CN114705611A - A flow cytometer based on confocal optical path design - Google Patents

Abstract

A flow cytometer based on confocal optical path design belongs to the technical field of life analysis scientific detection equipment. The flow cytometer includes a liquid flow system, an optical system and a detection system; the liquid flow system includes an injection pipeline and a sheath liquid pipeline respectively connected with the quartz flow cell, and the injection pipeline injects into the central sheath liquid channel of the quartz flow cell Cell sample, the cell sample is wrapped and squeezed by the sheath flow injected into the sheath liquid channel in the center of the quartz flow cell to form a single cell flow; the optical system includes an excitation light path and a fluorescence collection light path, and the laser beam of the excitation light path is focused on the quartz flow The central sheath flow channel of the cell is used to excite the cell samples in the central sheath flow channel of the quartz flow cell to generate fluorescence, and the fluorescence is collected by the fluorescence collection optical path; the detection system includes a photodetector, a data acquisition card and an upper computer connected in sequence. The flow cytometer of the invention has a compact structure, simple and efficient focusing of the optical path, and is convenient to use.

Description

A flow cytometer based on confocal optical path design

technical field

The invention relates to the technical field of life analysis scientific detection equipment, in particular to a flow cytometer based on a confocal optical path design.

Background technique

Cells are the most basic unit that constitutes life and completes life activities, and there is general heterogeneity between cells. Therefore, characterizing life activities at the single-cell level has become a life science research such as proteomics and metalloomics. The technical means necessary in the medical field such as the diagnosis and treatment of diseases and diseases. Flow cytometry is currently the most widely used high-throughput single-cell multiparameter analysis technology. Flow cytometer can obtain quantitative information of intracellular biomolecules such as fluorescently labeled proteins, and information such as cell structure and morphology through the optical and fluorescent properties of single cells in sheath flow when they pass through a light source (usually a laser beam). Conduct research on cell viability, immunophenotype, and physiological functions such as endocytosis. The target analytes for flow cytometry have also now expanded from cells to smaller sized particles such as microorganisms, nuclei, chromosomal preparations, and nanoparticles.

A flow cytometer usually includes a fluidic system, an optical system, and a detection system. The liquid flow system is to introduce the cells in the form of suspension and use the sheath flow focusing technology to focus and arrange the cells, and pass through the detection area of the optical system according to a fixed path. The sheath flow focusing technology is that after the sheath flow and the sample flow converge in the microchannel of the flow cell, relying on the flow rate difference between the sheath flow and the sample flow, the sample flow forms stable cells of a certain width under the wrapping and extrusion of the sheath liquid. Streamline, so that the chaotic and scattered cells are arranged in a row and pass through the detection area of the optical system one by one. This technology can ensure that the cells in the suspension follow the same path through the excitation light source to illuminate the center of the spot, ensure that the cells passing through the detection area are irradiated by the same excitation light source, and greatly reduce the probability of cells passing through the detection area. Single-cell fluorescence detection signal. It can be seen that the efficient and stable focusing of the cells by the liquid flow system is the prerequisite for the flow cytometer to obtain accurate single-cell detection signals. In order to obtain stable focusing effect, traditional flow cytometers use a complex integrated flow cell, which not only requires high processing precision, but also is expensive and difficult to maintain.

The optical system of the flow cytometer includes an excitation light path and a fluorescence collection light path. The excitation light path includes a laser, a laser beam quality adjustment module (such as beam expander, beam reduction, filter, etc.), a laser focusing module, etc. The fluorescence collection light path includes fluorescence collection, Modules such as collimation, filtering, and fluorescence photoelectric conversion and signal amplification. The traditional flow cytometer adopts the mode of orthogonal optical path design, that is, the excitation optical path and the fluorescence collection optical path are perpendicular to each other. The orthogonal optical path can effectively reduce the background interference of the excitation light and improve the sensitivity, but this mode requires the excitation light converging lens and the fluorescence collection lens to focus on the cell streamline from two directions. It will greatly reduce the detection sensitivity, so professional engineers are usually required to complete the optical path focus adjustment. In addition, it is difficult to realize a compact instrument in the orthogonal optical path mode, and the instrument is relatively bulky.

