelength between 384-444nm.

[0080] The laser is used to generate the laser light source to obtain light scattering images and fluorescence spectrum information.

[0081] The second focusing objective lens 10 is used to collect a bright field image parallel to the bright field light source and a light scattering image and fluorescence spectrum information perpendicular to the laser.

[0082] The receiving end of the optical system divides the mixed light collected by the second focusing objective lens 10 into fluorescence spectrum information, light scattering image, and bright field image through a dichroic mirror and a fluorescence filter, and specifically includes the second focusing objective lens 10, a first long-pass dichroic mirror 11, a first short-pass dichroic mirror 12, a second long-pass dichroic mirror 13, a third long-pass dichroic mirror 14, a reflecting mirror 15, a second band-pass filter 16, a third band-pass filter 17, a fourth band-pass filter 18, a third focusing objective lens 19, a fourth focusing objective lens 20, and a fifth focusing objective lens 21.

[0083] The second focusing objective lens 10 collects the fluorescence information and light scattering image emitted by the sample perpendicular to the laser, as well as the bright field image parallel to the mercury lamp.

[0084] The color separation mirror is used to separate different parts of the cell sample information collected by the objective lens to obtain results including bright field images, light scattering images and fluorescence spectrum information.

[0085] Specifically, the collected light first passes through the first long-pass dichroic mirror 11 with a wavelength of 505nm to separate the bright field image, corresponding to a wavelength of 384-444nm; the light passing through the first long-pass dichroic mirror 11 passes through the first short-pass dichroic mirror 12 to separate the light scattering with a wavelength less than 550nm and the fluorescence with a wavelength greater than 550nm, and the fluorescence spectrum information containing the fluorescence and the light scattering image result containing the light scattering cells are obtained from the light separated from the bright field image. In this embodiment, a light scattering image of 532nm is obtained.

[0086] Next, the fluorescence channel passes through the second long-pass dichroic mirror 13, the third long-pass dichroic mirror 14 and the reflector 15 to separate three beams of parallel light with different wavelengths, and separates the fluorescence into fluorescence with a wavelength of 550-605nm, fluorescence with a wavelength of 605-659nm, and fluorescence with a wavelength above 659.

[0087] The three parallel beams of light pass through the second bandpass filter 16, the third bandpass filter 17, and the fourth bandpass filter 18 to obtain fluorescence signals of three bands. The fluorescence signal is focused into a point shape by the collecting objective lens. The second bandpass filter 16 is a bandpass filter with a central wavelength of 580nm and a bandwidth of 10nm, the third bandpass filter 17 is a bandpass filter with a central wavelength of 635nm and a bandwidth of 10nm, and the fourth bandpass filter 18 is a bandpass filter with a central wavelength of 680nm and a bandwidth of 10nm. The three focusing objective lenses are 4× objective lenses with a numerical aperture of 0.1.

[0088] The focusing lens at the collecting end (including the third focusing lens 19, the fourth focusing lens 20, and the fifth focusing lens 21) is used to focus the fluorescence of different wavelength bands separated by the dichroic mirror. The bandpass filter (including the second bandpass filter 16, the third bandpass filter 17, and the fourth bandpass filter 18) is used to obtain fluorescence of a specific bandwidth.

[0089] In the above optical system, the first focusing objective lens is used to focus the bright field light source; the cylindrical lens is used to focus the laser light source; the second focusing objective lens is used to collect the light of the irradiated sample; the dichroic mirror is used to separate different parts of the cell sample information collected by the collecting objective lens to obtain results including bright field images, light scattering images and three-channel fluorescence signals; the collecting end focusing objective lens is used to focus fluorescence of different bands; and the spatial filter is used to obtain fluorescence of a specific bandwidth.

