CN114705611A - Flow cytometry based on confocal light path design - Google Patents
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Abstract
A flow cytometry analyzer based on confocal optical path design belongs to the technical field of life analysis scientific detection equipment. The flow cytometry analyzer comprises 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 connected in sequence. The flow cytometry analyzer disclosed by the invention has the advantages of compact structure, simple and efficient focusing of a light path and convenience in use.
Description
Technical Field
The invention relates to the technical field of life analysis scientific detection equipment, in particular to a flow cytometry analyzer based on confocal optical path design.
Background
Since cells are the most basic units constituting and completing living activities, and there is a general heterogeneity between cells, characterization of living activities at the single cell level is an essential technical means in the field of life science research such as proteomics and metallomics, and in the medical field such as diagnosis and treatment of diseases. Flow cytometry is currently the most widely used high-throughput single-cell multiparameter analysis technique. The flow cytometer can obtain quantitative information of biomolecules such as fluorescent labeled proteins in cells and information such as cell structures and morphologies through optical and fluorescent characteristics generated when single cells in a sheath flow pass through a light source (usually laser beams), and can perform related researches on physiological functions such as cell activity, immunophenotype and endocytosis. Target analytes for flow cytometry have also now been expanded from cells to smaller sized particles such as microorganisms, nuclei, chromosome preparations, and nanoparticles, among others.
Flow cytometers typically include a flow system, an optical system, and a detection system. The liquid flow system is used for introducing cells in the form of suspension, carrying out focusing arrangement on the cells by using a sheath flow focusing technology, and passing through an optical system detection area according to a fixed path. The sheath flow focusing technology is that after the sheath flow and the sample flow are converged in a micro-channel of a flow cell, the sample flow forms a stable cell streamline with a certain width under the wrapping and extrusion of sheath liquid by depending on the flow speed difference between the sheath flow and the sample flow, so that cells which are dispersed in a disordered way are arranged in a line and pass through a detection area of an optical system one by one. The technology can ensure that cells in suspension irradiate the central position of a light spot through an excitation light source according to the same path, ensure that the cells passing through a detection area are irradiated by the consistent excitation light source, and greatly reduce the probability that the cells overlap to pass through the detection area, thereby obtaining accurate single-cell fluorescence detection signals. Therefore, the efficient and stable focusing of the liquid flow system on the cells is the premise that the flow cytometer obtains accurate single cell detection signals. In the traditional flow cytometer, an integrated flow cell with a complex design is adopted to obtain a stable focusing effect, and the design has the disadvantages of high processing precision requirement, high cost and difficult maintenance.
The optical system of the flow cytometer comprises an excitation light path and a fluorescence collection light path, wherein the excitation light path comprises a laser, a laser beam quality adjusting module (such as beam expanding, beam shrinking, light filtering and the like), a laser focusing module and the like, and the fluorescence collection light path comprises fluorescence collection, collimation, light filtering, fluorescence photoelectric conversion, signal amplification and the like. The traditional flow cytometer adopts a mode of orthogonal light path design, i.e. the excitation light path and the fluorescence collection light path are perpendicular to each other. The orthogonal light path can effectively reduce the background interference of the exciting light and improve the sensitivity, but in the mode, the exciting light converging lens and the fluorescence collecting lens are required to be focused on a cell flow line from two directions, the focusing of the light path is complicated, and the detection sensitivity can be greatly reduced by slight deviation of the exciting light converging lens and the fluorescence collecting lens, so that a professional engineer is required to complete the focusing adjustment of the light path. In addition, the orthogonal optical path mode is difficult to realize a compact instrument, and the instrument is bulky.
The detection system of the flow cytometer comprises a photoelectric conversion module, a signal acquisition module and a data processing module, wherein the photoelectric conversion module is usually a photomultiplier tube and the like, converts a weak fluorescent signal into an electrical signal and amplifies the electrical signal, the electrical signal output by the photoelectric conversion module is acquired and recorded by the data acquisition module, and a mass of data points are processed by data processing software to obtain a final cell analysis result. The data processing of the conventional flow cytometer is usually on-line processing, which is fast in processing speed and capable of real-time observation, but the complicated and difficult-to-understand gate threshold setting makes the operation of the flow cytometer require long training and practice.
