A sorting control system and sorting method for flow cytometer

Abstract

The present invention relates to the field of cell sorting technology, and in particular to a sorting control system and sorting method for a flow cytometer. The system includes a data acquisition unit and a control unit. The method includes: acquiring several breakpoint images of a droplet to determine whether the droplet forming process is qualified based on the pixel variance of the breakpoints; based on an unqualified droplet forming process, determining whether the oscillation stability is qualified based on the oscillation area of ​​the oscillation curve of the piezoelectric ceramic to determine whether to obtain the sheath liquid flow rate; determining the matching degree based on the flow rate curve of the sheath liquid flow rate and the frequency curve of the droplet to increase the sheath liquid pressure based on the fluctuation intensity of the fluorescence signal; determining the target droplet based on the fluorescence detection result to determine the droplet delay based on the target droplet; and determining whether the target droplet can land in the collection tube based on the initial offset angle of the target droplet to adjust the inter-plate voltage of the deflection plate. The present invention improves sorting accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of cell sorting, and in particular to a sorting control system and a sorting method of a flow cytometer. Background Art

[0002] Traditional sorting systems use preset piezoelectric oscillation frequency and sheath fluid flow rate parameters to compensate for droplet breakup time differences through fixed delays. However, the droplet generation process is susceptible to environmental interference (such as temperature fluctuations, changes in sample viscosity) or aging of mechanical components (such as attenuation of piezoelectric ceramic response), resulting in drift of the droplet breakage point or poor formation. For example, high-viscosity samples (such as buffers containing DNA or protein) will delay liquid flow breakage. If the piezoelectric frequency or sheath fluid pressure is not dynamically adjusted, the droplet spacing will increase and the sorting signal synchronization will fail. At the same time, existing technologies usually independently adjust the sheath fluid flow rate, piezoelectric frequency or deflection voltage, and lack a multi-parameter coordinated feedback mechanism. For example, if the deflection voltage is fixed, it cannot compensate for the droplet trajectory deviation caused by electric field unevenness or air resistance, resulting in sorting contamination.

[0003] Chinese patent application publication number CN116263397A discloses a flow cytometer sorting control system, sorting method, and storage medium. The sorting control system includes an interface for interacting with user software, an analog-to-digital conversion module, a data processing module, a sorting identification module, a sorting delay module, and a sorting actuator. The invention also provides a method for cell sorting using the flow cytometer sorting control system provided by the present invention. This method includes novel sorting identification and delay methods, significantly reducing computation time and increasing sorting speed when performing complex gating operations on hardware with the same computational speed.

[0004] The following problems also exist in the existing technology: during the cell sorting process, the flow cytometer may have insufficient droplet generation stability due to objective factors, resulting in failure of sorting signal synchronization and deviation of the sorting trajectory, thus leading to low sorting accuracy. Summary of the Invention

[0005] To this end, the present invention provides a sorting control system and a sorting method for a flow cytometer to overcome the problem in the prior art that droplet generation stability is insufficient, resulting in failure of sorting signal synchronization, deviation of the sorting trajectory, and thus low sorting accuracy.

[0006] To achieve the above object, the present invention provides a flow cytometer sorting control system, comprising:

[0007] A data acquisition unit, comprising a camera for acquiring a breakpoint image of a liquid droplet, a flow sensor for acquiring a flow rate of the sheath liquid, and a laser Doppler vibrometer for acquiring an oscillation curve of the piezoelectric ceramic;

[0008] A control unit, connected to the data acquisition unit, comprising:

[0009] a breakpoint determination module, configured to determine a length gradient of adjacent droplets and a frequency curve of the droplets based on the breakpoint image, to determine whether a droplet is a breakpoint based on the length gradient, and to determine a pixel variance based on the number of pixels at the breakpoint to determine whether the droplet forming process is qualified;

