Flow-type sorting droplet delay determination method and system and flow-type cell sorter

Abstract

The invention provides a flow-type separation droplet delay determination method and system and a flow-type cell sorter, and the method comprises the following steps: enabling calibration particles to pass through a detection area and be identified by a laser detector, and generating a trigger signal; acquiring a first induction signal and a second induction signal; obtaining a first time difference based on the trigger signal and the first induction signal; obtaining a second time difference based on the first induction signal and the second induction signal; acquiring an image of a liquid column between the position of the second sensor and a liquid drop breaking point, and determining a period number of a waveform formed by a liquid drop between the second sensor and the liquid drop breaking point based on the image; the total liquid drop delay time is obtained, the total delay is decomposed into the time difference capable of being directly measured with high precision and the waveform period capable of being directly counted, the problems that a traditional trial-and-error method is low in efficiency, depends on experience and is insufficient in precision are solved, and rapid, direct, accurate and automatic calibration of the liquid drop delay time is achieved.

Description

Technical Field

[0001] This invention relates to the field of biological detection and cell analysis technology, and in particular to a method, system and flow cytometer for determining droplet delay in flow cytometry. Background Technology

[0002] Flow cytometry sorting technology enables high-speed, multi-parameter identification, analysis, and sorting of specific cells within a mixed cell population. One of its core principles is breaking down a flow containing target cells into tiny droplets, charging these droplets to deflect them in an electric field, thus collecting them. In this process, the "droplet delay time" is a crucial parameter; it refers to the time difference between when a target cell is detected by a laser and when it is enveloped in the center of a precisely broken droplet, typically on the order of microseconds. Only by precisely calibrating this delay time can the instrument apply charge to the target droplet at the correct moment, achieving accurate sorting.

[0003] Currently, the commonly used calibration method in the industry is the "calibration particle trial-and-error method." The specific operation involves: before formal sorting, calibrated particles of uniform size and optical properties are used for simulated sorting. The operator presets a delay time based on experience. Under this setting, the instrument sorts the calibrated particles to the target container. By counting the difference between the actual number of sorted calibrated particles and the expected number, the accuracy of the preset delay time is determined, and manual iterative adjustments are made until the sorted number approaches the expected value. This method has significant drawbacks: 1) Low efficiency: The entire process requires repeated trials and is time-consuming, especially when the system is unstable or the operator lacks experience; 2) Strong subjective dependence: The selection of adjustment step size and the judgment of results heavily rely on the operator's personal experience, making standardization and automation difficult; 3) Limited accuracy: This method is essentially an indirect, statistically based approximation method, unable to directly and accurately measure the physical value of the delay time.

[0004] Therefore, there is an urgent need for a direct, accurate, rapid and highly automated method for measuring droplet delay time to improve the stability, accuracy and ease of use of flow cytometry. Summary of the Invention

[0005] This invention provides a method, system, and flow cytometer for measuring droplet delay in flow cytometry, which addresses the shortcomings of existing droplet delay calibration methods, such as low efficiency, reliance on experience, and low accuracy. It enables a direct, accurate, rapid, and highly automated method for measuring droplet delay time, thereby improving the stability, accuracy, and ease of use of flow cytometry.

[0006] This invention provides a method for determining the delay time of droplets in flow cytometry, comprising the following steps.

[0007] The calibration particles are passed through the detection zone of the flow cytometer and identified by the laser detector, generating a trigger signal; The first and second sensing signals generated when the calibration particles pass sequentially through the first and second sensors located on the downstream liquid flow path of the nozzle are acquired. Based on the trigger signal and the first sensing signal, a first time difference is obtained; Based on the first sensing signal and the second sensing signal, a second time difference is obtained; Acquire an image of the liquid column between the position of the second sensor and the droplet break point, and determine the number of cycles of the droplet formation waveform between the second sensor and the droplet break point based on the image; The total droplet delay time is obtained based on the first time difference, the second time difference, the number of cycles, and the droplet oscillation frequency.

[0008] According to the present invention, a method for determining the delay time of flow sorting droplets is provided, wherein the first sensor and the second sensor are inductive sensors or capacitive sensors.

[0009] According to the present invention, a method for determining the delay time of flow-sorted droplets is provided, wherein the calibration particles are magnetic particles or conductive coated particles.

