Method and system for determining flow cytometer flow rate, latency
By measuring and calculating the pulse count of each cell or particle in the flow cytometer and dynamically adjusting the droplet delay, the problem of large sorting error in high-throughput sample analysis of flow cytometers is solved, achieving high-precision sorting and improved system stability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
- Filing Date
- 2026-02-11
- Publication Date
- 2026-05-12
AI Technical Summary
In existing technologies, flow cytometry, in high-throughput sample analysis, suffers from large sorting errors due to the droplet delay measurement method being based on the average value of the entire particle, making it difficult to meet the requirements of high-precision sorting.
By numbering each cell or particle, measuring the number of pulses it passes through the same laser beam, and calculating the individual droplet delay and flow rate based on the number of pulses, the sorting delay is dynamically adjusted to avoid errors caused by the average delay value.
It achieves precise individual calibration, improves sorting accuracy and system stability, reduces hardware modification costs, is suitable for high-throughput complex sample analysis scenarios, and improves system repeatability and overall stability.
Smart Images
Figure CN121678491B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of flow cytometry cell sorting technology, and particularly relates to the field of sorting accuracy improvement technology in high-throughput sample analysis scenarios, specifically a method and system for determining the flow rate and delay of a flow cytometer. Background Technology
[0002] In flow cytometry cell sorting systems, the accuracy of droplet delay directly determines the accuracy of the sorting results, including key indicators such as sorting purity and sorting yield. In high-speed sorting systems, the spacing between particles is extremely small, thus requiring extremely high accuracy in delay.
[0003] Traditional methods for measuring droplet delay mainly include channel imaging, optical signal methods, and trial-and-error methods. These methods measure the droplet delay during the calibration process, but the obtained values are based on the average value of the entire particle count. However, in actual sorting, the flow velocity of each particle varies, leading to differences in its corresponding droplet delay. Furthermore, the fluid flow system fluctuates over time due to factors such as sheath fluid pressure and level. Using a uniform average delay value for sorting operations can easily result in incorrect sorting, reducing sorting purity and yield, and failing to meet the requirements for high-precision sorting. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of existing technologies, such as large sorting errors caused by using average delay values, and to provide a system and method for real-time correction of the sorting delay of each particle, realizing a paradigm shift from "group averaging" to "individual precision", and improving the sorting accuracy, system stability and repeatability of flow cytometers without increasing hardware costs.
[0005] The technical solution to achieve the objective of this invention is as follows: On one hand, a method for determining the flow rate and delay of a flow cytometer is provided, the method comprising the following steps:
[0006] Step 1: Number the cells or particles being detected and measure the number of pulses passing through the same laser beam for each cell or particle;
[0007] Step 2: For each cell or particle, calculate the corresponding droplet delay based on its pulse count using the following formula:
[0008]
[0009] In the formula, This indicates the droplet delay corresponding to the Nth cell or particle. This represents the pulse number corresponding to the Nth cell or particle. This represents a custom-defined droplet delay baseline value. This represents a custom-defined pulse count baseline value.
[0010] Step 3: For each cell or particle, calculate its flow rate based on its pulse count, which is negatively correlated with the pulse count;
[0011] Steps 2 and 3 are executed synchronously or sequentially.
[0012] Furthermore, in step 3, the flow rate is calculated based on the number of pulses, using the following formula:
[0013]
[0014] In the formula, This represents the flow rate of the Nth cell or particle. This represents the standard pulse number corresponding to the Nth cell or particle. This represents a custom-defined droplet delay baseline value. This represents a custom-defined pulse count baseline value. For droplet-related and The corresponding physical distance parameter is only related to the device.
[0015] Furthermore, the droplet delay reference value Pulse count reference value The method of obtaining it is:
[0016] The pulse count of a segment of particles was collected as described in step 1, and the pulse count distribution was calculated. The pulse count at the peak value was extracted as the pulse count baseline. The corresponding droplet delay is the delay value measured by flow cytometry, and this delay value is used as the droplet delay reference value. .
