Flow sorter droplet delay adjustment method and system based on real-time cell flow velocity

Abstract

The invention provides a flow sorter liquid drop delay adjusting method and system based on real-time cell flow velocity, relates to the field of flow sorting, and ensures that the liquid drop delay time can not be delayed even if factors such as temperature, nozzle state, air pressure and pipelines are changed in the sorting process by monitoring the cell flow velocity in real time and dynamically adjusting the liquid drop delay time. And the charge loading time of the liquid drop can be accurately controlled. Meanwhile, the liquid drop delay time is dynamically adjusted in real time, so that the random distribution of the cell flow velocity during high-speed sample loading can be effectively dealt with, and the cells at different positions can be accurately sorted. The automatic calibration and self-stabilization functions are achieved, a large number of experiment articles and a complex optimization process are not needed, accurate control over liquid drop delay can be completed only through real-time monitoring and adjustment on the basis of liquid drop delay initial value setting, the separation efficiency is improved, the experiment cost and the operation difficulty are reduced, and the separation efficiency is improved. And the cell sorting requirements in various biomedical fields are met.

Description

Technical Field

[0001] This invention relates to the field of flow cytometry technology, and in particular to a method and system for adjusting droplet delay in a flow cytometer based on real-time cell flow rate. Background Technology

[0002] Flow cytometry is an important tool for cell analysis and sorting. Its working principle involves a cell suspension sample entering a flow chamber. Cells are focused under the influence of the sheath fluid and sequentially pass through a detection area, allowing for the acquisition of cell characteristic information one by one. High-frequency vibrations generated by a piezoelectric crystal in the flow chamber break the stable liquid flow into a series of extremely uniform microdroplets. These cell-containing droplets are then charged, and under the influence of a subsequent electric field, they are deflected, thus achieving cell sorting. The droplet charging time is determined by the droplet delay time, which is the time difference between the cell passing through the laser detection point and the droplet being charged at the break point. The accuracy of the droplet delay directly affects the cell sorting yield and purity.

[0003] Currently, accurate determination of droplet delay time is typically achieved through imaging data processing or feedback verification of sorting results. A common approach is to perform statistical analysis on the information contained in the sorted samples to optimize the droplet delay time. This method, which verifies the accuracy of the delay through the final result, offers high precision but is time-consuming and requires experimental samples such as microspheres.

[0004] The main problem with existing technologies is the inability to obtain droplet delay time in real time. Droplet delay is not constant; it is affected by factors such as sheath fluid pressure, nozzle diameter, and cell sample characteristics. During flow cytometry sorting, any fluctuation in flow rate caused by any factor, such as temperature changes, nozzle status changes, unstable gas pressure, or minor blockages in the tubing, will lead to changes in droplet delay. Furthermore, during sample loading, because the sample flow distribution has a certain width, the cell position distribution within the sample flow is random, and the flow velocity of cells at different positions is not the same. Using a single, fixed delay value will lead to a decrease in sorting yield and purity, especially at high-speed sample loading, where this effect is more pronounced.

[0005] Therefore, there is an urgent need for a method that can dynamically adjust droplet delay during the sorting process to improve the reliability and efficiency of sorting. Summary of the Invention

[0006] To achieve the above-mentioned objectives and other advantages of the present invention, a first objective of the present invention is to provide a method for adjusting droplet delay in a flow cytometer based on real-time cell flow rate, comprising the following steps: Determine the initial droplet delay time value and the corresponding average particle velocity of the flow cytometer; During the sorting process of the flow cytometer, the real-time flow rate of the particles to be sorted passing through the sensing zone is detected in real time; Based on the real-time flow rate, the droplet delay time value corresponding to the current particle is dynamically adjusted according to the initial droplet delay time value and the average particle flow rate. Based on the adjusted droplet delay time value, the charging operation of the droplet containing the particle is controlled to perform sorting.

[0007] Furthermore, the step of determining the initial droplet delay time value of the flow cytometer and its corresponding average particle velocity includes: Inject calibration microsphere samples into the flow separator; Adjust and determine the initial droplet delay time value that optimizes the sorting deflection effect; Under the condition of determining the initial droplet delay time value, the flow rate of multiple calibration microspheres through the sensing area is detected and obtained, and the average flow rate of the particles is calculated.

[0008] Furthermore, the step of adjusting and determining the initial droplet delay time value that optimizes the sorting deflection effect specifically includes: Adjust the droplet delay time and acquire fluorescence intensity images of the deflection region and waste liquid region; Calculate the ratio of the brightness of the deflection zone to the sum of its brightness and that of the waste liquid zone; The droplet delay time corresponding to when the fluorescence brightness ratio reaches its maximum value is determined as the initial droplet delay time value.

[0009] Furthermore, the formula for calculating the droplet delay time value D corresponding to the current particle is as follows: ,in, This is the initial droplet delay time value. The average velocity of the particles. This is the real-time flow rate.

[0010] Furthermore, the method for dynamically adjusting the droplet delay time value based on the real-time flow rate is any one of the following: For each detected particle, its corresponding droplet delay time value is adjusted individually based on its real-time flow rate. The real-time flow velocity of multiple particles is periodically measured and the average flow velocity is calculated. This average flow velocity is then used as the real-time flow velocity in the calculation to update the droplet delay time value that is common to all particles over a subsequent period of time.

[0011] Furthermore, the method also includes a droplet breakpoint stabilization step: Acquire images of droplet breakage; Extract a first feature parameter representing the location of the fracture point and a second feature parameter representing the width of the fracture gap from the image; The first and second characteristic parameters are compared with preset target values, and the frequency and / or amplitude of the driving piezoelectric oscillator are adjusted to keep the first and second characteristic parameters near the target values ​​to ensure that the reference distance of droplet delay is constant.