The detection system of the flow cytometer includes a photoelectric conversion module, a signal acquisition module and a data processing module. The photoelectric conversion module is usually a photomultiplier tube, etc., which converts the weak fluorescent signal into an electrical signal and amplifies it. The electrical signal output by the photoelectric conversion module It is collected and recorded by the data acquisition module, and the massive data points are processed by the data processing software to obtain the final cell analysis result. The data processing of traditional flow cytometers is usually online, which is fast and can be observed in real time, but at the same time, the complicated and incomprehensible gate threshold settings make the instrument operation require long-term training and practical practice.

SUMMARY OF THE INVENTION

In order to solve the technical problems of the existing flow cytometer, such as low sensitivity, complex optical path adjustment, cumbersome use process and test parameter setting, and high price, the present invention provides a flow cytometer based on confocal optical path design. Analyzer.

In order to achieve the above object, the technical scheme of the present invention is:

A flow cytometer based on confocal optical path design, comprising a liquid flow system, an optical system and a detection system;

The liquid flow system includes an injection pipeline and a sheath fluid pipeline that are respectively communicated with the quartz flow cell. The injection pipeline injects the cell sample into the central sheath fluid channel of the quartz flow cell, and the cell sample flows to the quartz in the sheath fluid pipeline. Single-cell flow is formed under the wrapping and extrusion of the sheath flow injected by the sheath fluid channel in the center of the pool;

The optical system includes an excitation light path and a fluorescence collection light path. The laser beam of the excitation light path is focused on the central sheath flow channel of the quartz flow cell, and is used to excite the cell sample in the central sheath flow channel of the quartz flow cell to generate fluorescence, and the fluorescence passes through the central sheath flow channel of the quartz flow cell. Fluorescence collection optical path collection;

The detection system includes a photodetector, a data acquisition card and a host computer which are connected in sequence. The photodetector detects the fluorescence collected by the fluorescence collection optical path, and sends the data to the host computer for storage through the data acquisition card.

Further, the liquid flow system further includes a flushing pipeline, which is communicated with the quartz flow cell and is used for flushing the central sheath liquid channel of the quartz flow cell and discharging air bubbles in the liquid path.

Further, the injection pipeline includes a syringe pump, a sample injection syringe and a capillary that are communicated in sequence, and the outlet end of the capillary extends into the first port of the spool, passes through the third port of the spool, and is inserted into the capillary. to the entrance of the central sheath liquid channel of the quartz flow cell;

The sheath liquid pipeline includes a sheath liquid bottle communicated with the second port of the spool, and a first switch valve is provided on the pipeline communicated with the second port of the spool;

The flushing pipeline includes a flushing syringe that communicates with the fourth port of the four-way, and a second on-off valve is arranged on the pipeline that communicates with the fourth port of the four-way.

Further, the inlet end of the quartz flow cell is communicated with the third port of the spool, and the outlet end of the quartz flow cell is communicated with the waste liquid bottle.

Preferably, the capillary is a quartz capillary, the outer diameter is 0.1-0.4 mm, the inner diameter is 20-200 μm, and the outlet end of the capillary is ground into a 15°-45° conical tip.

Preferably, the quartz flow cell is a four-sided transparent quartz flow cell, the outer length, width and height are 4mm×4mm×10mm～6mm×6mm×20mm, and the central sheath fluid channel is 0.1mm×0.1mm～0.4mm×0.4mm rectangular channel.