[0017] Furthermore, the droplet delay reference value The calculation formula is:
[0018]
[0019] In the formula, For the physical distance parameters related to droplet sorting, The pulse width of the standard particle. The velocity of the standard particle.
[0020] Furthermore, the flow rate of the standard particles The calculation formula is:
[0021]
[0022] In the formula, These represent the pulse width and flow rate of a standard particle under specific conditions, respectively.
[0023] Furthermore, the flow rate of each cell or particle in step 3 can be obtained through laser time delay, and the calculation formula is as follows:
[0024]
[0025] In the formula, Let be the distance between any two laser beams. This refers to the laser delay between the two laser beams.
[0026] The droplet delay reference value The calculation formula is:
[0027]
[0028] In the formula, The physical distance parameters related to droplet sorting.
[0029] Furthermore, the laser beam mentioned in step 1 can be any laser beam in a flow cytometer, or be output through a specific laser, and its spot size is much larger than the size of a cell or particle.
[0030] Furthermore, the method also includes execution after step 1 and before step 2:
[0031] The pulse count is corrected to ensure that it accurately reflects the time it takes for the pulse to pass through the light spot.
[0032] Furthermore, the number of pulses is corrected through optimal trigger extension.
[0033] On the other hand, a system for determining the flow rate and delay of a flow cytometer is provided, the system comprising:
[0034] The first module is used to: number the detected cells or particles and measure the number of pulses passing through the same laser beam for each cell or particle;
[0035] The second module is used to calculate the corresponding droplet delay for each cell or particle based on its pulse count, using the following formula:
[0036]
[0037] In the formula, This indicates the droplet delay corresponding to the Nth cell or particle. This represents the pulse number corresponding to the Nth cell or particle. This represents a custom-defined droplet delay baseline value. This represents a custom-defined pulse count baseline value.
[0038] The third module is used to calculate the flow rate for each cell or particle based on its pulse count, which is negatively correlated with the pulse count.
[0039] Compared with the prior art, the significant advantages of this invention are:
[0040] (1) Individual precision correction: By collecting the number of sampling pulses of each particle when it passes through a specific laser, combined with the known physical distance of the fluid channel, the actual flow velocity of the individual particle is calculated in real time, and the corresponding sorting delay is dynamically adjusted accordingly, thus avoiding the sorting error caused by the traditional average delay value.
[0041] (2) No hardware modification required: It can be achieved by using the photoelectric detection signal that is common to flow cytometry systems, without the need to modify the original system, thus reducing application costs.
[0042] (3) Easy to integrate: This invention can be embedded in existing signal processing modules to improve sorting accuracy without increasing hardware costs.
[0043] (4) Wide range of applicable scenarios: It is especially suitable for complex sample analysis scenarios with high throughput and multiple parameters, effectively reducing sorting errors caused by flow rate fluctuations and improving the overall stability and repeatability of the system.
[0044] (5) Adaptive optimization: By continuously monitoring the dynamic behavior of each particle, the system can adaptively optimize the sorting parameters so that each sorting decision is based on the real fluid dynamic state.
[0045] The present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description
[0046] Figure 1 This is a flowchart of a method for determining flow rate and delay in a flow cytometer in one embodiment.
[0047] Figure 2 This is a flowchart of a method for determining flow rate and delay in a flow cytometer in one embodiment. Detailed Implementation
[0048] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.
[0049] It should be noted that if the embodiments of the present invention involve descriptions such as "first" and "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" and "second" may explicitly or implicitly include at least one of those features. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.
[0050] In one embodiment, combined Figure 1 and Figure 2 A method for determining the flow rate and delay of a flow cytometer is provided, the method comprising the following steps:
[0051] Step 1: Number the cells or particles being detected and measure the number of pulses passing through the same laser beam for each cell or particle;
[0052] Step 2: For each cell or particle, calculate the corresponding droplet delay based on its pulse count using the following formula:
[0053]
[0054] In the formula, This indicates the droplet delay corresponding to the Nth cell or particle. This represents the pulse number corresponding to the Nth cell or particle. This represents a custom-defined droplet delay baseline value. This represents a custom-defined pulse count baseline value.