[0010] According to the present invention, a method for determining the delay time of droplets in flow cytometry includes the following steps in determining the number of cycles: Identify the periodic waveforms of droplet formation in the image; Locate the corresponding position of the second sensor in the image; Starting from this position, count the number of complete waveforms along the direction of liquid flow up to the point where the droplet breaks, where the number of cycles is an integer or a half-integer.

[0011] According to the present invention, a method for determining the delay time of droplets in flow cytometry further includes a system stability verification step. The second time difference was measured multiple times under stable sheath fluid pressure and oscillation frequency; If the fluctuation range of the second time difference measured multiple times exceeds the preset threshold, the fluid flow system is determined to be unstable and a warning is issued.

[0012] According to the present invention, a method for determining the delay time of droplets in flow cytometry further includes an accuracy verification step: The average velocity of the calibration particles is calculated based on the second time difference and the known distance between the first sensor and the second sensor; Based on the known distance between the second sensor and the droplet break point and the average velocity, the theoretical flight time from the second sensor to the droplet break point is calculated. The visual time calculated based on the number of cycles is compared with the theoretical flight time, and the number of cycles or the image analysis process is corrected according to the comparison results.

[0013] According to the present invention, a method for determining the delay time of droplets in flow cytometry further includes a final calibration step: The measurement method is repeated multiple times to obtain multiple total droplet delay times; After removing outliers, the average or median of the total droplet delay times is calculated as the final calibrated droplet delay time.

[0014] The present invention also provides a flow cytometry droplet delay time measurement system, comprising the following modules: The laser detection module is used to detect calibration particles and generate trigger signals; The sensing module includes a first sensor and a second sensor disposed along the liquid flow path for generating a sensing signal as the calibration particles pass through; The image acquisition module is used to acquire images of the liquid column between the second sensor and the droplet break point; The processing control module is communicatively connected to the laser detection module, the sensing module, and the image acquisition module, and is configured to perform the above-described method for determining the delay time of flow cytometry-sorted droplets.

[0015] According to the present invention, a flow cytometry droplet delay time measurement system is provided, wherein the processing control module includes: A timing unit is used to record the timestamps of the trigger signal and the sensing signal with high precision. An image analysis unit is used to process the liquid column image to identify the waveform and count the number of cycles; The calculation unit is used to calculate the first time difference, the second time difference, and the total droplet delay time; The calibration output unit is used to output the final calibrated droplet delay time to the sorting control system.

[0016] The present invention also provides a flow cytometer that integrates the above-mentioned flow cytometer droplet delay time measurement system.

[0017] The present invention provides a flow cytometry method, system, and flow cytometer for measuring droplet delay. By having calibration particles pass through the detection zone of the flow cytometer and be identified by a laser detector, a trigger signal is generated. First and second sensing signals are acquired as the calibration particles sequentially pass through a first sensor and a second sensor positioned downstream of the nozzle. A first time difference is obtained based on the trigger signal and the first sensing signal. A second time difference is obtained based on the first and second sensing signals. An image of the liquid column between the second sensor position and the droplet break point is acquired, and the number of cycles of the droplet-forming waveform between the second sensor and the droplet break point is determined based on the image. The total droplet delay time is obtained based on the first time difference, the second time difference, the number of cycles, and the droplet oscillation frequency. The total delay is decomposed into directly measurable time differences and directly countable waveform cycles. This solves the problems of low efficiency, reliance on experience, and insufficient accuracy of traditional trial-and-error methods, achieving rapid, direct, accurate, and automated calibration of droplet delay time. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is a schematic flowchart of the flow cytometry method for determining droplet delay provided by the present invention.

[0020] Figure 2 This is a schematic diagram of the flow cytometer provided by the present invention.

[0021] Figure 3 This is a schematic diagram of the flow cytometry droplet delay measurement system provided by the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0023] In flow cytometry cell sorting, the instrument needs to break the fluid stream containing target cells into extremely small droplets and deflect these droplets by applying an electric charge, thus completing the sorting process. The most critical aspect of this process is the precise calibration of the droplet delay time. There is an extremely short time difference (on the order of microseconds) between when a cell passes through the laser detection point and when the cell is precisely encased in a droplet about to break apart. The instrument must know this time difference precisely in order to charge the droplet at the correct moment.