Further, the excitation light path includes a laser, a laser beam reduction mirror, a first reflection mirror, a first filter, a dichroic mirror and an objective lens arranged in sequence, and the laser beam emitted by the laser passes through the laser beam reduction mirror, After the first reflection mirror, the first filter, the dichroic mirror and the objective lens, focus on the central sheath flow channel of the quartz flow cell; the fluorescence collection optical path includes the objective lens, the dichroic mirror and the second reflection mirror arranged in sequence , the second filter, the aspheric lens and the pinhole plate, the cell sample in the central sheath flow channel of the quartz flow cell generates fluorescence and is collected by the objective lens, and passes through the dichroic mirror, the second reflector, the second filter in turn After the film, the aspheric lens and the pinhole plate are detected, it is detected by the photodetector.

Further, the first filter, the dichroic mirror, the second reflector, the second filter, the aspherical lens and the pinhole plate are all packaged inside the cassette, and the laser beam-reducing mirror is fixed to the laser. At the beam exit, the laser, the first reflecting mirror and the objective lens are all arranged on the top of the cassette, the cassette is fixed on the optical plate, the first reflecting mirror and the second reflecting mirror are respectively fixed on the beam turning frame, the The spool and the quartz flow cell are fixed above the objective lens through a three-dimensional translation stage.

Compared with the prior art, the beneficial effects of the present invention are:

1. The present invention adopts the confocal optical path design, which can effectively reduce the stray light interference, improve the detection sensitivity, and greatly reduce the difficulty of focusing the optical path, and the user can adjust the focus by himself.

2. The liquid flow system of the present invention adopts a modular design, and the quartz flow cell, capillary, sheath liquid pipeline and flushing pipeline are independent of each other, which is convenient to use and easy to maintain, and the modular design can greatly reduce the processing accuracy requirements of parts, cut costs.

3. The sheath liquid channel of the present invention is coaxial with the sampling capillary and the central channel of the quartz flow cell, and the sheath liquid channel entrance is far away from the convergence point of the sheath liquid and the sample, which effectively avoids the sheath liquid pulsation disturbance or when the sheath liquid enters the focusing cell. The phenomenon of focus instability caused by the perturbation of the cell reduces the detection error caused by cell adhesion or overlapping.

4. In the ultra-high-throughput single-cell analysis workstation of the present invention, its instrument control and data acquisition system and data processing and analysis system can operate independently of each other, and the collected data is complete fluorescence intensity-time spectrum data, which users can directly obtain The raw data is processed offline by itself, and the data processing threshold setting of the data processing and analysis system is simple and easy to understand. Users can flexibly process and filter data according to their needs.

5. The present invention is based on a highly sensitive, time-resolved optical system and an efficient and stable liquid flow system, which can achieve high-throughput and high-accuracy single-cell analysis of at least 100 cells/s, and through the rapid and accurate analysis of raw data. Offline processing can obtain the peak height and peak area information of the fluorescent signal spectrum peak of the target analyte in a single cell, realize semi-quantitative analysis, and realize multi-variate display of detection data through statistical analysis.

6. The present invention is installed on an optical flat plate, with compact structure and small volume, and can be moved at will, and the present invention is a modular and open design, the liquid flow system and the detection system are independent of each other, and the user can modify the instrument or It can be used in combination with sample pre-processing modules such as microfluidic chips to meet the needs of personalized single-cell analysis.

Description of drawings

1 is a schematic structural diagram of a flow cytometer based on a confocal optical path design provided by the present invention;

FIG. 2 is a schematic diagram of the liquid flow system provided by the present invention.

in,

1-syringe pump, 2-injection syringe, 3-capillary, 4-four-way, 5-sheath liquid bottle, 6-quartz flow cell, 7-flushing syringe, 8-first on-off valve, 9-second on-off valve , 10-waste bottle, 11-laser, 12-laser beam-contractor, 13-first reflector, 14-first filter, 15-dichroic mirror, 16-objective lens, 17-second reflector , 18-second filter, 19-aspherical lens, 20-pinhole plate, 21-photodetector, 22-data acquisition card, 23-host computer, A-fluid system, B-optical system, C -Detection Systems.