[0012] Furthermore, the real-time detection of the flow rate of the particles to be sorted through the sensing area is achieved in any of the following ways: A laser spot much larger than the particle size is set in the sensing area. The pulse signal generated when the particle passes through the laser spot is collected. The real-time flow velocity is calculated based on the pulse width of the pulse signal. At least two laser detection spots with a known distance are set along the flow direction in the sensing area. The time difference of the signal generated when the particles pass through each spot in sequence is collected. The real-time flow velocity is calculated based on the distance and the time difference.

[0013] A second objective of this invention is to provide a droplet delay adjustment system for a flow cytometer based on real-time cell flow rate, for implementing the above-mentioned method, comprising: The detection module is used to excite the particles and collect the light signals they generate; The droplet generation module is used to drive the sheath fluid to oscillate, causing the fluid flow to break into uniform droplets; The droplet delay measurement module is used to determine the initial droplet delay time value during the initialization phase; The flow rate measurement module is used to measure the average flow rate of particles corresponding to the initial droplet delay time value during the initialization phase, and to detect the real-time flow rate of the particles to be sorted through the sensing zone during the sorting process. The control processing module is electrically connected to the flow velocity measurement module and is used to dynamically calculate the droplet delay time value corresponding to the current particle based on the real-time flow velocity, the initial droplet delay time value, and the average particle flow velocity. The sorting execution module is electrically connected to the control processing module and is used to apply charge to the droplets at the corresponding time according to the droplet delay time value output by the control processing module, so as to achieve sorting.

[0014] Furthermore, it also includes a droplet stabilization control module, which comprises: A fracture location monitoring unit is used to acquire images of droplet fracture. The stabilization control unit is electrically connected to the fracture location monitoring unit and is used to extract a first feature parameter and a second feature parameter from the image, and to maintain the first feature parameter and the second feature parameter stable by controlling the driving frequency and / or amplitude of the piezoelectric oscillator in the droplet generation module.

[0015] Furthermore, the system is also configured with a sorting mode module, which is used to execute different sorting logics, including: A single droplet is divided into n equal parts in the spatial dimension to locate the relative position of the particles in the droplet; Preset yield mask, purification mask, and phase mask; Based on the purity mode, enrichment mode, or single-cell mode selected by the user, different mask combinations are invoked, and combined with the droplet delay time value dynamically adjusted by the control processing module, the final sorting decision and execution logic are jointly determined.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method and system for adjusting droplet delay in a flow cytometer based on real-time cell flow rate, improving the purity and yield of sorted samples. By monitoring cell flow rate in real time and dynamically adjusting the droplet delay time, it ensures accurate control of the time for applying charge to the droplets during the sorting process, even when factors such as temperature changes, nozzle status changes, unstable air pressure, or minor blockages in the tubing change. Simultaneously, real-time dynamic adjustment of the droplet delay time effectively addresses the randomness of cell flow rate during high-speed sample loading, ensuring accurate sorting of cells at different locations. This not only improves the sorting purity of target cells but also significantly increases the sorting yield and reduces sample loss.

[0017] This invention achieves automatic calibration and self-stabilization: on the one hand, it provides automatic stabilization of droplet breakpoint positions to ensure the stability of droplet delay; on the other hand, it incorporates a personalized droplet delay adjustment strategy for each particle, thus solving the drift and uncertainty problems in cell sorting through a two-pronged approach. The system can automatically and continuously set, monitor, and adjust the delay time using internal circuitry and programming, thereby allowing for fully automated and self-stabilizing sorting operations. This automation not only simplifies the operation process but also ensures stability during long-term operation.

[0018] This invention is highly efficient and practical: it requires no large amount of experimental supplies or complex optimization processes. By simply setting an initial droplet delay value and then monitoring and adjusting it in real time, precise control of the droplet delay can be achieved in a short time. This method not only improves sorting efficiency but also reduces experimental costs and operational complexity, making it suitable for cell sorting needs in various biomedical fields.

[0019] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0020] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 This is a schematic diagram of a droplet delay adjustment system for a flow cytometer based on real-time cell flow rate. Figure 2 A schematic diagram of the yield mask; Figure 3 This is a diagram illustrating the purification mask. Figure 4 This is a schematic diagram of a phase mask; Figure 5 Flowchart for real-time droplet delay adjustment; Figure 6 Flowchart of a droplet delay adjustment method for a flow cytometer based on real-time cell flow rate; Figure 7 A flow chart was created to determine the initial droplet delay time value and the corresponding average particle velocity for the flow cytometer. Figure 8 Flowchart for determining the initial droplet delay time value using the droplet delay measurement module; Figure 9 This is the first flow rate testing scheme. Figure 10 Scheme 2 for flow rate testing; Figure 11 This is a schematic diagram of the deflection zone and the waste liquid zone; Figure 12 Flowchart for maintaining the stabilization of droplet breakpoint locations; Figure 13 Diagram of droplet formation; Figure 14 The deflection fluid flow state under different W values; Figure 15 Flowchart for maintaining stability of the centroid of the first fractured droplet; Figure 16 Flowchart for maintaining stable spacing between the first fractured droplets; Figure 17 A schematic diagram of computer equipment; Figure 18 This is a schematic diagram of a computer-readable storage medium. Detailed Implementation

[0021] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0022] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0023] The drawing numbers in this application are only used to distinguish the steps in the scheme and are not used to limit the execution order of the steps. The specific execution order is as described in the specification.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0025] To address the following technical problems in existing technologies: Fixed droplet delay time leads to poor sorting results: In existing technologies, the droplet delay time is usually fixed and cannot adapt to changes in the environment or individual factors in real time. When environmental conditions such as temperature changes, nozzle status changes, or unstable air pressure occur, this can lead to inaccurate droplet delay time, thereby affecting the yield and purity of cell sorting.