[0090] The signal acquisition system includes a first PMT22, a second PMT23, a third PMT24, a data acquisition card 25, a first CMOS camera 26, a second CMOS camera 27, and a data analysis system 28. The signal acquisition system can simultaneously acquire bright field image information, light scattering image information, and fluorescence information. The bright field image information is reflected to the second CMOS camera 27 by the first long-pass dichroic mirror reflector 11; the light scattering information is transmitted to the first CMOS camera 26 by the reflector 12; the fluorescence information of the three bands is focused in a point shape by three focusing objective lenses and then transmitted to the third PMT24, the second PMT23, and the first PMT22 light windows. The fluorescence information is photoelectrically converted and amplified by the PMT and then outputs an analog voltage signal. The three PMT output lines are respectively connected to the three analog voltage input channels of the data acquisition card 25, and the data acquisition card converts the analog signal into a digital signal and transmits it to the computer through the USB interface. The voltage signal output by the data acquisition card is collected and processed using the acquisition software. The program includes: an analog voltage acquisition program for collecting signals; a file saving function for saving data information; and a high-frequency filter for filtering out high-frequency noise signals.

[0091] In the present embodiment, the above-mentioned device can simultaneously collect single-particle fluorescence spectra, light scattering images and bright field images through two light sources. When detecting fluorescent markers, it can provide clearer specific imaging information while collecting fluorescence spectrum information, realize single particle size recognition and classify and identify mixed particles.

[0092] The working process of the above device:

[0093] The laser beam emitted by the laser 1 is focused by the cylindrical lens 6 into a sheet-shaped light source and irradiated into the microfluidic channel 7, exciting the sample to produce a light scattering image and a fluorescence spectrum signal. The mercury lamp light source 2 passes through the fluorescent illuminator 3, passes through the first bandpass filter 4 and the first focusing objective lens 5 to form a short-wavelength point-shaped light spot and irradiates the same irradiation area of ​​the microfluidic channel 7, irradiating the sample to produce a bright field image.

[0094] After the sample is excited by the two light sources, the second focusing objective lens 10 collects the bright field image, light scattering image and fluorescence signal of the sample. The bright field image reflected by the first long-pass dichroic mirror 11 is transmitted to the second CMOS camera 27, and the light scattering image is transmitted to the first CMOS camera 26 through the first short-pass dichroic mirror 12. The reflected fluorescence spectrum signal is separated into three fluorescences of different wavelengths through the second long-pass dichroic mirror 13, the third long-pass dichroic mirror 14 and the reflector 15. The fluorescences of the three fluorescences of different wavelengths are separated through the fourth band-pass filter 18, the third band-pass filter 17 and the reflector 18. The second bandpass filter 16 transmits fluorescence of a specific band, which is focused by the fifth focusing lens 21, the fourth focusing lens 20, and the third focusing lens 19 and then transmitted to the third PMT 24, the second PMT 23, and the first PMT 22 respectively. The data of the three PMTs are output to the data acquisition card 25. The signal is converted into digital-to-analog by the data acquisition card, and the digital signal is transmitted to the digital analysis system 28 through the USB interface. The first CMOS camera 26 and the second CMOS camera 27 transmit the image information to the digital analysis system 28 through the USB interface for data processing and analysis.

[0095] Fluorescence signal acquisition and data analysis require programming. The program consists of four parts, namely channel setting, timing setting, recording setting, and data acquisition. The channel setting can be used to configure the terminal. The terminal configuration used by this device is single-ended. The physical channel configuration is used by this device for the first three analog voltage channels of the data acquisition card. The minimum and maximum voltages of the data acquisition card used by this device are -5V and +5V. The timing setting can set the sampling rate and number of samples. Different sampling rates and numbers of samples can be set according to the measured samples and the required display time. The recording setting can save the collected data file, select the file recording mode, and the file saving path. The acquisition mode of the data acquisition is set. The acquisition mode used by this device is analog voltage multi-channel continuous sampling. In the acquisition mode, a stop acquisition button is set to stop at any time. Its multi-channel signal is connected to a waveform graph for original signal display. Due to the existence of noise, a filter is added after the original signal is acquired for noise elimination. The filter frequency is selected according to the experimental environment. The filtered voltage signal is then displayed through a waveform graph to compare with the original signal. The filtered signal is separated into single signals through a multiplexing device, and a waveform is added after each single signal for signal display.