Detailed ways

In order to solve the problems existing in the prior art, as shown in FIG. 1 and FIG. 2 , the present invention provides a flow cytometer based on confocal optical path design, including a liquid flow system A, an optical system B and a detection system C three part.

The liquid flow system A includes an injection pipeline and a sheath fluid pipeline that are respectively communicated with the quartz flow cell 6. The injection pipeline injects the cell sample into the central sheath liquid channel of the quartz flow cell 6, and the cell sample is in the sheath liquid pipeline to the center of the quartz flow cell 6. The single-cell flow is formed under the wrapping and extrusion of the sheath flow injected into the sheath liquid channel; the optical system B includes an excitation light path and a fluorescence collection light path. The laser beam of the excitation light path is focused on the central sheath flow channel of the quartz flow cell 6 to excite the quartz flow. The cell sample in the sheath flow channel in the center of the cell 6 generates fluorescence, and the fluorescence is collected by the fluorescence collection optical path; the detection system C includes a photodetector 21, a data acquisition card 22 and a host computer 23 connected in sequence, and the photodetector 21 detects the fluorescence collected by the optical collection path. The fluorescence is sent to the upper computer 23 through the data acquisition card 22 for storage.

The liquid flow system A further includes a flushing pipeline, which is communicated with the quartz flow cell 6 and is used for flushing the central sheath liquid channel of the quartz flow cell 6 and discharging air bubbles in the liquid path. The injection pipeline includes a syringe pump 1, a sample injection syringe 2 and a capillary tube 3 that are connected in sequence. The outlet end of the capillary tube 3 extends into the first port of the spool 4, passes through the third port of the spool 4, and is inserted into the spool 4. At the entrance of the central sheath liquid channel of the quartz flow cell 6; the sheath liquid pipeline includes a sheath liquid bottle 5 communicated with the second port of the spool 4, and the sheath liquid bottle 5 communicated with the second port of the spool 4 is provided with a The first on-off valve 8; the flushing pipeline includes a flushing syringe 7 communicating with the fourth port of the spool 4, and a second on-off valve 9 is provided on the pipeline connecting the flushing syringe 7 with the fourth port of the spool 4. The inlet end of the quartz flow cell 6 communicates with the third port of the spool 4 , and the outlet end of the quartz flow cell 6 communicates with the waste liquid bottle 10 .

Specifically, the liquid flow system A includes a syringe pump 1, a sample injection syringe 2, a capillary 3, a spool 4, a sheath liquid bottle 5, a quartz flow cell 6, a flushing syringe 7, a waste liquid bottle 10, a syringe pump 1, and a sample injection syringe 2. The capillary 3 is communicated in turn, and the spool 4 includes a first port, a second port, a third port and a fourth port. The outlet end of the capillary 3 extends into the spool 4 from the first port, and is inserted into the spool 4 after passing through the spool 4. At the entrance of the central sheath liquid channel of the quartz flow cell 6; the quartz flow cell 6 is communicated with the third port, the outlet end of the quartz flow cell 6 is communicated with the waste liquid bottle 10, the second port is communicated with the sheath liquid bottle 5, and the fourth port is communicated with the flushing The syringe 7 is communicated; a first switch valve 8 is arranged between the second port and the sheath liquid bottle 5 ; a second switch valve 9 is arranged between the fourth port and the flushing syringe 7 . The liquid flow system A and the optical system C are independent of each other, and the liquid flow system A is a modular design. The quartz flow cell 6, the capillary tube 3, the sheath fluid pipeline and the flushing pipeline are independent of each other, which greatly reduces the requirements for the machining accuracy of parts. Thus reducing costs.