[0026] Random cell flow rate during sample loading increases sorting error: During sample loading, the sample flow distribution has a certain width, and the cell positions within the sample flow are random, with varying flow rates at different locations. Using a single, fixed delay value leads to a decrease in sorting yield and purity, especially at high-speed loading, where this effect is more pronounced.

[0027] Current technologies cannot adjust droplet delay time in real time: Existing technologies typically rely on feedback to optimize droplet delay time, which is time-consuming, requires experimental materials such as microspheres, and cannot be adjusted in real time. Therefore, there is an urgent need for a method that can adjust droplet delay time in real time during the sorting process to improve the reliability and efficiency of sorting.

[0028] This invention provides a method and system for dynamically adjusting droplet delay in a flow cytometer based on real-time cell flow rate. This method allows for real-time monitoring of cell flow rate and dynamic adjustment of droplet delay time, ensuring that target cells are accurately sorted into designated collection tubes, thereby improving sorting yield and purity. The specific solution is as follows:

[0029] Example 1 A droplet delay adjustment system for a flow cytometer based on real-time cell flow rate, such as Figure 1 As shown, it includes: The detection module is used to excite the particles and collect the light signals they generate; The droplet generation module is used to drive the sheath fluid to oscillate, causing the fluid flow to break into uniform droplets; The droplet delay measurement module is used to determine the initial droplet delay time value during the initialization phase; The flow rate measurement module is used to measure the average flow rate of particles corresponding to the initial droplet delay time value during the initialization phase, and to detect the real-time flow rate of the particles to be sorted through the sensing zone during the sorting process. The control processing module is electrically connected to the flow velocity measurement module and is used to dynamically calculate the droplet delay time value corresponding to the current particle based on the real-time flow velocity, the initial droplet delay time value, and the average particle flow velocity. The sorting execution module is electrically connected to the control processing module and is used to apply charge to the droplets at the corresponding time according to the droplet delay time value output by the control processing module, so as to achieve sorting.

[0030] This invention ensures accurate control over the time it takes to charge the droplets during the sorting process by real-time monitoring of cell flow rate and dynamic adjustment of droplet delay time. This control is achieved even when factors such as temperature changes, nozzle status variations, unstable gas pressure, or minor blockages in the tubing change. Furthermore, real-time dynamic adjustment of the droplet delay time effectively addresses the randomness of cell flow rate during high-speed sample loading, ensuring accurate sorting of cells at different locations. This solution overcomes the problem of poor sorting results caused by a fixed droplet delay time in existing technologies, thus improving the accuracy and reliability of droplet sorting.

[0031] In some embodiments, the detection module includes an excitation unit and an optical path collection unit. The excitation unit uses various lasers to provide monochromatic light of a specific wavelength and shapes the light spot into a fixed shape and size, which then illuminates the flow chamber.

[0032] When cells carrying fluorescent markers pass through the laser detection point, they are excited to produce scattered light and fluorescence. These signals are guided and filtered by a series of lenses, spectrometers, and filters in the optical path collection unit, and are ultimately distributed to different detection channels. The signal detector is connected to a photodetector at its end. Photomultiplier tubes detect side-scattered light and fluorescence; photodiodes are typically used to detect stronger forward-scattered light.

[0033] In some embodiments, the droplet generation module includes a flow unit, a fluid pool unit, and a droplet generation control board. The flow unit uses sheath fluid to guide cells in a single file through the detection area. Various cell position focusing techniques can be employed, such as hydrodynamic focusing, acoustic focusing, and inertial focusing. Position focusing ensures that the laser irradiates only one cell at a time. The fluid pool unit includes a piezoelectric oscillator. The droplet generation control board generates oscillations applied to the sheath fluid. The sheath fluid passes through a nozzle, and the outflowing flow breaks into uniform droplets, preparing for charge sorting. The droplet generation control board generates a voltage signal to drive the piezoelectric oscillator.

[0034] In some embodiments, a droplet stabilization control module is further included, the droplet stabilization control module comprising: A fracture location monitoring unit is used to acquire images of droplet fracture. Specifically, the fracture location monitoring unit includes a monitoring camera and a stroboscopic light source, used to provide illumination signals and acquire images of the droplet fracture. The stroboscopic light source includes light-emitting diodes (LEDs), and the switching frequency of the light source matches the droplet generation frequency. By setting appropriate exposure time and brightness, clear imaging can be ensured.

[0035] The stabilization control unit is electrically connected to the fracture location monitoring unit and is used to extract a first feature parameter and a second feature parameter from the image, and to maintain the first feature parameter and the second feature parameter stable by controlling the driving frequency and / or amplitude of the piezoelectric oscillator in the droplet generation module.

[0036] This embodiment monitors key parameters of the droplet fracture image (the position S of the centroid of the first fractured droplet and the width W of the fracture gap) through a stability control unit, and ensures the stability of the fracture point position by controlling the driving frequency and amplitude of the piezoelectric oscillator.

[0037] To ensure the accuracy of real-time droplet delay adjustment, it is necessary to stabilize key parameters of droplet breakage, which is a prerequisite for guaranteeing the initial droplet delay value. This invention provides automatic stabilization of the droplet breakpoint position to ensure droplet delay stability, and incorporates a personalized droplet delay adjustment strategy for each particle, thus addressing drift and uncertainty issues in cell sorting through a two-pronged approach. The system can automatically and continuously set, monitor, and adjust the delay time using internal circuitry and programming, allowing for fully automated and self-stabilizing sorting operations. This automation not only simplifies the operation process but also ensures stability during long-term operation.