Disclosure of Invention
The invention provides a flow cytometry analyzer based on confocal optical path design, aiming at solving the technical problems of low sensitivity, complex optical path adjustment, fussy use flow and test parameter setting, high price and the like of the existing flow cytometry analyzer.
In order to realize the purpose, the technical scheme of the invention is as follows:
a flow cytometry analyzer based on confocal optical path design comprises 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.
Furthermore, the liquid flow system also comprises a flushing pipeline, and the flushing pipeline is communicated with the quartz flow cell and is used for flushing a central sheath liquid channel of the quartz flow cell and discharging bubbles in a liquid path.
Furthermore, the injection pipeline comprises an injection pump, a sample injection injector and a capillary tube which are sequentially communicated, wherein the outlet end of the capillary tube extends into the four-way valve from the first port of the four-way valve, penetrates out of the third port of the four-way valve and then is inserted into the inlet of the central sheath liquid channel of the quartz flow cell;
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.
Furthermore, the inlet end of the quartz flow cell is communicated with the third port of the four-way valve, and the outlet end of the quartz flow cell is communicated with the waste liquid bottle.
Preferably, the capillary tube is a quartz capillary tube, the outer diameter of the capillary tube is 0.1-0.4 mm, the inner diameter of the capillary tube is 20-200 mu m, and the outlet end of the capillary tube is polished into a conical tip with the angle of 15-45 degrees.
Preferably, the quartz flow cell is a four-side transparent quartz flow cell, the length, width and height of the outer part of the quartz flow cell are 4mm multiplied by 10mm to 6mm multiplied by 20mm, and the central sheath fluid channel is a rectangular channel with 0.1mm multiplied by 0.1mm to 0.4mm multiplied by 0.4 mm.
Furthermore, the excitation light path comprises a laser, a laser beam reducing mirror, a first reflecting mirror, a first optical filter, a dichroic mirror and an objective lens which are sequentially arranged, and a laser beam emitted by the laser sequentially passes through the laser beam reducing mirror, the first reflecting mirror, the first optical filter, the dichroic mirror and the objective lens and then is focused on the central sheath flow channel 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.
Furthermore, 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 in the dark box, the laser beam shrinking mirror is fixed at a beam outlet of the laser, the first reflecting mirror and the objective lens are all arranged at the top of the dark box, the dark box is fixed on the optical flat plate, the first reflecting mirror and the second reflecting mirror are respectively fixed on the beam turning frame, and the four-way joint and the quartz flow cell are fixed above the objective lens through the three-dimensional translation table.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention adopts the design of a confocal light path, can effectively reduce stray light interference, improves detection sensitivity, greatly reduces the focusing difficulty of the light path, and can be automatically focused by a user.
2. The liquid flow system adopts a modular design, the quartz flow cell, the capillary tube, the sheath liquid pipeline and the flushing pipeline are mutually independent, the use is convenient, the maintenance is convenient, the modular design can greatly reduce the processing precision requirement of parts, and the cost is reduced.
3. The sheath fluid channel is coaxial with the sample introduction capillary and the central channel of the quartz flow cell, and the distance between the inlet of the sheath fluid channel and the convergent point of the sheath fluid and the sample is longer, so that the phenomenon of unstable focusing caused by the pulsation disturbance of the sheath fluid or the disturbance when the sheath fluid enters the focusing cell is effectively avoided, and the detection error caused by cell adhesion or overlapping is reduced.
4. According to the ultrahigh-flux single cell analysis workstation, the instrument control and data acquisition system and the data processing and analysis system can operate independently, acquired data are complete fluorescence intensity-time spectrogram data, a user can directly obtain original data and perform off-line processing automatically, the data processing threshold of the data processing and analysis system is set simply and easily to be understood, and the user can perform flexible data processing and screening according to requirements.
5. The invention can realize single cell analysis with high flux and high accuracy of at least 100cells/s based on a high-sensitivity and time-resolved optical system and a high-efficiency and stable liquid flow system, can obtain the peak height and peak area information of a fluorescence signal spectrum peak of a target analyte in a single cell by fast and accurate off-line processing of original data, realizes semi-quantitative analysis, and realizes multi-element display of detection data through statistical analysis.