[0010] an oscillation evaluation module, configured to determine an oscillation area based on the oscillation curve to determine whether the oscillation stability is qualified;

[0011] a cell viscosity determination module, configured to establish a flow velocity curve based on the sheath fluid flow velocity, determine the eligibility of the cell viscosity based on a matching degree between the flow velocity curve and the frequency curve, and increase the sheath fluid pressure based on the fluctuation intensity of the fluorescence signal if the cell viscosity is unqualified;

[0012] The sorting control module is used to determine the target droplet according to the fluorescence detection result, determine the droplet delay according to the shape of the target droplet, and determine whether the target droplet can fall into the collection tube according to the initial deviation angle of the target droplet, so as to adjust the inter-plate voltage of the deflection plate.

[0013] Furthermore, the breakpoint determination module determines that the droplet is a breakpoint based on a comparison result that the length gradient is greater than or equal to a preset gradient.

[0014] Furthermore, the breakpoint determination module determines the number of pixels of the breakpoint to calculate the pixel variance under the condition that the droplet is determined to be a breakpoint, and determines that the droplet forming process is unqualified based on the comparison result that the pixel variance is greater than a preset variance.

[0015] Furthermore, when the oscillation evaluation module determines that the droplet forming process is unqualified, the oscillation curve and the standard oscillation curve are overlapped with the coordinate origin as the reference point, so as to determine the area enclosed by the union of the oscillation curve and the standard oscillation curve and the coordinate axis as the oscillation area.

[0016] Furthermore, the oscillation evaluation module determines that the oscillation stability is qualified based on a comparison result that the oscillation area is less than or equal to a standard oscillation area.

[0017] Furthermore, the cell viscosity determination module overlaps the flow rate curve and the frequency curve at intervals of time difference, determines the slopes corresponding to the same horizontal coordinates, determines the slope standard deviation based on the slopes, and determines the slope standard deviation as the matching degree.

[0018] Furthermore, the cell viscosity determination module determines that the cell viscosity is unqualified based on the comparison result that the matching degree is greater than a preset matching degree.

[0019] Furthermore, the cell viscosity determination module determines the fluctuation intensity of the fluorescence signal based on the judgment result that the cell viscosity is unqualified, compares the fluctuation intensity with a preset intensity to obtain a corresponding comparison result, and sets several adjustment coefficients corresponding to the corresponding comparison results to increase the sheath fluid pressure.

[0020] Furthermore, the sorting control module determines that the target droplet cannot fall into the collection tube based on the comparison result that the initial offset angle is less than the first preset angle or greater than the second preset angle, and subtracts the initial offset angle from the preset angle to obtain a number of angle differences, and sets a number of correction coefficients corresponding to the angle differences to increase or decrease the inter-plate voltage.

[0021] On the other hand, the present invention also provides a flow cytometer sorting method, comprising:

[0022] Acquire a plurality of breakpoint images of the droplet to determine whether the droplet forming process is qualified according to the pixel variance of the breakpoints;

[0023] Based on the unqualified droplet forming process, the oscillation stability is determined according to the oscillation area of ​​the oscillation curve of the piezoelectric ceramic to obtain the sheath liquid flow rate:

[0024] determining a matching degree based on a flow rate curve of the sheath fluid flow rate and a frequency curve of the droplet to increase the sheath fluid pressure according to the fluctuation intensity of the fluorescence signal;

[0025] The target droplet is determined according to the fluorescence detection result to determine the droplet delay according to the target droplet, and whether the target droplet can fall into the collection tube is determined according to the initial deviation angle of the target droplet to adjust the inter-plate voltage of the deflection plate.