[0038] In some embodiments, the droplet delay measurement module includes a high-voltage electrode and an imaging monitoring unit. The high-voltage electrode generates a high-voltage electrostatic field, which deflects charged droplets when they pass through it. The imaging monitoring unit includes a laser source and a monitoring camera. The laser illuminates the liquid column, and an image is acquired through an imaging window, dividing the image area into a waste liquid area and a deflection area. Microspheres are loaded at a low flow rate for calibration. These microspheres are excited by the laser and emit fluorescence of a specific wavelength. The fluorescence is collected by an imaging device (such as a CCD) through a filter. When the droplet delay is correct, droplets containing target cells are deflected; when the droplet delay is incorrect, some or all target particles may not be deflected. The optimal droplet delay is determined by the maximum ratio of the brightness of the deflection area to the sum of the brightness of the waste liquid area.

[0039] In some embodiments, the flow rate measurement module employs an optical method to detect the particle velocity in the fluid. The control processing module determines the initial droplet delay time value by calibrating the low-speed loading of microspheres. Corresponding average particle velocity During the formal sorting process, the droplet delay value D for each particle is adjusted by detecting the real-time flow rate V of each particle. The adjustment is made according to the following formula: Sorting is performed based on the adjusted droplet delay value.

[0040] The control and processing module converts the optical signal into analyzable digital information. The analog electrical signal generated by the photomultiplier tube is pre-amplified and then converted into a digital signal by an analog-to-digital converter. The embedded software processes these signals and, based on a preset gate logic strategy, determines the characteristics of each cell in real time and makes sorting decisions.

[0041] The sorting execution module, based on the sorting decision from the control processing module and the droplet delay value, applies a momentary electrostatic charge to the liquid flow just before the droplet breaks apart. A high-voltage deflector plate is positioned below the droplet break point. As the charged droplet passes through a pair of deflector plates carrying several kilovolts of voltage, it is deflected in the electrostatic field. Droplets with different charges deflect at different distances, thus sorting different target cells into different collection tubes. Uncharged droplets fall into the waste liquid container.

[0042] In other embodiments, during sorting, a momentary electrostatic charge is applied to the liquid flow just before each droplet breaks apart. The charged droplets are deflected by the electrostatic field, and according to the sorting decision of the control processing module, target droplets are not charged. Target droplets fall into the collection tube in the middle position, while non-target droplets fall into the adjacent waste container. This method effectively avoids loading the target cells with a charge, ensuring higher biological activity and function of the cells.

[0043] Since cells are encapsulated in droplets for sorting, sorting decisions involving multiple cells within the droplet need to be carefully considered. In some embodiments, different sorting modes are defined, including purity, enrichment, and single-cell modes, with the purity mode further divided into purity A and purity B modes. Specifically, the system also includes a sorting mode module, which executes different sorting logic, including: A single droplet is divided into n equal parts in the spatial dimension to locate the relative position of the particles in the droplet; By dividing each droplet into n equal parts, the location of target or non-target cells within the droplet can be determined.

[0044] Preset yield mask, purification mask, and phase mask; Among them, such as Figure 2 As shown, the yield mask sets the distance from the droplet edge in increments of 1 / n droplets, defining equal regions before and after the target droplet at half the yield mask value. When the target cell is within the mask, droplets before and after the target cell are additionally sorted based on the mask's position. When the yield mask is set to zero, only droplets containing the target cell are deflected. When the mask is set to n, two droplets are always deflected. Yield masks between 0 and n will sort one or two droplets, depending on the target cell's position within the droplet. For example, when n=32, the yield mask is set to 16, and the target cell is within 8 / 32 of the droplet's start, the previous droplet will be sorted. If the target cell is within 8 / 32 of the droplet's end, subsequent droplets will be sorted.

[0045] like Figure 3 As shown, the purification mask defines the proximity of non-target cells to target cells in increments of 1 / n droplets. The purification mask sets the distance from the droplet edge in increments of 1 / n droplets, defining equal regions near the target droplet at half the purification mask value. If a non-target cell is located within the mask or within the target droplet, it will be discarded. If a non-target cell is located outside the mask, it will be selected. For any purity mask greater than zero, the detected droplet must be free of contaminant particles; otherwise, the droplet will not be selected. If the purity mask is set to zero, droplets containing the target event will be selected regardless of contaminant particles. For example, when n=32 and the purity mask is set to 16, if a non-target particle falls within the last 8 / 32 of the preceding droplet or the first 8 / 32 of the trailing droplet, the target will not be selected.

[0046] like Figure 4 As shown, when a cell is at the edge of a droplet, the uncertainty of the droplet containing the target cell increases due to the randomness of droplet breakage. When the target cell is too close to the edge of the droplet, the phase mask restricts droplet deflection. An equal region is defined at the front and back ends of all droplets, using 1 / n droplets as increments and half the phase mask value. The phase mask is used to improve sorting accuracy and sideflow quality at the expense of yield. For example, when n=32 and the phase mask is set to 16, the detected droplet is only sorted if the target particle falls outside the phase mask. The phase mask cannot be used in conjunction with the yield mask. Therefore, when the phase mask is greater than zero, the yield mask automatically reverts to zero.

[0047] Based on the purity mode, enrichment mode, or single-cell mode selected by the user, different mask combinations are invoked, and combined with the droplet delay time value dynamically adjusted by the control processing module, the final sorting decision and execution logic are jointly determined.