6. The invention is arranged on an optical flat plate, has compact structure and small volume, can be carried and moved at will, is in a modularized open design, has a liquid flow system and a detection system which are mutually independent, and can be modified by a user according to requirements or combined with sample pretreatment modules such as a microfluidic chip and the like, thereby meeting the requirements of personalized single cell analysis.
Drawings
FIG. 1 is a schematic structural diagram of a flow cytometer based on confocal optical path design according to the present invention;
FIG. 2 is a schematic view of a fluid flow system provided by the present invention.
Wherein,
the system comprises a 1-injection pump, a 2-sample injection syringe, a 3-capillary tube, a 4-four-way joint, a 5-sheath liquid bottle, a 6-quartz flow cell, a 7-flushing syringe, an 8-first switch valve, a 9-second switch valve, a 10-waste liquid bottle, an 11-laser, a 12-laser beam shrinking mirror, a 13-first reflector, a 14-first optical filter, a 15-dichroic mirror, a 16-objective lens, a 17-second reflector, an 18-second optical filter, a 19-aspheric lens, a 20-pinhole plate, a 21-photoelectric detector, a 22-data acquisition card, a 23-upper computer, an A-liquid flow system, a B-optical system and a C-detection system.
Detailed Description
In order to solve the problems in the prior art, as shown in fig. 1 and fig. 2, the present invention provides a flow cytometer based on a confocal optical path design, which includes a liquid flow system a, an optical system B, and a detection system C.
The liquid flow system A comprises an injection pipeline and a sheath liquid pipeline which are respectively communicated with the quartz flow cell 6, the injection pipeline injects a cell sample into a central sheath liquid channel of the quartz flow cell 6, 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 6 by the sheath liquid pipeline; the optical system B comprises an excitation light path and a fluorescence collection light path, wherein a laser beam of the excitation light path is focused on the central sheath flow channel of the quartz flow cell 6 and is used for exciting the cell sample in the central sheath flow channel of the quartz flow cell 6 to generate fluorescence, and the fluorescence is collected through the fluorescence collection light path; the detection system C comprises a photoelectric detector 21, a data acquisition card 22 and an upper computer 23 which are connected in sequence, wherein the photoelectric detector 21 detects fluorescence collected by a fluorescence collection optical path and sends the fluorescence to the upper computer 23 for storage through the data acquisition card 22.
The liquid flow system A further comprises a flushing pipeline which is communicated with the quartz flow cell 6 and used for flushing a central sheath liquid channel of the quartz flow cell 6 and discharging bubbles in a liquid path. The injection pipeline comprises an injection pump 1, a sample injection injector 2 and a capillary tube 3 which are sequentially communicated, wherein the outlet end of the capillary tube 3 extends into the cross joint from the first port of the cross joint 4, penetrates out from the third port of the cross joint 4 and then is inserted into the inlet of the central sheath liquid channel of the quartz flow cell 6; the sheath liquid pipeline comprises a sheath liquid bottle 5 communicated with the second port of the cross joint 4, and a first switch valve 8 is arranged on the pipeline of the sheath liquid bottle 5 communicated with the second port of the cross joint 4; the flushing pipeline comprises a flushing injector 7 communicated with the fourth port of the four-way valve 4, and a second switch valve 9 is arranged on the pipeline of the flushing injector 7 communicated with the fourth port of the four-way valve 4. The inlet end of the quartz flow-through cell 6 is communicated with the third port of the four-way valve 4, and the outlet end of the quartz flow-through cell 6 is communicated with the waste liquid bottle 10.