[0026] Compared with the prior art, the beneficial effect of the present invention is that the present invention collects the breakpoint image of the droplet through the image sensor, and the length of the breakpoint position will suddenly change. Therefore, the breakpoint can be determined according to the length gradient of the adjacent droplets. Under the oscillation of the piezoelectric ceramic, the sheath liquid is ejected from the nozzle, and there is a process of changing from a liquid column to a droplet. The stability of this process is directly related to the determination of the charging timing. Cell sorting makes a decision to charge the target droplet based on the fluorescence information detected at the detection point. There is a droplet delay between the charging decision and the charging timing. Under the condition of using a fixed delay to compensate for the droplet breakage time difference, the better the stability of the droplet forming process, the higher the synchronization of the charging timing with the target droplet. Therefore, under the condition of determining the breakpoint, the larger the pixel variance, the better the pixel variance. The smaller the value is, the more stable the length of the droplet generation process is, the more accurate the fixed delay compensation droplet breakage time difference is, and thus the higher the signal synchronization of the sorting decision is; under the condition of precise charging of the target droplet, the offset process of the droplet must also be monitored. The charging charge of the droplet is determined by the fluorescence detection signal of the detection point. The target droplet cannot adjust the charge again according to the real-time offset trajectory during the offset process after being charged. However, the droplet itself has uncertainty, and the preset charge cannot accurately make the target droplet fall into the collection tube according to the established trajectory. Therefore, the falling trajectory of the droplet can be predicted according to the initial offset angle of the target droplet, and the inter-plate voltage of the deflection plate can be adjusted according to the prediction result, thereby further improving the sorting accuracy.

[0027] Furthermore, the present invention determines the oscillation stability of the piezoelectric ceramic under the condition that the droplet forming process is determined to be unqualified. The oscillation stability of the piezoelectric ceramic will be affected by many factors. Factors such as internal lattice fatigue, electrode oxidation, and voltage fluctuations of the piezoelectric ceramic will affect the oscillation stability of the piezoelectric ceramic. The piezoelectric ceramic emits a sine wave of a certain frequency and amplitude to turn the sheath liquid column into droplets one by one. The poor oscillation stability of the piezoelectric ceramic will lead to an unstable droplet forming process; the oscillation stability can be accurately judged based on the oscillation area of ​​the oscillation curve of the piezoelectric ceramic and the standard oscillation curve. After the oscillation curve and the standard oscillation curve overlap, the larger the area of ​​the union, the greater the degree of offset of the two curves, and the worse the oscillation stability of the piezoelectric ceramic. The oscillation stability of the piezoelectric ceramic is judged to determine whether the unqualified droplet forming process is caused by the piezoelectric ceramic, thereby further improving the sorting accuracy.

[0028] Furthermore, the cell viscosity is determined under the condition that the oscillation stability of the piezoelectric ceramic is qualified. The droplet formation process is not only related to the oscillation stability of the piezoelectric ceramic, but excessive cell viscosity will also lead to unqualified droplet formation process. During the cell sorting process, some cells will aggregate or settle during the sorting process due to their own characteristics, resulting in increased cell viscosity. High viscosity will increase the cohesive force of the liquid, making it more difficult for droplets to break, reducing the droplet generation frequency and increasing the number of pixels at the breakpoint position, resulting in a decrease in the accuracy of the fixed delay compensation droplet breakage time difference, thereby resulting in poor synchronization of the sorting signal; the cell viscosity is determined by the matching degree of the sheath fluid flow rate curve and the frequency curve. Under the condition that the cell viscosity is qualified, the fluctuation of the sheath fluid flow rate curve is consistent with the fluctuation of the droplet generation frequency, but excessive cell viscosity will cause deviations in the fluctuation of the two. Under the condition that the cell viscosity is determined to be unqualified, the sheath fluid pressure is appropriately increased according to the fluctuation intensity of the fluorescence signal to improve the droplet formation stability, thereby further improving the sorting accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 1 is a structural block diagram of a sorting control system of a flow cytometer according to an embodiment of the present invention;

[0030] Figure 2 A flow chart of determining whether a droplet forming process is qualified according to an embodiment of the present invention;