The sample is pressed by the syringe pump 1 through the injection syringe 2 from the inlet end of the capillary 3, the capillary 3 extends from the first port of the spool 4 and is fixedly sealed, and the outlet end of the capillary 3 penetrates the spool 4 and then is inserted into the quartz flow cell 6. At the entrance of the central sheath liquid channel, the third port of the spool 4 is connected and sealed with the quartz flow cell 6, the second port is connected to the sheath liquid bottle 5 through a hose, and the sheath liquid flows into the quartz through the spool 4 under the action of gravity and static pressure. The central sheath fluid channel of the flow cell 6 forms a sheath flow and merges with the sample flow, and the sample flow flowing out from the tip of the outlet of the capillary 3 is arranged under the wrapping and extrusion of the sheath fluid to form a single-cell flow; the fourth port of the spool 4 passes through the soft The tube is connected to the flushing syringe 7 for flushing the central sheath liquid channel of the quartz flow cell 6 and the air bubbles in the discharge liquid path. The outlet end of the quartz flow cell 6 is connected to the waste liquid bottle 10 through a hose.

Specifically, the capillary 3 is a quartz capillary with an outer diameter of 0.1 to 0.4 mm and an inner diameter of 20 to 200 μm. The outlet end of the capillary 3 is polished into a 15° to 45° conical tip; the quartz flow cell 6 is a quartz flow cell that transmits light on all sides , the external length, width and height are 4mm×4mm×10mm～6mm×6mm×20mm, and the central sheath fluid channel is a rectangular channel of 0.1mm×0.1mm～0.4mm×0.4mm; the syringe pump 1 sets the sample injection flow rate to 2～200μL /min, and the sheath fluid flow rate was 100 to 2000 μL/min.

The excitation light path includes a laser 11, a laser beam reduction mirror 12, a first reflection mirror 13, a first filter 14, a dichroic mirror 15 and an objective lens 16 arranged in sequence; the fluorescence collection light path includes an objective lens 16, a dichroic Mirror 15, second mirror 17, second filter 18, aspheric lens 19 and pinhole plate 20; first filter 14, dichroic mirror 15, second mirror 17, second filter 18. The aspherical lens 19 and the pinhole plate 20 are encapsulated inside the cassette, the laser beam reducing mirror 12 is fixed at the beam exit of the laser 11, the laser 11, the first reflecting mirror 13 and the objective lens 16 are arranged on the top outside the cassette, and the cassette is fixed On the optical plate, the first reflecting mirror 13 and the second reflecting mirror 17 are respectively fixed on the beam turning frame to adjust the incident angle of the laser beam and the fluorescent beam, the pinhole plate 20 is used to shield the stray light, and the liquid flow system The spool 4 and the quartz flow cell 6 in A are fixed above the objective lens 16 by a three-dimensional translation stage.

The excitation light path and fluorescence collection light path of optical system B are in confocal mode. The setting method of the confocal mode is as follows: the laser beam emitted by the laser 11 passes through the laser beam reducing mirror 12, the first reflecting mirror 13, the first filter 14, the dichroic mirror 15 and the objective lens 16 in sequence, and then is focused on the quartz flow cell. In the central sheath flow channel of 6, the laser excites the cell sample in the central sheath flow channel of the quartz flow cell 6 to generate fluorescence, and the fluorescence is collected by the objective lens 16 and then passes through the dichroic mirror 15, the second reflection mirror 17, and the second filter 18. , the aspherical lens 19 and the pinhole plate 20 are then detected by the photodetector 21 . The optical system adopts a confocal optical path, that is, the excitation optical path and the emission optical path are collinear, the excitation light is concentrated on the cell sample through the objective lens 16, and the generated fluorescence is collected by the same objective lens 16, so that the analyzer of the present application only needs to adjust the laser exit objective lens The angle of 16 and the single-cell flow and the focus of the objective lens 16 can be adjusted to complete the optical path adjustment, which greatly reduces the difficulty of optical path adjustment, and the optical path is more compact, reducing the size of the instrument.