Specifically, the liquid flow system A comprises an injection pump 1, a sample injection syringe 2, a capillary tube 3, a cross joint 4, a sheath liquid bottle 5, a quartz flow cell 6, a flushing syringe 7 and a waste liquid bottle 10, wherein the injection pump 1, the sample injection syringe 2 and the capillary tube 3 are sequentially communicated, the cross joint 4 comprises a first port, a second port, a third port and a fourth port, the outlet end of the capillary tube 3 extends into the cross joint 4 from the first port, penetrates through the cross joint 4 and then is inserted into the inlet of a central sheath liquid channel of the quartz flow cell 6; the quartz flow cell 6 is communicated with a third port, the outlet end of the quartz flow cell 6 is communicated with a waste liquid bottle 10, a second port is communicated with a sheath liquid bottle 5, and a fourth port is communicated with a flushing injector 7; a first switch valve 8 is arranged between the second port and the sheath liquid bottle 5; a second on-off valve 9 is provided between the fourth port and the flush syringe 7. The liquid flow system A and the optical system C are mutually independent, the liquid flow system A is in a modular design, and the quartz flow cell 6, the capillary tube 3, the sheath liquid pipeline and the flushing pipeline are mutually independent, so that the requirement on the processing precision of parts is greatly reduced, and the cost is reduced.
A sample is pressed in from the inlet end of a capillary tube 3 by an injection pump 1 through a sample injection syringe 2, the capillary tube 3 extends into and is fixedly sealed from the first port of a cross joint 4, the outlet end of the capillary tube 3 penetrates through the cross joint 4 and then is inserted into the inlet of a central sheath liquid channel of a quartz flow cell 6, the third port of the cross joint 4 is connected and sealed with the quartz flow cell 6, the second port is connected with a sheath liquid bottle 5 through a hose, sheath liquid flows into the central sheath liquid channel of the quartz flow cell 6 through the cross joint 4 under the action of gravity and static pressure to form sheath flow and is converged with sample flow, and the sample flow flowing out from the outlet tip of the capillary tube 3 is arranged under the wrapping and extrusion of the sheath liquid to form single cell flow; the fourth port of the cross joint 4 is connected with a flushing injector 7 through a hose and is used for flushing a central sheath liquid channel of the quartz flow cell 6 and discharging bubbles in a liquid path, and the outlet end of the quartz flow cell 6 is connected with a waste liquid bottle 10 through a hose.
Specifically, the capillary tube 3 is a quartz capillary tube, the outer diameter is 0.1-0.4 mm, the inner diameter is 20-200 mu m, and the outlet end of the capillary tube 3 is polished into a conical tip with an angle of 15-45 degrees; the quartz flow cell 6 is a quartz flow cell with four transparent surfaces, the length, width and height of the outer part are 4mm multiplied by 10 mm-6 mm multiplied by 20mm, and the central sheath liquid channel is a rectangular channel with 0.1mm multiplied by 0.1 mm-0.4 mm multiplied by 0.4 mm; the injection pump 1 sets the sample injection flow rate to be 2-200 mu L/min and the sheath fluid flow rate to be 100-2000 mu L/min.
The excitation light path comprises a laser 11, a laser beam reducing mirror 12, a first reflecting mirror 13, a first optical filter 14, a dichroic mirror 15 and an objective lens 16 which are arranged in sequence; the fluorescence collecting optical path comprises an objective lens 16, a dichroic mirror 15, a second reflecting mirror 17, a second optical filter 18, an aspheric lens 19 and a pinhole plate 20 which are arranged in sequence; the first optical filter 14, the dichroic mirror 15, the second reflector 17, the second optical filter 18, the aspheric lens 19 and the pinhole plate 20 are packaged in the cassette, the laser beam reducing mirror 12 is fixed at the light beam outlet of the laser 11, the first reflector 13 and the objective lens 16 are arranged at the top outside the cassette, the cassette is fixed on an optical flat plate, the first reflector 13 and the second reflector 17 are respectively fixed on a light beam turning frame and used for adjusting the incident angles of laser beams and fluorescent light beams, the pinhole plate 20 is used for shielding stray light rays, and the four-way 4 and the quartz flow cell 6 in the fluid flow system A are fixed above the objective lens 16 through a three-dimensional translation table.