[0031] Figure 3 A flow chart for determining whether oscillation stability is qualified according to an embodiment of the present invention;

[0032] Figure 4 The figure is a flow chart of a sorting method using a sorting control system according to an embodiment of the present invention. DETAILED DESCRIPTION

[0033] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0034] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0035] It should be noted that, in the description of the present invention, terms such as "up", "down", "left", "right", "inside", and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0036] Furthermore, it should be noted that, in the description of the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integral connections; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0037] See also Figure 1-Figure 4 As shown, Figure 1 1 is a structural block diagram of a sorting control system of a flow cytometer according to an embodiment of the present invention; Figure 2 A flow chart of determining whether a droplet forming process is qualified according to an embodiment of the present invention; Figure 3 A flow chart for determining whether oscillation stability is qualified according to an embodiment of the present invention; Figure 4 The figure is a flow chart of a sorting method using a sorting control system according to an embodiment of the present invention.

[0038] An embodiment of the present invention provides a sorting control system for a flow cytometer, comprising:

[0039] A data acquisition unit, comprising an image sensor for acquiring a breakpoint image of a liquid droplet, a flow sensor for acquiring a flow rate of the sheath liquid, and a laser Doppler vibrometer for acquiring an oscillation curve of the piezoelectric ceramic;

[0040] A control unit, connected to the data acquisition unit, comprising:

[0041] a breakpoint determination module, configured to determine a length gradient of adjacent droplets and a frequency curve of the droplets based on the breakpoint image, to determine whether a droplet is a breakpoint based on the length gradient, and to determine a pixel variance based on the number of pixels at the breakpoint to determine whether the droplet forming process is qualified;

[0042] an oscillation evaluation module, configured to determine an oscillation area based on the oscillation curve to determine whether the oscillation stability is qualified;

[0043] a cell viscosity determination module, configured to establish a flow velocity curve based on the sheath fluid flow velocity, determine the eligibility of the cell viscosity based on a matching degree between the flow velocity curve and the frequency curve, and increase the sheath fluid pressure based on the fluctuation intensity of the fluorescence signal if the cell viscosity is unqualified;

[0044] The sorting control module is used to determine the target droplet according to the fluorescence detection result, determine the droplet delay according to the shape of the target droplet, and determine whether the target droplet can fall into the collection tube according to the initial deviation angle of the target droplet, so as to adjust the inter-plate voltage of the deflection plate.

[0045] It is understandable that when a flow cytometer is used for cell sorting, it is first necessary to prepare a cell suspension of appropriate concentration, which is generally 1×10 6 -1×10 7 cells / ml; secondly, fluorescently labeled antibodies are selected based on the antigens on the surface of the target cells. The cells are incubated in the dark for 15-30 minutes, centrifuged, and resuspended in PBS (fetal bovine serum) to remove unbound antibodies. The cell suspension is transferred to the flow chamber at an appropriate uptake rate, which should be less than 1 / 4 of the nozzle frequency. For example, if an 87kHz nozzle is selected, the uptake rate can be 20,000 events / s. The nozzle should be selected to be larger than 5 times the diameter of the target cells. The cell suspension is then wrapped with sheath fluid to form a single cell column, which then passes through the laser detection point in sequence. At this point, fluorescently labeled antibodies (such as CD3-PE and PD-L1-APC) on the cell surface or within the cell are excited by the laser, generating a fluorescence signal of a specific wavelength. After photoelectric conversion, the fluorescence signal is used to select target cells through multi-parameter gating, triggering the droplet delay mechanism to charge the target droplets, causing them to be deflected by the high-voltage electric field into the designated collection tube, thereby completing the cell sorting.

[0046] Specifically, the droplet delays the spatiotemporal matching relationship between the laser detection window and the droplet breakpoint. After the cell passes through the laser detection point, it takes the liquid flow propagation time to reach the droplet breakpoint (that is, the position where the droplet breaks away from the sheath fluid to form an independent droplet). This time difference is expressed in the number of pixels (not seconds / milliseconds).