[0096] The process of simultaneously collecting bright field images, light scattering images, and fluorescence signals is as follows:

[0097] (1) Open the camera software, adjust the exposure time and gain according to the image quality, set the number of bright field and scattered light images, and select the file saving path;

[0098] (2) Open the fluorescence signal acquisition program, adjust the filter cutoff frequency according to the quality of the acquired signal, set the appropriate sampling frequency, and select the file saving path;

[0099] (3) Click the three acquisition program start acquisition buttons at the same time to start acquisition, and stop acquisition at the same time after the appropriate acquisition time.

[0100] The collection flow chart is as follows Figure 2 shown.

[0101] Verification Example 1:

[0102] In order to verify that the present invention has the function of label-free imaging and can perform particle size recognition and morphology recognition, 2 μm non-fluorescent polystyrene beads are used to verify the label-free detection function, and light scattering and bright field imaging are performed simultaneously.

[0103] Specific steps:

[0104] (1) Take an appropriate amount of non-fluorescent ball stock solution and dilute it with ultrapure water. The ball suspension is used as the sample solution and the ultrapure water is used as the sheath solution.

[0105] (2) Turn on the mercury lamp, sample injection pump, sheath injection pump, light scattering camera and image acquisition software, and bright field camera and image acquisition software;

[0106] (3) First, adjust the camera exposure time and gain according to the imaging quality. When the imaging is appropriate, adjust the sample liquid speed to 5 μL / h and the sheath liquid speed to 250 μL / h, which can form a focused single particle flow. When the liquid flow is stable, adjust the sheath flow device and the collection lens position to make the light scattering and bright field images in a focused state. Then adjust the camera position to make the light scattering image in a defocused state.

[0107] (4) Set the acquisition quantity and save path of the two image acquisition software, and then click to start acquisition at the same time;

[0108] (5) Analyze the collected images through a computer.

[0109] The experimental results of this embodiment are as follows Figure 3 As shown, Figure 3This is a one-to-one correspondence diagram between light scattering images and bright field images. When making the correspondence, the correspondence is made through the time difference between the appearance of two particles. First, a light scattering image containing particles is selected, and the corresponding bright field image is found according to the sampling time. Then, the difference in the number of light scattering images of the next particle and the previous image is counted, and the bright field image is checked to see if there is a corresponding bright field image after the same number of images. If so, the correspondence is correct. This embodiment selects the light scattering image and the corresponding bright field image of the particles that pass through a continuous period of time. It can be seen from the figure that the light scattering image of the 2μm non-fluorescent sphere has five stripes, and corresponds one-to-one with the bright field image. The bright field image can display the morphological information of the particles. It proves that the device can perform label-free imaging detection.

[0110] Verification Example 2:

[0111] In order to verify that the present invention can simultaneously realize the acquisition of trimodal information, polystyrene beads are used for experimental verification and device inspection. In the present invention, standard fluorescent polystyrene beads with maximum excitation and emission wavelengths of 535nm and 575nm, respectively, and a particle size of 2μm are selected as samples, and fluorescence information, light scattering images, and bright field images are simultaneously obtained under hydrodynamic focusing.

[0112] Specific steps:

[0113] (1) Take an appropriate amount of polystyrene fluorescent ball stock solution and dilute it with ultrapure water, using the ball suspension as the sample solution and ultrapure water as the sheath solution;

[0114] (2) Turn on the mercury lamp, laser, sample injection pump, sheath injection pump, data acquisition card, light scattering camera and image acquisition software, bright field camera and image acquisition software;

[0115] (3) Adjust the sample liquid speed to 5 μL / h and the sheath liquid speed to 250 μL / h. When the liquid flow is stable, adjust the sheath flow meter and the collection lens position to make the fluorescence signal intensity appropriate, set the filter cutoff frequency for filtering, and then adjust the camera position to make the bright field in a focused state and the light scattering in a defocused state;

[0116] (4) Set the sampling rate and saving path of the fluorescence acquisition program, set the acquisition quantity and saving path of the two image acquisition software, and then click to start acquisition at the same time.