The excitation light path and the fluorescence collection light path of the optical system B adopt a confocal mode. The setting mode of the confocal mode is as follows: laser beams emitted by a laser 11 sequentially pass through a laser beam reducing mirror 12, a first reflecting mirror 13, a first optical filter 14, a dichroic mirror 15 and an objective lens 16 and are focused on a central sheath flow channel of the quartz flow cell 6, the laser excites a 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 sequentially passes through the dichroic mirror 15, a second reflecting mirror 17, a second optical filter 18, an aspheric lens 19 and a pinhole plate 20 and is detected by a photoelectric detector 21. The optical system adopts a confocal light path, namely an excitation light path and an emission light path are collinear, the excitation light is converged on a cell sample through the objective lens 16, and generated fluorescence is collected through the same objective lens 16, so that the analyzer only needs to adjust the angle of the laser emission objective lens 16 and focus the single cell flow and the objective lens 16 to complete light path adjustment, the difficulty of light path adjustment is greatly reduced, the light path is more compact, and the volume of the analyzer is reduced.
Specifically, the laser 11 is a solid 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 and is used for reducing the diameter of a 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 flat field 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 to 800 nm. The aspheric lens 19 has a focal length of 1-30 mm. The clear aperture of the pinhole plate 20 is 0.1-1 mm. The optical flat plate is an aluminum alloy flat plate with the side length of 300mm and the thickness of 13mm, the surface array is M6 threaded holes, and the hole distance is 25 mm. The stroke of the three-dimensional translation table is 4-20 mm; the adjusting angle of the light beam turning frame is 2-10 degrees.
The detection system C comprises a photoelectric detector 21, a data acquisition card 22 and an upper computer 23, wherein the photoelectric detector 21 is fixed on an optical flat plate, the photoelectric detector 21 is used for detecting fluorescence of a fluorescence collection light path passing through the pinhole plate 20, the photoelectric detector 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 provided with signal acquisition and data processing software. The signal acquisition and data processing software is an ultrahigh-flux single-cell analysis workstation, and the ultrahigh-flux single-cell analysis workstation comprises an instrument control and data acquisition system and a data processing and analysis system. The detection system C converts the fluorescence signal into an electric signal and stores the electric signal to obtain complete fluorescence time sequence data, so that data processing and data acquisition can be performed off-line.
The photoelectric detector 21 is fixed on the optical flat plate, the fluorescent signal is converted into an electric signal by the photoelectric detector 21, the electric signal is transmitted to the upper computer 23 through a data line after being collected by the data acquisition card 22, and the data is displayed, stored and processed in an off-line way in real time by the ultrahigh-flux single cell analysis workstation. The ultrahigh-flux single cell analysis workstation comprises two modules, namely an instrument control and data acquisition system and a data processing and analysis system, wherein the two modules can be installed on the same upper computer 23 and can also be installed on different upper computers 23 to independently operate, and the instrument control and data acquisition system is used for adjusting data acquisition parameters, controlling the operating voltage of the photoelectric detector 21 and acquiring and storing data; the data processing and analyzing system is used for identifying and screening the single-cell pulse signals of the collected original data, and finally obtaining the peak height, the peak area and the peak width information of the spectrum peak of the single-cell pulse signals and the frequency distribution statistical result corresponding to the peak height, the peak area and the peak width information.