[0047] Specifically, the breakpoint image range is the range from the nozzle to a completely independent droplet.

[0048] Specifically, the image sensor is an industrial camera that meets the acquisition requirements, which is not specifically limited; the flow sensor is, for example, an ultrasonic flowmeter, which is not specifically limited; the specific model of the laser Doppler vibrometer is not limited.

[0049] Specifically, the breakpoint determination module determines that the droplet is a breakpoint based on a comparison result that the length gradient is greater than or equal to a preset gradient;

[0050] The breakpoint determination module determines that the droplet is not a breakpoint based on a comparison result that the length gradient is less than the preset gradient.

[0051] Specifically, the length of the droplet is related to the oscillation frequency of the piezoelectric ceramic. The greater the frequency, the smaller the droplet length, and the smaller the frequency, the longer the droplet length. For example, when 87kHz is selected, the droplet length range is 15-20 microns, and the preset gradient value range is set to [3 microns, 5 microns]. 3 microns is preferred in the embodiment of the present invention.

[0052] Specifically, the breakpoint determination module determines the number of pixels of the breakpoint to calculate the pixel variance under the condition that the droplet is determined to be a breakpoint, and determines that the droplet forming process is unqualified based on a comparison result that the pixel variance is greater than a preset variance;

[0053] The droplet forming process is determined to be qualified based on the comparison result that the pixel variance is less than or equal to the preset variance.

[0054] It can be understood that the smaller the pixel variance is, the better the quality of the droplet forming process is.

[0055] Specifically, the value range of the preset variance is set to [0.1, 0.4], and 0.2 is preferably used in the embodiment of the present invention.

[0056] Specifically, under the condition that the droplet forming process is determined to be unqualified, the oscillation evaluation module overlaps the oscillation curve and the standard oscillation curve with the coordinate origin as the reference point, so as to determine the area enclosed by the union of the oscillation curve and the standard oscillation curve and the coordinate axis as the oscillation area.

[0057] Specifically, the oscillation evaluation module determines that the oscillation stability is qualified based on a comparison result that the oscillation area is less than or equal to a standard oscillation area;

[0058] The oscillation evaluation module determines that the oscillation stability is unqualified based on a comparison result that the oscillation area is greater than the standard oscillation area.

[0059] It can be understood that the larger the oscillation area, the worse the oscillation stability.

[0060] Specifically, the standard oscillation area is the maximum area enclosed by the oscillation curve and the coordinate axis within the allowable oscillation fluctuation range, such as <±0.5 kHz, that is, the area enclosed by the vibration curve and the coordinate axis within the maximum allowable fluctuation range.

[0061] Specifically, the cell viscosity determination module overlaps the flow rate curve and the frequency curve at intervals of time difference, determines the slopes corresponding to the same horizontal coordinates, determines the slope standard deviation based on the slopes, and determines the slope standard deviation as the matching degree.

[0062] Specifically, the time difference is the droplet delay, and the sheath liquid flow rate and the droplet frequency are different. Therefore, the judgment needs to be made after eliminating the time difference.

[0063] It can be understood that if the cell viscosity is qualified, the sheath fluid flow rate is high, the droplet generation frequency is high, and if the sheath fluid flow rate is low, the droplet generation frequency is low, and the fluctuation of the flow rate curve and the frequency curve are consistent; if the cell viscosity is unqualified, the sheath fluid flow rate is high, the increase in the droplet generation frequency is small or even unchanged, and the fluctuation of the flow rate curve and the frequency curve are inconsistent.