[0048] Users can define different combinations of droplet masks to specify purity, enrichment, and single-cell sorting modes according to their needs. Below is one feasible combination mode.

[0049] Purity Mode: Ensures the highest purity of sorted cells. Purity A Mode: Simultaneously uses yield and purification masks to ensure the target particle is sorted from the droplet, and further sorts empty droplets based on the target droplet's location to ensure the target particle is sorted even at the droplet edge, thus improving yield. Purity B Mode: Uses only a purification mask to ensure the target particle is sorted from the droplet, without additionally sorting empty droplets, ensuring a minimal sorting system.

[0050] Enrichment Mode: Uses only a yield mask. Recovery is optimized, but at the cost of purity. This mode can be used as the first round of classification for target particle enrichment, followed by a purity classification. It is also suitable for sorting low-proportion cells, prioritizing obtaining a sufficient number of cells.

[0051] Single-cell mode: The purity mask is set to its maximum value, so only droplets free of contaminating particles are sorted. The phase mask is set to half of its maximum value, so only particles located near the center of sorted droplets are sorted. Droplet trajectory and counting accuracy are optimized at the expense of yield. This mode is recommended for well plate sorting or situations requiring precise counting.

[0052] Considering the randomness of cell encapsulation in droplets, and the uncertainty of a droplet containing multiple cells and the position of cells at the droplet breakage edge, this invention establishes different sorting modes, including purity, enrichment, and single-cell modes. The purity mode is further divided into purity A and purity B modes. Dividing each droplet into n equal parts allows for the location of target or non-target cells within the droplet. Three droplet masks are defined: a yield mask, a purification mask, and a phase mask. Different combinations of masks achieve different sorting modes to meet diverse user needs.

[0053] Based on the above system, automatic droplet delay adjustment is completed. For a detailed description of the adjustment method, please refer to the corresponding description in the following method embodiments, which will not be repeated here.

[0054] This invention specifically relates to a system and method for real-time adjustment of droplet delay time, as well as a sorting device, primarily used in flow cytometry cell sorters and related sorting equipment to improve cell sorting yield and purity. For example, in medical diagnostic and therapeutic applications, this technology can be used to efficiently sort and separate specific cell types, such as tumor cells and immune cells, thereby providing high-quality cell samples for disease diagnosis and personalized treatment. Furthermore, this technology can also be applied to biological research fields, such as separating sperm carrying X and Y chromosomes for animal breeding, sorting chromosomes for genetic analysis, and isolating specific organisms from complex biological populations. By dynamically adjusting the droplet delay in real time, this system can significantly improve sorting yield and purity, enhance sorting reliability, and is suitable for various cell sorting applications.

[0055] Example 2 A method for adjusting droplet delay in a flow cytometer based on real-time cell flow rate is disclosed. Based on the system described above, a detailed description of the system can be found in the corresponding description in the system embodiments described above, and will not be repeated here. This method is suitable for scenarios where droplet delay needs to be adjusted according to the flow rate of each particle, such as during high-speed sample loading. Figure 5 , Figure 6 As shown, the method includes the following steps: S100. Determine the initial droplet delay time value of the flow cytometer and its corresponding average particle velocity. In this embodiment, during the system initialization phase, an initial droplet delay time value is set. This initial value needs to be relatively accurate, and it is generally set using the result observation method to ensure that the initial sorting value is accurate.

[0056] Specifically, particle velocity detection points are set in the flow path to detect the signals generated when particles pass through these points. There can be one or more detection points. As particles flow through the flow path, the signals generated at the detection points are received by the control system and converted into particle velocities. The average velocity is calculated from the velocities of multiple particles, and this average velocity corresponds to the initial droplet delay value. By detecting the real-time velocity of each particle, the droplet delay value corresponding to each particle is adjusted. Sorting is then performed based on the adjusted droplet delay value.

[0057] In some embodiments, such as Figure 7 As shown, the step of determining the initial droplet delay time value and the corresponding average particle velocity of the flow cytometer includes: S110. Inject calibration microsphere samples into the flow separator; S120. Adjust and determine the initial droplet delay time value that optimizes the sorting deflection effect through the droplet delay measurement module; S130. Under the condition of determining the initial droplet delay time value, the flow velocity of multiple calibration microspheres passing through the sensing area is detected and obtained by the flow velocity measurement module, and the average flow velocity of the particles is calculated.

[0058] The droplet delay measurement module includes an imaging monitoring unit, such as... Figure 8 As shown, determining the initial droplet delay time value through the droplet delay measurement module specifically includes: S121. Adjust the droplet delay time and acquire fluorescence brightness images of the deflection zone and waste liquid zone through the imaging monitoring unit; S122. Calculate the ratio of the brightness of the deflection zone to the sum of its brightness and that of the waste liquid zone; S123. The droplet delay time corresponding to the fluorescence brightness ratio reaching its maximum value is determined as the initial droplet delay time value.

[0059] Specifically, upon detecting the target cell, a charge is applied to the droplet at the breakpoint after a droplet delay. When the droplet passes the high-voltage plate, it is deflected under the influence of the electrostatic field. An imaging window is positioned below the high-voltage plate, and a laser beam illuminates the liquid column. When the liquid column deflects, it is simultaneously illuminated by the laser. Images are acquired through the imaging window, and the image area is divided into a waste liquid area and a deflection area, such as... Figure 11 As shown. Microspheres are calibrated by loading samples at a low flow rate. These microspheres are excited by a laser and emit fluorescence of a specific wavelength. The fluorescence is collected by an imaging device (such as a CCD) through a filter. When the droplet delay is correct, droplets containing target cells are deflected; if the droplet delay is incorrect, some or all target particles may not be deflected. The correctness of the droplet delay is determined by the ratio of the brightness of the deflected area to the sum of the brightness of the waste liquid area. The droplet delay value is adjusted so that the ratio of the brightness of the deflected area to the sum of the brightness of the waste liquid area reaches its maximum, typically greater than 90%. This droplet delay value is the initial droplet delay value. After the initial values ​​are set, the initial flow rate corresponding to the current droplet delay value is calculated through flow rate detection. .