[0024] Figure 1 This is one of the flowcharts of the flow cytometry method for determining droplet delay provided by the present invention, such as... Figure 1 As shown, the method includes the following: like Figure 2 As shown, the flow cytometer of this application has two sensors, a first sensor and a second sensor, positioned at an appropriate location between the nozzle and the droplet break point. When calibration particles used to determine droplet delay pass through the sensors, the inductance changes, generating a precise time point signal. The first and second sensors are inductive or capacitive sensors, and can be arranged in a ring shape. The calibration particles are magnetic particles or conductive coated particles. The two sensors divide the total droplet delay into three parts: the first part of the droplet delay is obtained by the time difference between the laser detection point and the first sensor; the second part is obtained by the time difference between the first sensor and the second sensor; and the third part is obtained by the time difference between the second sensor and the break point, obtained by the camera capturing a clear liquid column image and counting the droplet cycle.

[0025] Step S101: The calibration particles are passed through the detection area of ​​the flow cytometer and identified by the laser detector, generating a trigger signal.

[0026] Step S102: Obtain the first sensing signal and the second sensing signal generated when the calibration particles pass through the first sensor and the second sensor set on the downstream liquid flow path of the nozzle in sequence.

[0027] Step S103: Based on the trigger signal and the first sensing signal, obtain the first time difference T1.

[0028] The time difference between the calibration particle passing through the laser detection point and the first sensing ring is marked as T1, which is the flight time T1 of the calibration particle from the laser detection point to the first sensing ring.

[0029] Step S104: Based on the first sensing signal and the second sensing signal, obtain the second time difference T2.

[0030] The time difference T2 between the two sensors is obtained by timing the calibrated particles as they pass through the two sensors. When the sheath fluid pressure and oscillation frequency remain constant, this time difference T2 is a fixed value. Therefore, the stability of the fluid flow system can be determined using the obtained time difference T2 each time, which is fundamental to successful sorting. The total droplet delay, which is difficult to measure directly, is transformed into three directly and accurately measurable quantities, improving the reliability of the entire delay calculation chain. The results are direct, stable, and is isomorphic to the "droplet thinking" of the instrument control, allowing for the integration of image analysis tools for automatic waveform recognition and counting.

[0031] Step S105: Obtain an image of the liquid column between the position of the second sensor and the droplet break point, and determine the number of cycles N of the droplet formation waveform between the second sensor and the droplet break point based on the image.

[0032] The step of determining the number of periods N includes: identifying the periodic waveforms formed by the droplets in the image; locating the corresponding position of the second sensor in the image; and counting the number of complete waveforms from that position along the direction of liquid flow to the point where the droplets break off, wherein the number of periods N is an integer or a half-integer.

[0033] Using a high-speed camera or stable stroboscopic illumination, acquire clear images of the liquid column and droplet breakage. The images clearly show the periodic "neckback" waveforms on the liquid column caused by piezoelectric vibrations, also known as "droplet formation ripples." Locate the physical position P_ring of the second sensor on the image. Using image analysis or direct observation, count the number of complete waveform cycles N along the liquid flow from point P_ring towards the droplet breakage point. One waveform cycle corresponds to one droplet about to form. That is, count how many complete droplet waveforms exist between the position of the second sensor and the droplet breakage point; the droplet flight time during this stage is marked as T3.

[0034] Preferably, the method further includes an accuracy verification step: Based on the second time difference T2 and the known distance S2 between the first sensor and the second sensor, calculate the average velocity V = S2 / T2 of the calibration particles; Based on the known distance S3 between the second sensor and the droplet break point and the average velocity V, the theoretical flight time T3' = S3 / V from the second sensor to the droplet break point is calculated. The visual time T3 = N / f calculated based on the number of cycles N is compared with the theoretical flight time T3', and the number of cycles N or the image analysis process is corrected according to the comparison results.