[0064] Specifically, after the flow velocity curve and the frequency curve are overlapped, the two slopes corresponding to the same horizontal coordinate are determined respectively, and the two slopes are subtracted to obtain the slope difference. The slope standard deviation is calculated according to the slope difference and standard deviation formula. The size of the slope standard deviation represents the degree of dispersion of the slope difference. If the volatility of the flow velocity curve and the frequency curve is consistent, the slope difference fluctuates little and the data dispersion is small. If the volatility of the flow velocity curve and the frequency curve is inconsistent, the slope difference fluctuates greatly and the data dispersion is large.

[0065] Specifically, the cell viscosity determination module determines that the cell viscosity is unqualified based on the comparison result that the matching degree is greater than a preset matching degree;

[0066] The cell viscosity determination module determines that the cell viscosity is unqualified based on the comparison result that the matching degree is less than or equal to a preset matching degree.

[0067] Specifically, the preset matching degree value range is set to [0.3, 0.5], and 0.4 is preferred in the embodiment of the present invention.

[0068] Specifically, the cell viscosity determination module determines the fluctuation intensity of the fluorescence signal based on the judgment result that the cell viscosity is unqualified, compares the fluctuation intensity with a preset intensity to obtain a corresponding comparison result, and sets several adjustment coefficients corresponding to the corresponding comparison results to increase the sheath fluid pressure.

[0069] It is understandable that during the cell sorting process, if the sheath fluid temperature is lower than room temperature, the arrangement of the cell membrane lipid bilayer tends to be ordered, the membrane fluidity decreases, and the cell viscosity increases; stressors such as mechanical pressure and charge changes during the sorting process can activate intracellular signaling pathways (such as MAPK / ERK), upregulate the expression of integrin family proteins (such as LFA-1), enhance cell-cell or cell-matrix adhesion, and thus lead to changes in cell viscosity; dead cells generated during the sorting process release intracellular substances such as DNA and histones, forming a network structure to wrap living cells, significantly increasing the overall viscosity of the cell suspension.

[0070] It is understandable that an increase in cell viscosity will result in a larger or smaller fluorescence signal intensity.

[0071] Specifically, the cell viscosity determination module overlaps the fluorescence spectrum with the standard spectrum, and records the percentage of the overlapping area to the area of ​​the standard spectrum and the absolute difference as the fluctuation intensity, wherein the standard spectrum is the fluorescence spectrum of the same target cell in the historical detection process of qualified cell viscosity.

[0072] It is understandable that when screening target cells, there may be situations where multiple parameters are set, so there will be multiple graphs in the test results. In this case, any fluctuation intensity is calculated according to the above method, and the fluctuation intensities are added up, and the sum result is recorded as the fluctuation intensity.

[0073] Specifically, the cell viscosity determination module determines to increase the sheath fluid pressure by a first adjustment coefficient based on a comparison result that the fluctuation intensity is greater than the preset intensity;

[0074] The cell viscosity determination module determines to increase the sheath fluid pressure by a second adjustment coefficient based on a comparison result that the fluctuation intensity is less than or equal to the preset intensity.

[0075] Specifically, the value range of the preset intensity is set to [10%, 30%], and 15% is preferred in the embodiment of the present invention; the value range of the first adjustment coefficient is set to [1.003, 1.005], and 1.0035 is preferred in the embodiment of the present invention; the value range of the second adjustment coefficient is set to [1.001, 1.0029], and 1.0015 is preferred in the embodiment of the present invention.

[0076] Specifically, the sorting control module determines that the target droplet cannot fall into the collection tube based on the comparison result that the initial offset angle is less than the first preset angle or greater than the second preset angle, and subtracts the initial offset angle from the preset angle to obtain several angle differences, and sets several correction coefficients corresponding to the angle differences to increase or decrease the inter-plate voltage.

[0077] Specifically, the initial offset angle is the angle between a line connecting the position of the target droplet just entering the deflection plate and the position of the target droplet when it is charged, and a vertical line.

[0078] It can be understood that the first preset angle is the minimum deviation angle that enables the target droplet to enter the collection tube, which is usually 10°, and the second preset angle is the maximum deviation angle that enables the target droplet to enter the collection tube, which is usually 30°.