[0117] (5) Analyze the collected data through computer

[0118] The experimental results of this embodiment are as follows Figure 4(a)-Figure 4(b)As shown, Figure 4 (a) is the three-channel fluorescence information of a 2μm fluorescent ball, and Figure 4 (b) is the bright field image and light scattering image of a 2μm fluorescent ball. It can be seen from the figure that the three-channel intensity and spectrum of the ball under 532nm laser excitation correspond to each other, and the voltage intensity of the same ball is uniform, which proves that the fluorescence spectrum acquisition module has good acquisition quality and small voltage width differences, which proves that the single-cell flow focusing effect is good. If there are multiple balls at the same position, the voltage amplitude will increase significantly. When performing the three-modal information correspondence of the ball, first select a period of three-modal data, calculate the appearance time of the first ball voltage waveform according to the sampling time, and the number of image acquisitions, and perform the correspondence, and convert the voltage time interval of the next ball into the number of image acquisitions, and check from the light scattering image and bright field image whether there is a corresponding image of the second ball after the first ball image, so as to perform the three-modal correspondence. As shown in the figure, the voltage waveform within 1s has four peaks from left to right corresponding to the bright field image and light scattering image of four fluorescent balls. It can be seen from the third peak that the third peak does not collect a single ball. Then, according to the bright field and light scattering images, it can be confirmed that the first peak and the second peak are single 2μm fluorescent balls, while the third peak is two fluorescent balls. The fourth peak is two fluorescent balls in close positions, and the light intensity of the two fluorescent balls is about twice that of a single fluorescent ball. If the two fluorescent balls are too close, the fluorescence intensity of the two balls will overlap and appear as one peak.

[0119] Verification Example 3:

[0120] Using a flow device that simultaneously collects bright field and light scattering imaging and multi-color fluorescence, 2μm standard fluorescent polystyrene, 3.87μm and 4.19μm standard non-fluorescent polystyrene mixed beads are identified and classified. In this experiment, three fluorescent bead suspensions of specific concentrations were prepared, and then the three were mixed in a certain proportion. The appropriate sheath flow and sample liquid flow rate ratio was adjusted to collect three-modal information, and the mixed beads were classified based on the collected information.

[0121] Specific steps:

[0122] (1) Take three kinds of microspheres and dilute them with ultrapure water to a suitable concentration, then mix them in a certain proportion, with the mixed microsphere suspension as the sample liquid and ultrapure water as the sheath liquid;

[0123] (2) Turn on the mercury lamp and laser, sample injection pump, sheath injection pump, and data acquisition card;

[0124] (3) Adjust the sample liquid speed to 5 μL / h and the sheath liquid speed to 300 μL / h. When the liquid flow is stable, adjust the sheath flow meter and the collection lens position to make the fluorescence signal intensity appropriate, set the filter cutoff frequency for filtering, and then adjust the camera position to make the bright field in a focused state and the light scattering in a defocused state;

[0125] (4) Open the CMOS camera and the fluorescence signal acquisition program at the same time to collect three-channel data, and classify the beads according to the saved information.

[0126] The experimental results of this embodiment are as follows Figure 5(a)-Figure 5(b) As shown in the figure, the 2μm fluorescent beads in the mixed beads will generate fluorescent signals, while the 3.87μm and 4.19μm beads will not generate fluorescent signals, but all three can generate bright field images and light scattering images, and there are differences between the bright field images and the light scattering images. When classifying the beads, firstly, the fluorescent beads containing voltage signals are selected according to the time to establish the correspondence between the three signals, and the non-fluorescent beads are imaged by the acquisition time. Secondly, the three signal types of each bead are classified and identified. The 2μm fluorescent beads have fluorescent signals, the bright field image is small, and the light scattering image has five stripes. The 3.87μm non-fluorescent beads have no fluorescent signals, the bright field image is large, and the light scattering image has nine stripes. The 4.19μm non-fluorescent beads have no fluorescent signals, the bright field image is large, and it is similar to the bright field image of the 3.87μm non-fluorescent beads, and the light scattering image has ten stripes. According to the three signal characteristics of each bead, different types of beads can be distinguished well. And the size of the beads can be identified by light scattering, and the bright field image shows morphological information. As shown in Figure 5(a), it is a fluorescence signal waveform diagram, in which each wave corresponds to one or more 2μm fluorescent spheres. Figure 5(b) is the bright field image and light scattering image of the sphere. The left side of Figure 5(b) is the light scattering image and bright field image of a 2μm fluorescent sphere indicated by the fluorescence waveform diagram of Figure 5(a). It can be seen that the light scattering image has five stripes and the bright field image is smaller. The middle of Figure 5(b) is the light scattering image and bright field image of a 3.89μm non-fluorescent sphere. It can be seen from the figure that its light scattering image has nine stripes, and the bright field image is much larger than that of the 2μm fluorescent sphere. The right side of Figure 5(b) is the light scattering image and bright field image of a 4.19μm non-fluorescent sphere. The light scattering image has ten stripes, and the bright field image is similar to that of the 3.87μm non-fluorescent sphere.