[0055] Step 3: For each cell or particle, calculate its flow rate based on its pulse count, which is negatively correlated with the pulse count;
[0056] Steps 2 and 3 are executed synchronously or sequentially.
[0057] Preferably, in some embodiments, the laser beam in step 1 is any laser beam in the flow cytometer, and no special limitation is made here.
[0058] Preferably, in some embodiments, the laser beam described in step 1 can also be output by a specific laser with a spot size much larger than that of a cell or particle.
[0059] The solution in this embodiment can make the pulse count as accurate as possible.
[0060] Furthermore, in one embodiment, the method further includes execution after step 1 and before step 2:
[0061] The pulse count is corrected to ensure that it accurately reflects the time it takes for the pulse to pass through the light spot.
[0062] Preferably, in some embodiments, the number of pulses is corrected by, but not limited to, optimal trigger spread (forward scattering angle signal). Here, it is preferred to use the forward scattering angle measurement channel as the trigger channel.
[0063] Furthermore, in one embodiment, the droplet delay reference value Pulse count reference value The acquisition methods include, but are not limited to:
[0064] The pulse count of a segment of particles was collected as described in step 1, and the pulse count distribution was calculated. The pulse count at the peak value was extracted as the pulse count baseline. The corresponding droplet delay is the delay value measured by flow cytometry, and this delay value is used as the droplet delay reference value. .
[0065] Preferably, in some embodiments, the formula for calculating the flow rate in step 3 is:
[0066]
[0067] In the formula, This represents the flow rate of the Nth cell or particle. This represents the standard pulse number corresponding to the Nth cell or particle. This represents a custom-defined droplet delay baseline value. This represents a custom-defined pulse count baseline value. For droplet-related and The corresponding physical distance parameter is only related to the device.
[0068] Preferably, in some embodiments, the droplet delay reference value The calculation formula is:
[0069]
[0070] In the formula, For the physical distance parameters related to droplet sorting, The pulse width of the standard particle. The velocity of the standard particle.
[0071] Preferably, in some embodiments, the flow rate of the standard particles The calculation formula is:
[0072]
[0073] In the formula, These represent the pulse width and flow rate of a standard particle under specific conditions, respectively.
[0074] Preferably, in some embodiments, the flow rate of each cell or particle in step 3 can also be obtained by, but is not limited to, laser delay, and the calculation formula is as follows:
[0075]
[0076] In the formula, Let be the distance between any two laser beams. This refers to the laser delay between the two laser beams.
[0077] The droplet delay reference value The calculation formula is:
[0078]
[0079] In the formula, The physical distance parameters related to droplet sorting.
[0080] In one embodiment, a system for determining flow rate and delay in a flow cytometer is provided, the system comprising:
[0081] The first module is used to: number the detected cells or particles and measure the number of pulses passing through the same laser beam for each cell or particle;
[0082] The second module is used to calculate the corresponding droplet delay for each cell or particle based on its pulse count, using the following formula:
[0083]
[0084] In the formula, This indicates the droplet delay corresponding to the Nth cell or particle. This represents the pulse number corresponding to the Nth cell or particle. This represents a custom-defined droplet delay baseline value. This represents a custom-defined pulse count baseline value.
[0085] The third module is used to calculate the flow rate for each cell or particle based on its pulse count, which is negatively correlated with the pulse count.
[0086] Specific limitations regarding the system for determining flow cytometry flow rate and delay can be found in the limitations of the methods for determining flow cytometry flow rate and delay described above, and will not be repeated here. Each module in the aforementioned system for determining flow cytometry flow rate and delay can be implemented entirely or partially through software, hardware, or a combination thereof. These modules can be embedded in or independent of the processor in a computer device, or stored in the memory of a computer device as software, so that the processor can call and execute the corresponding operations of each module.