[0079] Specifically, the sorting control module, based on a comparison result that the initial offset angle is smaller than the first preset angle, subtracts the first preset angle from the initial offset angle to obtain a first difference value;

[0080] The sorting control module determines to increase the inter-plate voltage by a first voltage correction coefficient based on a comparison result that the first difference is greater than a preset difference;

[0081] The sorting control module determines to increase the inter-plate voltage by a second voltage correction coefficient based on a comparison result that the first difference is less than or equal to the preset difference.

[0082] Specifically, the sorting control module, based on a comparison result that the initial offset angle is greater than the second preset angle, subtracts the initial offset angle from the second preset angle to obtain a second difference value;

[0083] The sorting control module determines to reduce the inter-plate voltage by a third voltage correction coefficient based on a comparison result that the second difference is greater than a preset difference;

[0084] The sorting control module determines to reduce the inter-plate voltage by a fourth voltage correction coefficient based on a comparison result that the second difference is less than or equal to the preset difference.

[0085] Specifically, the value range of the preset difference is set to [1°, 3°], and 3° is preferably selected in the embodiment of the present invention; the value range of the first voltage correction coefficient is set to [1.015, 1.03], and 1.02 is preferably selected in the embodiment of the present invention; the value range of the second voltage correction coefficient is set to [1.005, 1.014], and 1.01 is preferably selected in the embodiment of the present invention; the value range of the third voltage correction coefficient is set to [0.97, 0.985], and 0.98 is preferably selected in the embodiment of the present invention; the value range of the fourth voltage correction coefficient is set to [0.986, 0.995], and 0.99 is preferably selected in the embodiment of the present invention.

[0086] It is understandable that when an adjustment coefficient or correction coefficient is used to adjust a corresponding parameter in the embodiment of the present invention, the product of the adjustment coefficient or correction coefficient and the corresponding parameter to be adjusted is the adjusted parameter.

[0087] An embodiment of the present invention further provides a flow cytometer sorting method, comprising:

[0088] Step S1, acquiring a plurality of breakpoint images of the droplet to determine whether the droplet forming process is qualified according to the pixel variance of the breakpoints;

[0089] Step S2, based on the unqualified droplet forming process, determining the oscillation stability according to the oscillation area of ​​the oscillation curve of the piezoelectric ceramic to obtain the sheath liquid flow rate;

[0090] Step S3, determining a matching degree based on a flow rate curve of the sheath liquid flow rate and a frequency curve of the droplets to increase the sheath liquid pressure according to the fluctuation intensity of the fluorescence signal;

[0091] Step S4, determining the target droplet according to the fluorescence detection result to determine the droplet delay according to the target droplet, and determining whether the target droplet can fall into the collection tube according to the initial deviation angle of the target droplet, so as to adjust the inter-plate voltage of the deflection plates.

[0092] It is understandable that in the embodiment of the present invention, the number of pixels of the target droplet, that is, the droplet delay, is directly obtained based on the breakpoint image, and the charging timing is compensated according to the droplet delay to improve the sorting accuracy.

[0093] Thus far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art may make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will fall within the scope of protection of the present invention.