[0060] S200: During the sorting process of the flow cytometer, the real-time flow rate of the particles to be sorted passing through the sensing zone is detected in real time. Specifically, the real-time detection of the flow rate of the particles to be sorted through the sensing area is achieved in any of the following ways: like Figure 9 As shown, Method A: A laser spot with a size much larger than the particle size is set in the sensing area, and the pulse signal generated when the particle passes through the laser spot is collected. The real-time flow velocity is calculated based on the pulse width of the pulse signal. Method A uses a laser spot much larger than the cell size, ensuring that the time it takes for a cell to move completely into the spot and first leave the spot at its edge is significantly longer than the time it takes for the cell to enter and leave the spot. A difference of more than 10 times is recommended. The data acquisition system collects the pulse signals as the cell passes through the laser spot and calculates the pulse width, using pulse width to characterize the cell flow velocity.

[0061] like Figure 10 As shown, Method B: At least two laser detection spots with a known distance are set in the sensing area along the flow direction, and the time difference of the signal generated when the particles pass through each spot in sequence is collected. The real-time flow velocity is calculated based on the distance and the time difference.

[0062] Method B uses several laser detection spots, which can be used to detect fluorescence signals. The distance between the spots is much larger than the cell size. The cell flow rate is characterized by the time difference between the cells passing through different laser spots.

[0063] S300. Based on the real-time flow rate, the droplet delay time value corresponding to the current particle is dynamically adjusted according to the initial droplet delay time value and the average particle flow rate. After the initial values ​​are set, the real-time automatic adjustment of droplet delay begins. The droplet delay value D for each particle is adjusted by detecting the real-time flow velocity V of each particle; the adjustment is made according to the following formula: in, This is the initial droplet delay time value. The average velocity of the particles. This is the real-time flow rate.

[0064] The sorting process will then proceed based on the adjusted droplet delay value.

[0065] In some embodiments, the droplet delay time value is dynamically adjusted according to the real-time flow rate in any of the following ways: like Figure 5 As shown in (a), Method 1: For each detected particle, adjust its corresponding droplet delay time value individually according to its own real-time flow rate, which is suitable for high-speed sample loading; like Figure 5 As shown in (b), Method 2 involves periodically measuring the real-time flow velocity of multiple particles and calculating the average flow velocity. This average flow velocity is then used as the real-time flow velocity in the calculation to update the droplet delay time value common to all particles over a subsequent period. This method is suitable for scenarios where environmental factors change and the droplet delay needs to be adjusted based on the average flow velocity, i.e., it is suitable for overall flow fluctuations, such as when the temperature changes.

[0066] S400: Based on the adjusted droplet delay time value, control the charging operation of the droplet containing the particle to perform sorting.

[0067] In some embodiments, such as Figure 12 As shown, the process of executing the method also includes S500, a droplet breakpoint stabilization step: S510. Obtain droplet fracture images through the fracture location monitoring module; S520. Extract a first feature parameter representing the location of the fracture point and a second feature parameter representing the width of the fracture interval from the image. Stroboscopic imaging is performed on the droplet breakpoint. Stroboscopic imaging refers to matching the switching frequency of the light source with the droplet generation frequency, setting appropriate duty cycles and brightness of the light source to ensure a clear image. The droplet generation image is shown below. Figure 13 As shown. The characteristic parameters of the extracted droplet are included, such as the position S of the centroid of the first broken droplet and the width W of the breakage gap.

[0068] S530. The first characteristic parameter and the second characteristic parameter are compared with the preset target value, and the frequency and / or amplitude of the driving piezoelectric oscillator are adjusted to keep the first characteristic parameter and the second characteristic parameter near the target value to ensure that the reference distance of the droplet delay is constant.

[0069] Specifically, the fracture gap width W needs to be adjusted to a suitable value to ensure a proper phase relationship between the applied charge and droplet formation, thereby ensuring that the charge is applied according to the entire droplet cycle and that cross-droplet charge loading does not occur. This suitable value can be determined by monitoring the dispersion of the deflected liquid flow in the imaging region below, such as... Figure 14 As shown, the appropriate value of the break gap W is when the deflected liquid flow tightens. The position S of the centroid of the first broken droplet is related to the vibration amplitude of the piezoelectric element that breaks the continuous liquid column into discrete droplets; the higher the vibration amplitude, the closer the break point is to the nozzle. It is also related to the vibration frequency of the piezoelectric element that breaks the continuous liquid column into discrete droplets; the higher the vibration frequency, the more droplets are generated per second. The initial droplet delay is determined under specific S and W values. Therefore, in the real-time automatic adjustment of droplet delay, these S and W values ​​need to remain stable to ensure the consistency of the delay distance.

[0070] The stabilization of S and W values ​​can be achieved through the following methods: Figure 15 , Figure 16As shown, the distance F between the centroids of the two droplets is determined by the image. When only W deviates from the target value, the driving amplitude of the piezoelectric element is adjusted slightly to return it to its initial value. When S deviates from the target value, or when both S and W deviate from the target value simultaneously, the deviation of S from the target value is first determined. When the deviation exceeds F, the driving amplitude of the piezoelectric element is adjusted significantly to bring it back within the deviation F, and then the driving frequency of the piezoelectric element is adjusted to return it to its initial value. After S is adjusted, W is returned to its initial value by adjusting the driving amplitude of the piezoelectric element slightly.