[0127] Of course, the device provided by the present invention can also be used for cell analysis in other scenarios and is not limited to the usage scenarios of the embodiments.

[0128] Embodiment 2

[0129] The purpose of this embodiment is to provide a measurement method of a single particle fluorescence spectrum and label-free imaging multi-parameter measurement flow device, including:

[0130] Step (1): Turn on the light source, adjust the position of the sample chip so that the laser and mercury lamp irradiation areas overlap, adjust the optical element (second focusing lens 10) and the collection device so that their horizontal heights are equal and the mixed light can be collected by the collection device (first CMOS camera 26; second CMOS camera 27; first PMT22; second PMT23; third PMT24) through the optical path;

[0131] Step (2): preparing a sample suspension and introducing it into the sample chip through a syringe and a syringe pump, and adjusting the flow rate ratio of the sheath liquid and the sample liquid through the syringe pump so that the sample forms a stable focusing flow in the middle of the chip;

[0132] Step (3): Connect the acquisition card and the camera to the computer, open the camera software and the acquisition card acquisition program, adjust the bright field and light scattering focus according to the sample imaging, and then move the first CMOS camera 26 to make the light scattering image diverge, so as to form a suitable light scattering image;

[0133] Step (4): Select a suitable sampling rate and filter cutoff frequency according to the voltage signal displayed by the software, and when the signal is suitable, select the file saving path, set the appropriate acquisition quantity and saving path for the image acquisition software, and enable the three acquisition software to collect information at the same time, and stop the acquisition at the same time after a suitable time;

[0134] Step (5): Analyze the information collected by the three detectors to perform image recognition and sample classification.

[0135] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. Single particle fluorescence spectroscopy and label-free imaging multi-parameter measurement flow device, characterized in that: include: A liquid flow system, wherein the liquid flow system comprises a sheath flow device, wherein the sheath flow device contains a sheath fluid and a sample fluid, wherein the sample fluid contains a cell sample; An optical system, wherein the optical system comprises an excitation end and a receiving end, wherein the excitation end comprises a first light source and a second light source, wherein the first light source and the second light source jointly illuminate the cell sample to excite particles to generate fluorescence spectrum information and a light scattering image and a bright field image; The signal acquisition system is used to simultaneously acquire fluorescence spectrum signals, bright field images and light scattering images.

2. The single particle fluorescence spectroscopy and label-free imaging multi-parameter measurement flow device as claimed in claim 1, characterized in that: The liquid flow system also includes a sample injection pump and a sheath flow injection pump. The upper end of the sheath flow device is provided with an injection port, and the injection port is connected to the syringe clamped on the sample injection pump through a pipeline. The left and right ends of the sheath flow device are respectively provided with a first sheath flow port and a second sheath flow port, and the first sheath flow port and the second sheath flow port are respectively connected to the syringe clamped on the sheath flow injection pump through pipelines.

3. The single particle fluorescence spectroscopy and label-free imaging multi-parameter measurement flow device as claimed in claim 1, characterized in that: The sheath flow device is a rectangular glass channel. The speeds of the sample liquid and the sheath liquid are changed by the sample injection pump and the sheath flow injection pump, thereby controlling the position and flow speed of the cell sample in the observation area of ​​the sheath flow device.