Specifically, the laser 11 is a solid-state laser, the output wavelength is 200-800 nm, the fixed output power is 5-500 mW, and the beam diameter is 0.5-4 mm. The laser beam reducing mirror 12 is a 1-3 times beam reducing mirror, which is used to reduce the diameter of the laser beam. The first filter 14 and the second filter 18 are both band-pass dielectric film filters. The objective lens 16 is a 2.5-100 times plan achromatic objective lens, the working distance is 2-10 mm, and the numerical aperture is 0.08-0.8. The dichroic mirror 15 is a long-pass or short-pass dichroic mirror, and the initial wavelength is 200nm˜800nm. The focal length of the aspherical lens 19 is 1 to 30 mm. The clear aperture of the pinhole plate 20 is 0.1-1 mm. The optical plate is an aluminum alloy plate with a side length of 300mm and a thickness of 13mm. The surface is arrayed with M6 threaded holes and the hole spacing is 25mm. The stroke of the three-dimensional translation stage is 4-20mm; the adjustment angle of the beam turning frame is 2°-10°.

The detection system C includes a photodetector 21, a data acquisition card 22 and a host computer 23. The photodetector 21 is fixed on the optical plate, and the photodetector 21 is used to detect the fluorescence of the fluorescence collection optical path after passing through the pinhole plate 20. The photodetector 21 is connected with the data acquisition card 22, the data acquisition card 22 is connected with the upper computer 23, and the upper computer 23 is installed with signal acquisition and data processing software. The signal acquisition and data processing software is an ultra-high-throughput single-cell analysis workstation. The ultra-high-throughput single-cell analysis workstation includes an instrument control and data acquisition system and a data processing and analysis system. The detection system C converts fluorescent signals into electrical signals and stores them to obtain complete fluorescence time series data, so data processing and data acquisition can be performed offline.

The photodetector 21 is fixed on the optical plate, and the photodetector 21 converts the fluorescent signal into an electrical signal, which is collected by the data acquisition card 22 and transmitted to the upper computer 23 through the data line, and the data is real-time performed by the ultra-high-throughput single-cell analysis workstation. Display, store and post-process offline. The ultra-high-throughput single-cell analysis workstation includes two modules, the instrument control and data acquisition system and the data processing and analysis system. The data acquisition system is used to adjust the data acquisition parameters, control the operating voltage of the photodetector 21 and data acquisition and storage; the data processing and analysis system is used to identify and screen the single-cell pulse signal of the collected raw data, and finally obtain the single-cell pulse signal Peak height, peak area and peak width information of spectral peaks, as well as the corresponding frequency distribution statistics.

The instrument control and data acquisition system reads the fluorescence intensity data collected by the data acquisition card 22 in real time in the producer-consumer mode and stores it in the host computer in the TDMS file format to obtain the complete fluorescence intensity-time spectrum data, the maximum data The storage frequency is 10kS/s～100kS/s. The fluorescence intensity-time spectrum data is processed by the data processing and analysis system. The data processing and analysis process is divided into five steps: data import, parameter setting, spectral peak identification, statistical analysis, and result export. The spectral peak identification step is the core of the data processing and analysis system, and the purpose is to identify the single-cell pulse signal from the fluorescence intensity-time spectrum composed of a large number of data points. Single-cell pulse signal identification, that is, a kind of fluorescence intensity-time that uses the first derivative trend of the fluorescence intensity-time spectrum combined with the original fluorescence intensity-time spectrum to automatically identify the starting point, peak, and end point of the characteristic points of the spectrum peak. Spectral peak identification algorithm, the specific implementation method of spectral peak identification is as follows:

S01, iteratively calculates the original fluorescence intensity-time spectrum data to obtain a baseline value and a baseline noise value;

S02, derive the original fluorescence intensity-time spectrum data to obtain a first-order derivative time series array of fluorescence intensity;