[0087] In one embodiment, a computer device is provided, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor, when executing the computer program, implements:
[0088] Step 1: Number the cells or particles being detected and measure the number of pulses passing through the same laser beam for each cell or particle;
[0089] Step 2: For each cell or particle, calculate the corresponding droplet delay based on its pulse count using the following formula:
[0090]
[0091] In the formula, This indicates the droplet delay corresponding to the Nth cell or particle. This represents the pulse number corresponding to the Nth cell or particle. This represents a custom-defined droplet delay baseline value. This represents a custom-defined pulse count baseline value.
[0092] Step 3: For each cell or particle, calculate its flow rate based on its pulse count, which is negatively correlated with the pulse count.
[0093] For specific limitations on each step, please refer to the limitations on the methods for determining flow rate and delay in flow cytometry mentioned above, which will not be repeated here.
[0094] In one embodiment, a computer-readable storage medium is provided having a computer program stored thereon, the computer program being implemented when executed by a processor:
[0095] Step 1: Number the cells or particles being detected and measure the number of pulses passing through the same laser beam for each cell or particle;
[0096] Step 2: For each cell or particle, calculate the corresponding droplet delay based on its pulse count using the following formula:
[0097]
[0098] In the formula, This indicates the droplet delay corresponding to the Nth cell or particle. This represents the pulse number corresponding to the Nth cell or particle. This represents a custom-defined droplet delay baseline value. This represents a custom-defined pulse count baseline value.
[0099] Step 3: For each cell or particle, calculate its flow rate based on its pulse count, which is negatively correlated with the pulse count.
[0100] For specific limitations on each step, please refer to the limitations on the methods for determining flow rate and delay in flow cytometry mentioned above, which will not be repeated here.
[0101] As a specific example, the invention will be described in detail in some embodiments.
[0102] Example 1
[0103] This embodiment provides a specific implementation process for determining the flow rate and delay of a flow cytometer:
[0104] (1) Particle numbering and pulse count measurement: Start the flow cytometer and number the cells or particles to be tested one by one. Use the forward scattering angle measurement channel as the trigger channel to measure the number of pulses that each particle passes through the same laser beam. During the measurement process, the trigger extension algorithm is used to correct the pulse count to ensure that the pulse count accurately reflects the time it takes for the particle to pass through the light spot.
[0105] (2) Determination of reference parameters: Perform a trial sampling operation with a sampling duration of 1 minute, or collect 2000 particle pulse data. Perform statistical analysis on the collected pulse numbers, calculate the pulse number distribution curve, and take the pulse number W0 = 500 at the peak of the distribution curve as the calculation reference. At the same time, the droplet delay t0 = 100μs corresponding to this reference pulse number is obtained by system measurement.
[0106] (3) Individual delay calculation: For the particle numbered N=100, the measured pulse number W N = 550, according to the formula t N = t0 * W N / W0, calculate the droplet delay t of the particle. N = 100μs * 550 / 500 = 110μs. The system calculates t based on this. N The particle is then sorted.
[0107] (4) Flow rate correction: Standard particles are periodically added to the system. The known flow rate of the standard particles is v. 标= 10 m / s. The measured flow velocity v of the system is calculated using the formula v = S / t, where the laser delay t = 20 μs between the two laser beams is the distance between the two laser beams and the distance S = 200 μm is the distance between the two laser beams. 测 = 200μm / 20μs = 10m / s, consistent with the standard particle velocity, indicating that the system velocity correction is complete.
[0108] Example 2
[0109] This embodiment, based on Embodiment 1, uses a specific laser to assist in pulse number measurement:
[0110] A specific laser with a spot size of 100 μm is added to the flow cytometry system, which is much larger than the average size of the particles to be detected (5 μm). The number of pulses passing through the laser beam is measured using this specific laser. Because the spot size is much larger than the particle size, the influence of particle position differences within the spot on the pulse count measurement is reduced, further improving the accuracy of the pulse count measurement. The remaining operating steps are the same as in Example 1.