4. The single particle fluorescence spectroscopy and label-free imaging multi-parameter measurement flow device as claimed in claim 1, characterized in that: The first light source is a laser light source, and the second light source is a mercury lamp light source.

5. The single particle fluorescence spectroscopy and label-free imaging multi-parameter measurement flow device as claimed in claim 1, characterized in that: The excitation end also includes a fluorescent illuminator, a bandpass filter, a first focusing objective lens, and a cylindrical lens; The laser light emitted by the laser light source is shaped into a light sheet by a cylindrical lens and then used as an excitation light source for light scattering and fluorescence; The light emitted by the mercury lamp light source is focused by the fluorescent illuminator, the bandpass filter and the first focusing objective lens in sequence to serve as a bright field light source.

6. The single particle fluorescence spectroscopy and label-free imaging multi-parameter measurement flow device as claimed in claim 1, characterized in that: The receiving end of the optical system comprises a second focusing objective lens, a first long-pass dichroic mirror, a first short-pass dichroic mirror, and fluorescence spectrum information acquisition units of multiple channels, wherein the fluorescence spectrum information acquisition unit of each channel has the same structure, including a long-pass dichroic mirror, a band-pass filter and a focusing objective lens arranged in sequence; The second focusing objective lens collects the fluorescence spectrum information and light scattering image emitted by the cell sample perpendicular to the laser, as well as the bright field image parallel to the mercury lamp light source; The information collected by the second focusing objective lens is reflected by the first long-pass dichroic mirror to form a bright field image, and the light scattering image is transmitted through the first short-pass dichroic mirror. The fluorescence spectrum information reflected by the first short-pass dichroic mirror is first separated into multiple fluorescence bands by the long-pass dichroic mirror of the fluorescence spectrum information collection unit of multiple channels. The fluorescence of multiple different bands is respectively transmitted through band-pass filters to transmit the fluorescence of specific bands, and then respectively transmitted after being focused by the objective lens.

7. The single particle fluorescence spectroscopy and label-free imaging multi-parameter measurement flow device as claimed in claim 1, characterized in that: The signal acquisition system includes a plurality of PMTs, a data acquisition card, a first CMOS camera, a second CMOS camera, and a data analysis system; the number of the PMTs is consistent with the number of channels of the fluorescence spectrum information acquisition unit; The bright field image is reflected to the second CMOS camera after passing through the first long-pass dichroic mirror; The light scattering image is transmitted to the first CMOS camera through the first short-pass dichroic mirror; The fluorescence spectrum information of multiple bands is focused into point shape by multiple focusing lenses and then transmitted to the corresponding light window of PMT respectively; The output lines of the PMT are respectively connected to the analog voltage input channels of the data acquisition card. The data acquisition card converts the analog signal into a digital signal and transmits it to the data analysis system through an interface.

8. The single particle fluorescence spectroscopy and label-free imaging multi-parameter measurement flow device as claimed in claim 1, characterized in that: The fluorescence spectrum information acquisition unit has three channels.

9. A method for multi-parameter measurement of single particle fluorescence spectroscopy and label-free imaging, characterized by comprising: Prepare a sample suspension and introduce it into the sheath flow device, and adjust the flow rate ratio of the sheath liquid and the sample liquid so that the cell sample forms a stable focusing flow in the middle of the sheath flow device; Adjust the position of the sheath flow device so that the illumination areas of the two light sources on the sheath flow device overlap, adjust the bright field focus and light scattering defocus, and the two light sources jointly illuminate the cell sample to excite the particles to generate fluorescence spectrum information and light scattering images and bright field images; Select the appropriate sampling rate and filter cutoff frequency, then select the file save path to collect the generated information; The collected information is analyzed to perform image recognition and sample classification.

10. The single particle fluorescence spectrum and label-free imaging multi-parameter measurement method according to claim 9, characterized in that: When the information is collected and generated, the three working modes of fluorescence spectrum, light scattering imaging and bright field imaging can work separately or simultaneously, and the fluorescence spectrum, light scattering image and bright field image of the labeled single particle or one or two of the information can be detected simultaneously.

Images