S03, according to the fluctuation characteristics of the first-order derivative time series array combined with the baseline value and the noise value, identify the start point, peak value and end point of the single-cell pulse signal, and mark it in the original fluorescence intensity-time spectrum;

S04, perform integration, peak height and peak width calculations based on the marked single-cell pulse signal feature points, obtain the peak area, peak height and peak width information of the single-cell pulse signal, realize semi-quantitative analysis of the target analyte in a single cell, and can set the threshold value Perform signal screening, and finally achieve effective single-cell pulse signal count, proportion of overlapping cell pulse signals, frequency distribution histogram of single-cell pulse signal peak area, peak height and peak width, single-cell pulse signal peak area, peak height and peak width through statistical analysis display of the coefficient of variation.

Specifically, the photodetector 21 is a photomultiplier tube, and the frequency bandwidth is DC˜100 kHz. The maximum sampling frequency of the data acquisition card 22 is 10kS/s～100kS/s.

The flow cytometry analysis method based on confocal light path design, using the above-mentioned flow cytometer based on confocal light path design, includes the following steps:

Step 1, pre-flushing of the liquid flow system A: before each sample measurement, the liquid flow system A needs to be pre-flushed to discharge air bubbles and form a stable sheath flow in the liquid flow system A; first open the first on-off valve 8, Let the sheath liquid fill the liquid flow system A, then open the second on-off valve 9, introduce the sheath liquid into the flushing syringe 7, then close the first on-off valve 8, push the flushing syringe 7 lightly, and discharge the air bubbles in the liquid path; then close the second on-off valve 8. The valve 9 is switched on and off, the first switch valve 8 is opened, and the sheath liquid is filled with the entire liquid flow channel, and the operation is carried out for 3 minutes until the sheath flow reaches the preset flow rate and the flow rate is stable.

Step 2, data acquisition: turn on the instrument control and data acquisition system, set the operating voltage of the photodetector 21 and the data acquisition channel, and start the fluorescence intensity-time spectrum data acquisition.

Step 3, sample introduction: suck the cell suspension into the injection syringe 2, then set the flow rate of the syringe pump 1 to 5 μL/min for sample introduction, and the suitable concentration of the cell suspension is 1×10 ⁴ to 1×10 ⁶ /mL.

Step 4, data processing: import the collected fluorescence intensity-time spectrum data into the data processing and analysis system for processing, and obtain the peak height, peak area and peak width information of the single-cell pulse signal spectral peak for semi-quantitative analysis of the fluorescence signal, Statistical analysis was performed to obtain the frequency distribution results of the peak height, peak area and peak width of the single-cell pulse signal spectral peaks.

In addition, if the cell analyzer of the present application is moved and transported and there is obvious detection abnormality during use, it is necessary to adjust the focus of the optical path. The optical path focusing adjustment includes excitation optical path adjustment and fluorescence collection optical path adjustment. When adjusting the optical path of fluorescence collection, replace the cell suspension sample with fluorescent dye and collect data according to the normal use process, and then adjust the three-dimensional translation stage so that the focused fluorescent dye streamline is located at the focus of the laser beam converged by the objective lens 16, Finally, by adjusting the second reflecting mirror 17 , the fluorescent light beam collected by the objective lens 16 finally enters the pinhole of the pinhole plate 20 and is detected by the photodetector 21 . The entire optical path focusing adjustment process can feedback the focusing effect in real time through the collected fluorescence signal intensity, until the detected fluorescence signal intensity reaches the strongest and stable, then the adjustment is completed.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. within the range.