[0071] This invention achieves improved sorting accuracy and efficiency through dynamic adjustment of droplet delay time: The invention measures the velocity of particles passing through the sensing zone, monitors the flow rate of each cell in real time, and dynamically adjusts the droplet delay time accordingly, ensuring that the droplet delay time is always at its optimal state. This dynamic adjustment mechanism increases the ability to personalize the delay for each detection object, effectively responding to changes in environmental or individual factors, and improving sorting yield and purity. Compared to a fixed delay time, the dynamic adjustment mechanism significantly enhances the reliability of sorting.

[0072] This invention is highly efficient and practical: it requires no large amount of experimental supplies or complex optimization processes. By simply setting an initial droplet delay value and then monitoring and adjusting it in real time, precise control of the droplet delay can be achieved in a short time. This method not only improves sorting efficiency but also reduces experimental costs and operational complexity, making it suitable for cell sorting needs in various biomedical fields.

[0073] This invention achieves automatic calibration and self-stabilization: on the one hand, it provides automatic stabilization of droplet breakpoint positions to ensure the stability of droplet delay; on the other hand, it incorporates a personalized droplet delay adjustment strategy for each particle, thus solving the drift and uncertainty problems in cell sorting through a two-pronged approach. The system can automatically and continuously set, monitor, and adjust the delay time using internal circuitry and programming, thereby allowing for fully automated and self-stabilizing sorting operations. This automation not only simplifies the operation process but also ensures stability during long-term operation.

[0074] In summary, this invention offers advantages such as a highly flexible dynamic adjustment mechanism, significantly improved sorting accuracy and efficiency, and powerful automatic calibration and self-stabilization functions. These advantages make this technology widely applicable in flow cytometers and other sorting equipment, significantly enhancing the effectiveness and reliability of cell sorting.

[0075] Example 3 A computer device 500, such as Figure 17As shown, the system includes a memory 510, a processor 520, and a computer program 530 stored in the memory and executable on the processor. When the processor executes the computer program, it implements the steps of a flow cytometer droplet delay adjustment method based on real-time cell flow rate. For a detailed description of the method, please refer to the corresponding description in the above method embodiments; it will not be repeated here.

[0076] Example 4 A computer-readable storage medium, such as Figure 18 As shown, a computer program is stored thereon. When executed by a processor, the computer program implements the steps of a flow cytometer droplet delay adjustment method based on real-time cell flow rate. For a detailed description of the method, please refer to the corresponding description in the above method embodiments, which will not be repeated here.

[0077] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0078] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

[0079] The apparatus, computer device, and non-volatile computer storage medium and method provided in the embodiments of this specification are corresponding. Therefore, the apparatus, computer device, and non-volatile computer storage medium also have similar beneficial technical effects as the corresponding method. Since the beneficial technical effects of the method have been described in detail above, the beneficial technical effects of the corresponding apparatus, computer device, and non-volatile computer storage medium will not be repeated here.

[0080] Those skilled in the art will also know that, besides implementing the controller in the form of purely computer-readable program code, the same functions can be achieved by logically programming the method steps, making the controller take the form of logic gates, switches, application-specific integrated circuits (ASICs), programmable logic controllers (PLCs), and embedded microcontrollers. Therefore, such a controller can be considered a hardware component, and the devices included within it for implementing various functions can also be considered structures within that hardware component. Alternatively, the devices for implementing various functions can be considered as both software units implementing the method and structures within a hardware component.

[0081] The systems, apparatuses, or units described in the above embodiments can be implemented by computer chips or physical entities, or by products with certain functions. For ease of description, the above apparatuses are described separately as various units based on their functions. Of course, when implementing one or more embodiments of this specification, the functions of each unit can be implemented in one or more software and / or hardware.

[0082] Those skilled in the art will understand that the embodiments of this specification can be provided as methods, systems, or computer program products. Therefore, the embodiments of this specification can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, the embodiments of this specification can take the form of a computer program product implemented on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0083] This specification is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of this specification. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, generate instructions for implementing the flowchart illustrations and / or block diagrams. Figure 1 One or more processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0084] These computer program instructions may also be stored in a computer-readable storage medium that can direct a computer or other programmable data processing device to function in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of manufacture including instruction means, which are implemented in a process Figure 1 One or more processes and / or boxes Figure 1 The function specified in one or more boxes.

[0085] These computer program instructions may also be loaded onto a computer or other programmable data processing equipment to cause a series of operational steps to be performed on the computer or other programmable equipment to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable equipment for implementing the process. Figure 1 One or more processes and / or boxes Figure 1 The steps of the function specified in one or more boxes.

[0086] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0087] This specification may be described in the general context of computer-executable instructions, such as program units, that are executed by a computer. Generally, program units include routines, programs, objects, components, data structures, etc., that perform a specific task or implement a specific abstract data type. This specification may also be practiced in distributed computing environments, where tasks are performed by remote processing devices connected via a communication network. In distributed computing environments, program units may reside in local and remote computer storage media, including storage devices.

[0088] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to mutually. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions in the method embodiments.

[0089] The above description is merely an embodiment of this specification and is not intended to limit the scope of one or more embodiments of this specification. Various modifications and variations can be made to one or more embodiments of this specification by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of one or more embodiments of this specification should be included within the scope of the claims of one or more embodiments of this specification.