The instrument control and data acquisition system reads the fluorescence intensity data acquired by the data acquisition card 22 in real time in a producer-consumer mode and stores the data in an upper computer in a TDMS file format to obtain complete fluorescence intensity-time spectrogram data, and the maximum data storage frequency is 10 kS/s-100 kS/s. The fluorescence intensity-time spectrogram data is processed by using a data processing and analyzing system, and the flow of data processing and analyzing comprises five steps of data import, parameter setting, spectral peak identification, statistical analysis and result derivation. The invention relates to a single-cell pulse signal identification method, in particular to a spectrum peak identification algorithm of a fluorescence intensity-time spectrogram, wherein the spectrum peak identification step is the core of a data processing and analyzing system and aims to identify a single-cell pulse signal from the fluorescence intensity-time spectrogram consisting of massive data points, the single-cell pulse signal identification method is quickly and accurately carried out according to a first derivative auxiliary spectrum peak identification algorithm, namely, the first derivative trend of the fluorescence intensity-time spectrogram is combined with the original fluorescence intensity-time spectrogram to automatically identify the starting point, the peak value and the end point of a spectrum peak characteristic point, and the specific implementation method of the spectrum peak identification is as follows:
s01, carrying out iterative calculation on the original fluorescence intensity-time spectrogram data to obtain a baseline value and a baseline noise value;
s02, deriving the original fluorescence intensity-time spectrogram data to obtain a fluorescence intensity first derivative time sequence array;
s03, identifying the starting point, the peak value and the end point of the single-cell pulse signal according to the fluctuation characteristics of the first derivative time sequence array by combining the baseline value and the noise value, and marking the starting point, the peak value and the end point in the original fluorescence intensity-time spectrogram;
s04, performing integration, peak height and peak width calculation based on the marked single-cell pulse signal characteristic points to obtain the information of the peak area, the peak height and the peak width of the single-cell pulse signal, realizing semi-quantitative analysis of a target analyte in a single cell, performing signal screening through threshold setting, and finally realizing the display of the variation coefficients of the effective single-cell pulse signal count, the overlapping cell pulse signal occupancy, the single-cell pulse signal peak area, the frequency distribution histogram of the peak height and the peak width, the single-cell pulse signal peak area, the peak height and the peak width through statistical analysis.
Specifically, the photodetector 21 is a photomultiplier tube, and the frequency bandwidth is DC to 100 kHz. The maximum sampling frequency of the data acquisition card 22 is 10 kS/s-100 kS/s.
The flow cytometry analysis method based on the confocal optical path design adopts the flow cytometry analyzer based on the confocal optical path design, and comprises the following steps:
step one, pre-flushing a liquid flow system A: the liquid flow system A needs to be pre-flushed before each sample measurement, and is used for discharging bubbles and forming stable sheath flow; firstly, opening a first switch valve 8 to enable sheath liquid to be filled in the liquid flow system A, then opening a second switch valve 9 to introduce the sheath liquid into a flushing injector 7, then closing the first switch valve 8, slightly pushing the flushing injector 7, and discharging bubbles in a liquid path; then the second switch valve 9 is closed, the first switch valve 8 is opened, the sheath fluid is filled in the whole fluid channel, and the operation is carried out for 3 minutes until the sheath fluid reaches the preset flow rate and the flow rate is stable.
Step two, data acquisition: and opening the instrument control and data acquisition system, setting the operating voltage and data acquisition channel of the photoelectric detector 21, and starting fluorescence intensity-time spectrogram data acquisition.
Step three, sample introduction: sucking cell suspension into a sample injector 2, and introducing sample at flow rate of 5 μ L/min by setting an injection pump 1, wherein the appropriate concentration of cell suspension is 1 × 104～1×106/mL。
Step four, data processing: and importing the acquired fluorescence intensity-time spectrogram data into a data processing and analyzing system for processing to obtain information of peak height, peak area and peak width of a single-cell pulse signal spectral peak for semi-quantitative analysis of the fluorescence signal, and performing statistical analysis to obtain frequency distribution results of the information of the peak height, the peak area and the peak width of the single-cell pulse signal spectral peak.
In addition, if the cell analyzer of the present application is moved and transported and there is a significant abnormality in detection during use, it is necessary to perform optical path focusing adjustment. The optical path focusing adjustment includes an excitation optical path adjustment and a fluorescence collection optical path adjustment, and the excitation optical path adjustment adjusts the angle of the first reflecting mirror 13 through a knob of the beam turning frame, so that the laser is emitted out of the objective lens 16 vertically. When the fluorescence collection light path is adjusted, the cell suspension sample is replaced by fluorescent dye, data collection is carried out according to a normal use process, then the focused fluorescent dye streamline is positioned on a laser beam focus converged by the objective lens 16 by adjusting the three-dimensional translation table, and finally the fluorescent light beam collected by the objective lens 16 finally enters a pinhole of the pinhole plate 20 and is detected by the photoelectric detector 21 by adjusting the second reflecting mirror 17. The focusing effect of the whole light path can be fed back in real time through the acquired fluorescent signal intensity in the focusing adjustment process, and the adjustment is finished until the detected fluorescent signal intensity is strongest and stable.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
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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