[0035] The specific correction for T3 accuracy is as follows: The time difference T2 between the calibration particles passing through the first sensor detection point 1 and the second sensor detection point 2, and the fixed distance S2, allow us to calculate the average velocity V = S2 / T2 of the calibration particles in the air after passing the nozzle in segment S2. Under stable sheath fluid pressure, this average velocity of the fluid flow in the air is assumed to be constant over a short period. The location of the droplet break point can be observed using a camera. Therefore, based on the distance S3 between the second sensor detection point 2 and the break point, and the average velocity in segment S2 (assuming the average velocity of the fluid flow in the air in segments S2 and S3 is equal), the time T3' from the second sensor detection point 2 to the droplet break point can be deduced. T3' = S3 / V, i.e., T3' = (S3 / S2)*T2.

[0036] Compare the visual cycle counting time T3 with the calculated T3' value. If they are inconsistent, make appropriate corrections to T3 to improve measurement accuracy.

[0037] Step S106: Obtain the total droplet delay time T based on the first time difference T1, the second time difference T2, the number of cycles N, and the droplet oscillation frequency f.

[0038] Specifically, the sum of the times of the first three stages is the total droplet delay, i.e., T = T1 + T2 + T3.

[0039] Given that each waveform period (i.e., the formation period of each droplet) is T_drop = 1 / f, where f is the droplet oscillation frequency, the theoretical time from induction ring 2 to the droplet break point is T3 = N * T_drop = N / f. Sometimes the break point may fall on half a period, so N may be an integer or a half-integer (e.g., 4.5 periods). This can be obtained using the following formula: T = T1 + T2 + N / f Further, this can be converted into the number of droplets: (T1 +T2 + N / f) * f = (T1 +T2) * f + N.

[0040] It should be noted that (T1+T2)*f represents the distance from the laser detection point to the second sensor, equivalent to how many droplet cycles. N: represents the number of complete waveforms (droplets) between the second sensor and the droplet breakpoint. This is an integer or half-integer.

[0041] Adding the two together gives the total droplet delay from the detection point to the droplet breakage point. This is the time required from the moment the calibration particle is detected by the laser until it reaches the exact breakage point of the droplet. This droplet delay time is fixed when the sheath fluid pressure and vibration frequency remain constant. In the sorting system, the droplet delay time is first determined using dedicated calibration particles, and then this precise droplet delay time is used for sample cell sorting.

[0042] After the laser detects the target cell, the control system waits for a precise time T before applying a high-voltage charging pulse to the liquid flow. At this moment, the droplet containing the target particle is charged precisely at the break point and is then sorted by the deflecting electric field.

[0043] Preferably, the method further includes a system stability verification step: The second time difference T2 was measured multiple times under stable sheath fluid pressure and oscillation frequency. If the fluctuation range of the T2 value measured multiple times exceeds the preset threshold, the fluid flow system is determined to be unstable and a warning is issued.

[0044] Preferably, the method further includes a final calibration step: The measurement method was repeated multiple times to obtain multiple total droplet delay times T. After removing outliers, the average or median of the total droplet delay time T is calculated as the final calibrated droplet delay time.

[0045] The calibration and measurement procedure is as follows: Prepare a magnetic calibration microparticle suspension with uniform particle size and run it on the machine.

[0046] As the calibration particles pass through the detection zone, a laser signal triggers the start of the timing process.

[0047] The calibration particles pass through the first and second inductive reactors in sequence, generating two steep electrical pulse signals, and the timing circuit records the precise time.

[0048] A high-speed camera simultaneously captures images of the liquid column.

[0049] Control software to perform calculations: a) Calculate T1 (from laser trigger to the first signal) and T2 (from the first signal to the second signal).

[0050] b) Analyze the image and automatically locate the position (P_ring) of the second sensor and the droplet break point in the image using image recognition algorithms (such as edge detection and peak finding). Count the complete brightness or width change cycles from P_ring toward the break point to obtain N (e.g., 4.5 cycles).

[0051] c) Calculate the total delay using the formula T = T1 + T2 + N / f. Where f is directly obtained from the piezoelectric ceramic driving frequency set by the instrument.

[0052] d) Using the known ring spacing S2, calculate the speed V=S2 / T2, and then combine it with the known S3 to calculate T3=S3 / V. Compare it with N / f. If the deviation is greater than 1%, prompt to check the image or retake the shot.

[0053] Repeat the above steps approximately 10-20 times. After removing outliers, take the average value of T as the final calibrated droplet delay time and input it into the sorter control parameters.