[0111] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention without departing from its spirit and scope should be included within the protection scope of the present invention.
Claims
1. A method for determining the flow rate and delay of a flow cytometer, characterized in that, The method includes the following steps: Step 1: Number the cells or particles being detected and measure the number of pulses passing through the same laser beam for each cell or particle; Step 2: For each cell or particle, calculate the corresponding droplet delay based on its pulse count using the following formula: ； In the formula, This indicates the droplet delay corresponding to the Nth cell or particle. This represents the pulse number corresponding to the Nth cell or particle. This represents a custom-defined droplet delay baseline value. This represents a custom-defined pulse count baseline value. Step 3: For each cell or particle, calculate its flow rate based on its pulse count, which is negatively correlated with the pulse count; Steps 2 and 3 are executed synchronously or sequentially. In step 3, the flow rate is calculated based on the number of pulses. The calculation formula is as follows: ； In the formula, This represents the flow rate of the Nth cell or particle. This represents the standard pulse number corresponding to the Nth cell or particle. This represents a custom-defined droplet delay baseline value. This represents a custom-defined pulse count baseline value. For droplet-related and The corresponding physical distance parameter is only related to the device.
2. The method for determining flow rate and delay in a flow cytometer according to claim 1, characterized in that, The droplet delay reference value Pulse count reference value The method of obtaining it is: The pulse count of a segment of particles was collected as described in step 1, and the pulse count distribution was calculated. The pulse count at the peak value was extracted as the pulse count baseline. The corresponding droplet delay is the delay value measured by flow cytometry, and this delay value is used as the droplet delay reference value. .
3. The method for determining flow rate and delay in a flow cytometer according to claim 2, characterized in that, The droplet delay reference value The calculation formula is: ； In the formula, For the physical distance parameters related to droplet sorting, The pulse width of the standard particle. The velocity of the standard particle.
4. The method for determining flow rate and delay in a flow cytometer according to claim 3, characterized in that, The flow rate of the standard particles The calculation formula is: ； In the formula, These represent the pulse width and flow rate of a standard particle under specific conditions, respectively.
5. The method for determining flow rate and delay in a flow cytometer according to claim 1, characterized in that, The flow rate of each cell or particle in step 3 can be obtained through laser time delay, and the calculation formula is as follows: ； In the formula, Let be the distance between any two laser beams. This refers to the laser delay between the two laser beams. The droplet delay reference value The calculation formula is: ； In the formula, The physical distance parameters related to droplet sorting.
6. The method for determining flow rate and delay in a flow cytometer according to claim 1, characterized in that, The laser beam mentioned in step 1 can be any laser beam in a flow cytometer, or output through a specific laser, with a spot size much larger than the size of a cell or particle.
7. The method for determining flow rate and delay in a flow cytometer according to claim 1, characterized in that, The method also includes execution after step 1 and before step 2: The pulse count is corrected to ensure that it accurately reflects the time it takes for the pulse to pass through the light spot.
8. The method for determining flow rate and delay in a flow cytometer according to claim 7, characterized in that, The number of pulses is corrected using optimal trigger extension.
9. A system for determining flow cytometer flow rate and delay based on the method of any one of claims 1 to 8, characterized in that, The system includes: The first module is used to: number the detected cells or particles and measure the number of pulses passing through the same laser beam for each cell or particle; The second module is used to calculate the corresponding droplet delay for each cell or particle based on its pulse count, using the following formula: ； In the formula, This indicates the droplet delay corresponding to the Nth cell or particle. This represents the pulse number corresponding to the Nth cell or particle. This represents a custom-defined droplet delay baseline value. This represents a custom-defined pulse count baseline value. The third module is used to calculate the flow rate for each cell or particle based on its pulse count, which is negatively correlated with the pulse count.
10. A computer device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that, When the processor executes the computer program, it implements the method as described in any one of claims 1 to 8.