Claims

1. A flow cytometer sorting control system, characterized in that: include: A data acquisition unit, comprising an image sensor for acquiring a breakpoint image of a liquid droplet, a flow sensor for acquiring a flow rate of the sheath liquid, and a laser Doppler vibrometer for acquiring an oscillation curve of the piezoelectric ceramic; a control unit connected to the data acquisition unit, include, a breakpoint determination module, configured to determine a length gradient of adjacent droplets and a frequency curve of the droplets based on the breakpoint image, to determine whether a droplet is a breakpoint based on the length gradient, and to determine a pixel variance based on the number of pixels at the breakpoint to determine whether the droplet forming process is qualified; an oscillation evaluation module, configured to determine an oscillation area based on the oscillation curve to determine whether the oscillation stability is qualified; a cell viscosity determination module, configured to establish a flow velocity curve based on the sheath fluid flow velocity, determine the eligibility of the cell viscosity based on a matching degree between the flow velocity curve and the frequency curve, and increase the sheath fluid pressure based on the fluctuation intensity of the fluorescence signal if the cell viscosity is unqualified; The sorting control module is used to determine the target droplet according to the fluorescence detection result, determine the droplet delay according to the shape of the target droplet, and determine whether the target droplet can fall into the collection tube according to the initial deviation angle of the target droplet, so as to adjust the inter-plate voltage of the deflection plate.

2. The flow cytometer sorting control system according to claim 1, characterized in that: The breakpoint determination module determines that the droplet is a breakpoint based on a comparison result that the length gradient is greater than or equal to a preset gradient.

3. The flow cytometer sorting control system according to claim 2, characterized in that: The breakpoint determination module determines the number of pixels of the breakpoint to calculate the pixel variance when the droplet is determined to be a breakpoint, and determines that the droplet forming process is unqualified based on a comparison result that the pixel variance is greater than a preset variance.

4. The flow cytometer sorting control system according to claim 3, characterized in that: When determining that the droplet forming process is unqualified, the oscillation evaluation module overlaps the oscillation curve and the standard oscillation curve with the coordinate origin as a reference point, so as to determine the area enclosed by the union of the oscillation curve and the standard oscillation curve and the coordinate axis as the oscillation area.

5. The flow cytometer sorting control system according to claim 4, characterized in that: The oscillation evaluation module determines that the oscillation stability is qualified based on a comparison result that the oscillation area is less than or equal to a standard oscillation area.

6. The flow cytometer sorting control system according to claim 5, characterized in that: The cell viscosity determination module overlaps the flow rate curve and the frequency curve at intervals of time difference, determines the slopes corresponding to the same horizontal coordinates, determines the slope standard deviation based on the slopes, and determines the slope standard deviation as the matching degree.

7. The flow cytometer sorting control system according to claim 6, characterized in that: The cell viscosity determination module determines that the cell viscosity is unqualified based on the comparison result that the matching degree is greater than a preset matching degree.

8. The flow cytometer sorting control system according to claim 7, characterized in that: The cell viscosity determination module determines the fluctuation intensity of the fluorescence signal based on the determination result that the cell viscosity is unqualified, compares the fluctuation intensity with a preset intensity to obtain a corresponding comparison result, and sets a number of adjustment coefficients corresponding to the corresponding comparison results to increase the sheath fluid pressure.

9. The flow cytometer sorting control system according to claim 8, characterized in that: The sorting control module determines that the target droplet cannot fall into the collection tube based on the comparison result that the initial offset angle is less than the first preset angle or greater than the second preset angle, and subtracts the initial offset angle from the preset angle to obtain a number of angle differences, and sets a number of correction coefficients corresponding to the angle differences to increase or decrease the inter-plate voltage.

10. A sorting method applied to the sorting control system of the flow cytometer according to any one of claims 1 to 9, characterized in that: include: Acquire a plurality of breakpoint images of the droplet to determine whether the droplet forming process is qualified according to the pixel variance of the breakpoints; Based on the unqualified droplet forming process, the oscillation stability is determined according to the oscillation area of ​​the oscillation curve of the piezoelectric ceramic to obtain the sheath liquid flow rate; determining a matching degree based on a flow rate curve of the sheath fluid flow rate and a frequency curve of the droplet to increase the sheath fluid pressure according to the fluctuation intensity of the fluorescence signal; The target droplet is determined according to the fluorescence detection result to determine the droplet delay according to the target droplet, and whether the target droplet can fall into the collection tube is determined according to the initial deviation angle of the target droplet to adjust the inter-plate voltage of the deflection plate.

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