Claims (8)

1. A flow cytometry analyzer based on confocal optical path design is characterized by comprising a liquid flow system, an optical system and a detection system;

the liquid flow system comprises an injection pipeline and a sheath liquid pipeline which are respectively communicated with the quartz flow cell, the injection pipeline injects a cell sample into a central sheath liquid channel of the quartz flow cell, and the cell sample forms a single cell flow under the wrapping and extrusion of a sheath flow injected into the central sheath liquid channel of the quartz flow cell by the sheath liquid pipeline;

the optical system comprises an excitation light path and a fluorescence collection light path, wherein a laser beam of the excitation light path is focused on a central sheath flow channel of the quartz flow cell and is used for exciting a cell sample in the central sheath flow channel of the quartz flow cell to generate fluorescence, and the fluorescence is collected through the fluorescence collection light path;

the detection system comprises a photoelectric detector, a data acquisition card and an upper computer which are sequentially connected, wherein the photoelectric detector detects fluorescence collected by a fluorescence collection light path and sends the fluorescence to the upper computer for storage through the data acquisition card.

2. The confocal optical path design-based flow cytometer of claim 1, wherein the flow system further comprises a flushing line, the flushing line is in communication with the quartz flow cell for flushing the central sheath fluid channel of the quartz flow cell.

3. The flow cytometer based on confocal optical path design of claim 2, wherein the injection pipeline comprises an injection pump, a sample injector and a capillary tube which are sequentially communicated, and an outlet end of the capillary tube extends into the four-way valve from a first port of the four-way valve and is inserted into an inlet of a central sheath fluid channel of the quartz flow cell after penetrating out from a third port of the four-way valve;

the sheath liquid pipeline comprises a sheath liquid bottle communicated with the second port of the four-way joint, and a first switch valve is arranged on the pipeline of the sheath liquid bottle communicated with the second port of the four-way joint;

the flushing pipeline comprises a flushing injector communicated with the fourth port of the four-way valve, and a second switch valve is arranged on the pipeline communicated with the fourth port of the four-way valve.

4. The confocal optical path design-based flow cytometer analyzer as described in claim 3, wherein the inlet port of the quartz flow cell is in communication with the third port of the cross-piece, and the outlet port of the quartz flow cell is in communication with the waste liquid bottle.

5. A flow cytometer as described in claim 3, wherein said capillary tube is a quartz capillary tube with an outer diameter of 0.1-0.4 mm and an inner diameter of 20-200 μm, and the outlet end of the capillary tube is polished to a conical tip with a diameter of 15-45 °.

6. The confocal optical path design-based flow cytometer of claim 3, wherein the quartz flow cell is a four-sided transparent quartz flow cell, the external length, width and height are 4mm x 10mm to 6mm x 20mm, and the central sheath fluid channel is a rectangular channel of 0.1mm x 0.1mm to 0.4mm x 0.4 mm.

7. The flow cytometer as described in claim 3, wherein the excitation optical path includes a laser, a laser beam splitter, a first reflector, a first optical filter, a dichroic mirror, and an objective lens, which are sequentially disposed, and a laser beam emitted from the laser passes through the laser beam splitter, the first reflector, the first optical filter, the dichroic mirror, and the objective lens, and is focused on the center of the quartz flow cell; the fluorescence collection light path comprises an objective lens, a dichroic mirror, a second reflecting mirror, a second optical filter, an aspheric lens and a pinhole plate which are sequentially arranged, wherein the fluorescence generated by the cell sample of the central sheath flow channel of the quartz flow cell is collected through the objective lens and is detected by a photoelectric detector after sequentially passing through the dichroic mirror, the second reflecting mirror, the second optical filter, the aspheric lens and the pinhole plate.

8. The confocal optical path design-based flow cytometer assembly according to claim 7, wherein the first optical filter, the dichroic mirror, the second reflecting mirror, the second optical filter, the aspheric lens and the pinhole plate are all packaged inside a cassette, the laser beam shrinking mirror is fixed at a beam exit of the laser, the first reflecting mirror and the objective lens are all disposed on the top of the cassette, the cassette is fixed on an optical flat plate, the first reflecting mirror and the second reflecting mirror are respectively fixed on a beam turning frame, and the four-way junction and the quartz flow cell are fixed above the objective lens through a three-dimensional translation stage.

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