Claims

1. A method for adjusting droplet delay in a flow cytometer based on real-time cell flow rate, characterized in that, Includes the following steps: Determine the initial droplet delay time value and the corresponding average particle velocity of the flow cytometer; During the sorting process of the flow cytometer, the real-time flow rate of the particles to be sorted passing through the sensing zone is detected in real time; Based on the real-time flow rate, the droplet delay time value corresponding to the current particle is dynamically adjusted according to the initial droplet delay time value and the average particle flow rate. Based on the adjusted droplet delay time value, the charging operation of the droplet containing the particle is controlled to perform sorting.

2. The method for adjusting droplet delay in a flow cytometer based on real-time cell flow rate as described in claim 1, characterized in that, The steps for determining the initial droplet delay time value and the corresponding average particle velocity of the flow cytometer include: Inject calibration microsphere samples into the flow separator; Adjust and determine the initial droplet delay time value that optimizes the sorting deflection effect; Under the condition of determining the initial droplet delay time value, the flow rate of multiple calibration microspheres through the sensing area is detected and obtained, and the average flow rate of the particles is calculated.

3. The method for adjusting droplet delay in a flow cytometer based on real-time cell flow rate as described in claim 2, characterized in that, The step of adjusting and determining the initial droplet delay time value that optimizes the sorting deflection effect specifically includes: Adjust the droplet delay time and acquire fluorescence intensity images of the deflection region and waste liquid region; Calculate the ratio of the brightness of the deflection zone to the sum of its brightness and that of the waste liquid zone; The droplet delay time corresponding to when the fluorescence brightness ratio reaches its maximum value is determined as the initial droplet delay time value.

4. The method for adjusting droplet delay in a flow cytometer based on real-time cell flow rate as described in claim 1, characterized in that, The formula for calculating the droplet delay time value D corresponding to the current particle is as follows: ,in, This is the initial droplet delay time value. The average velocity of the particles. This is the real-time flow rate.

5. The method for adjusting droplet delay in a flow cytometer based on real-time cell flow rate as described in claim 1, characterized in that, The method for dynamically adjusting the droplet delay time value based on the real-time flow rate is any of the following: For each detected particle, its corresponding droplet delay time value is adjusted individually based on its real-time flow rate. The real-time flow velocity of multiple particles is periodically measured and the average flow velocity is calculated. This average flow velocity is then used as the real-time flow velocity in the calculation to update the droplet delay time value that is common to all particles over a subsequent period of time.

6. The method for adjusting droplet delay in a flow cytometer based on real-time cell flow rate as described in claim 1, characterized in that, The method also includes a droplet breakpoint stabilization step: Acquire images of droplet breakage; Extract a first feature parameter representing the location of the fracture point and a second feature parameter representing the width of the fracture gap from the image; The first and second characteristic parameters are compared with preset target values, and the frequency and / or amplitude of the driving piezoelectric oscillator are adjusted to keep the first and second characteristic parameters near the target values ​​to ensure that the reference distance of droplet delay is constant.

7. The method for adjusting droplet delay in a flow cytometer based on real-time cell flow rate as described in claim 1, characterized in that, The real-time detection of the flow rate of the particles to be sorted through the sensing zone is achieved using any of the following methods: A laser spot much larger than the particle size is set in the sensing area. The pulse signal generated when the particle passes through the laser spot is collected. The real-time flow velocity is calculated based on the pulse width of the pulse signal. At least two laser detection spots with a known distance are set along the flow direction in the sensing area. The time difference of the signal generated when the particles pass through each spot in sequence is collected. The real-time flow velocity is calculated based on the distance and the time difference.

8. A droplet delay adjustment system for a flow cytometer based on real-time cell flow rate, used to implement the method as described in any one of claims 1-7, characterized in that, include: The detection module is used to excite the particles and collect the light signals they generate; The droplet generation module is used to drive the sheath fluid to oscillate, causing the fluid flow to break into uniform droplets; The droplet delay measurement module is used to determine the initial droplet delay time value during the initialization phase; The flow rate measurement module is used to measure the average flow rate of particles corresponding to the initial droplet delay time value during the initialization phase, and to detect the real-time flow rate of the particles to be sorted through the sensing zone during the sorting process. The control processing module is electrically connected to the flow velocity measurement module and is used to dynamically calculate the droplet delay time value corresponding to the current particle based on the real-time flow velocity, the initial droplet delay time value, and the average flow velocity of the particle. The sorting execution module is electrically connected to the control processing module and is used to apply charge to the droplets at the corresponding time according to the droplet delay time value output by the control processing module, so as to achieve sorting.

9. The droplet delay adjustment system for a flow cytometer based on real-time cell flow rate as described in claim 8, characterized in that, It also includes a droplet stabilization control module, which comprises: A fracture location monitoring unit is used to acquire images of droplet fracture. The stabilization control unit is electrically connected to the fracture location monitoring unit and is used to extract a first feature parameter and a second feature parameter from the image, and to maintain the first feature parameter and the second feature parameter stable by controlling the driving frequency and / or amplitude of the piezoelectric oscillator in the droplet generation module.

10. The droplet delay adjustment system for a flow cytometer based on real-time cell flow rate as described in claim 8, characterized in that, The system is also configured with a sorting mode module, which is used to execute different sorting logics, including: A single droplet is divided into n equal parts in the spatial dimension to locate the relative position of the particles in the droplet; Preset yield mask, purification mask, and phase mask; Based on the purity mode, enrichment mode, or single-cell mode selected by the user, different mask combinations are invoked, and combined with the droplet delay time value dynamically adjusted by the control processing module, the final sorting decision and execution logic are jointly determined.

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