[0054] If the sample is replaced with a real cell sample, the instrument will use the precise delay time obtained from the above calibration to perform the sorting operation.

[0055] In the above-described flow cytometry method for determining droplet delay, a trigger signal is generated by passing calibration particles through the detection zone of the flow cytometer and being identified by a laser detector; a first sensing signal and a second sensing signal are acquired as the calibration particles sequentially pass through a first sensor and a second sensor located downstream of the nozzle; a first time difference is obtained based on the trigger signal and the first sensing signal; and a second time difference is obtained based on the first sensing signal and the second sensing signal. An image of the liquid column between the second sensor position and the droplet break point is acquired, and the number of cycles of the droplet formation waveform between the second sensor and the droplet break point is determined based on the image. The total droplet delay time is obtained according to the first time difference, the second time difference, the number of cycles, and the droplet oscillation frequency. The total delay is decomposed into a time difference that can be directly measured with high precision and a waveform cycle that can be directly counted. This solves the problems of low efficiency, reliance on experience, and insufficient accuracy of the traditional "trial and error method", and realizes fast, direct, accurate and automated calibration of droplet delay time.

[0056] It should be understood that, although Figure 1 The steps in the flowchart are shown sequentially as indicated by the arrows, but these steps are not necessarily executed in the order indicated by the arrows. Unless otherwise specified herein, there is no strict order in which these steps are executed, and they can be performed in other orders. Figure 1 At least some of the steps in the process may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same time, but can be executed at different times. The execution order of these sub-steps or stages is not necessarily sequential, but can be executed in turn or alternately with other steps or at least some of the sub-steps or stages of other steps. In one embodiment, such as Figure 2 As shown, a flow cytometry droplet delay measurement system is provided. The system includes: a laser detection module 21, a sensing module 22, an image acquisition module 23, and a processing and control module 24, wherein: Laser detection module 21 is used to detect calibration particles and generate trigger signals; Sensing module 22 includes a first sensor and a second sensor disposed along the liquid flow path for generating a sensing signal when the calibration particles pass through; Image acquisition module 23 is used to acquire images of the liquid column between the second sensor and the droplet break point; The processing control module 24 is communicatively connected to the laser detection module, the sensing module and the image acquisition module, and is configured to perform the flow cytometry droplet delay measurement method described in the above embodiments.

[0057] Furthermore, the sensing module 22 includes two inductive sensing rings coaxially sleeved outside the liquid flow sheath outside the nozzle, and the two inductive sensing rings have the same structural parameters.

[0058] Furthermore, the image acquisition module 23 includes a high-speed camera and a strobe light source or continuous light source for providing stable illumination.

[0059] Furthermore, the processing control module 24 includes: A timing unit is used to record the timestamps of the trigger signal and the sensing signal with high precision. An image analysis unit is used to process the liquid column image to identify the waveform and count the number of cycles; The calculation unit is used to calculate the first time difference, the second time difference, and the total droplet delay time; The calibration output unit is used to output the final calibrated droplet delay time to the sorting control system.

[0060] The flow cytometry droplet delay measurement system provided by the present invention is described below. The flow cytometry droplet delay measurement system described below can be referred to in correspondence with the flow cytometry droplet delay measurement method described above. Furthermore, this invention also provides a flow cytometer that integrates the aforementioned flow cytometer droplet delay time measurement system. The flow cytometer of this application has two sensors, a first sensor and a second sensor, positioned at an appropriate location between the nozzle and the droplet break point. When calibration particles used to determine droplet delay pass through the sensors, the inductance changes, generating a precise time point signal. The first and second sensors are inductive or capacitive sensors, and can be arranged in a ring shape. The calibration particles are magnetic particles or conductive coated particles. The two sensors divide the total droplet delay into three parts: the calibration particles obtain the first part of the droplet delay through the time difference between the laser detection point and the first sensor; the part from the first sensor to the second sensor; and the part from the second sensor to the break point, obtained by capturing a clear liquid column image through a camera and counting the droplet cycle to obtain the final part of the droplet delay.

[0061] The flow cytometry droplet delay time measurement system includes: The laser detection module is used to detect calibration particles and generate trigger signals; The sensing module includes a first sensor and a second sensor disposed along the liquid flow path for generating a sensing signal as the calibration particles pass through; The image acquisition module is used to acquire images of the liquid column between the second sensor and the droplet break point; The processing control module is communicatively connected to the laser detection module, the sensing module, and the image acquisition module, and is configured as the flow cytometry droplet delay measurement method described in the above embodiments.

[0062] The device embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this embodiment according to actual needs. Those skilled in the art can understand and implement this without any creative effort.

[0063] Through the above description of the embodiments, those skilled in the art can clearly understand that each embodiment can be implemented by means of software plus necessary general-purpose hardware platforms, and of course, it can also be implemented by hardware. Based on this understanding, the above technical solutions, in essence or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product can be stored in a computer-readable storage medium, such as ROM / RAM, magnetic disk, optical disk, etc., and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute the methods described in the various embodiments or some parts of the embodiments.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining the delay time of droplets in flow cytometry, characterized in that, Includes the following steps: The calibration particles are passed through the detection zone of the flow cytometer and identified by the laser detector, generating a trigger signal; The first and second sensing signals generated when the calibration particles pass sequentially through the first and second sensors located on the downstream liquid flow path of the nozzle are acquired. Based on the trigger signal and the first sensing signal, a first time difference is obtained; Based on the first sensing signal and the second sensing signal, a second time difference is obtained; Acquire an image of the liquid column between the position of the second sensor and the droplet break point, and determine the number of cycles of the droplet formation waveform between the second sensor and the droplet break point based on the image; The total droplet delay time is obtained based on the first time difference, the second time difference, the number of cycles, and the droplet oscillation frequency.

2. The method according to claim 1, characterized in that, The first sensor and the second sensor are inductive sensors or capacitive sensors.

3. The method according to claim 1, characterized in that, The calibration particles are magnetic particles or conductive coated particles.

4. The method according to claim 1, characterized in that, The steps for determining the number of cycles include: Identify the periodic waveforms of droplet formation in the image; Locate the corresponding position of the second sensor in the image; Starting from this position, count the number of complete waveforms along the direction of liquid flow up to the point where the droplet breaks, where the number of cycles is an integer or a half-integer.

5. The method according to claim 1, characterized in that, The method also includes a system stability verification step: The second time difference was measured multiple times under stable sheath fluid pressure and oscillation frequency; If the fluctuation range of the second time difference measured multiple times exceeds the preset threshold, the fluid flow system is determined to be unstable and a warning is issued.

6. The method according to claim 1, characterized in that, The method also includes an accuracy verification step: The average velocity of the calibration particles is calculated based on the second time difference and the known distance between the first sensor and the second sensor; Based on the known distance between the second sensor and the droplet break point and the average velocity, the theoretical flight time from the second sensor to the droplet break point is calculated. The visual time calculated based on the number of cycles is compared with the theoretical flight time, and the number of cycles or the image analysis process is corrected according to the comparison results.

7. The method according to claim 1, characterized in that, The method also includes a final calibration step: The measurement method is repeated multiple times to obtain multiple total droplet delay times; After removing outliers, the average or median of the total droplet delay times is calculated as the final calibrated droplet delay time.

8. A flow cytometry system for determining the delay time of liquid droplets, characterized in that, include: The laser detection module is used to detect calibration particles and generate trigger signals; The sensing module includes a first sensor and a second sensor disposed along the liquid flow path for generating a sensing signal as the calibration particles pass through; The image acquisition module is used to acquire images of the liquid column between the second sensor and the droplet break point; The processing control module is communicatively connected to the laser detection module, the sensing module and the image acquisition module, and is configured to perform the method according to any one of claims 1 to 7.

9. The system according to claim 8, characterized in that, The processing control module includes: A timing unit is used to record the timestamps of the trigger signal and the sensing signal with high precision. An image analysis unit is used to process the liquid column image to identify the waveform and count the number of cycles; The calculation unit is used to calculate the first time difference, the second time difference, and the total droplet delay time; The calibration output unit is used to output the final calibrated droplet delay time to the sorting control system.

10. A flow cytometer for cell sorting, characterized in that, It integrates a flow cytometry droplet delay time measurement system as described in any one of claims 8 to 9.

Images