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CN-121678491-B - Method and system for determining flow velocity and time delay of flow cytometer

CN121678491BCN 121678491 BCN121678491 BCN 121678491BCN-121678491-B

Abstract

The invention discloses a method and a system for determining flow velocity and time delay of a flow cytometer. The method comprises numbering the detected cells or particles, measuring the pulse number of each cell or particle passing through the same laser beam, calculating the corresponding drop delay according to the pulse number for each cell or particle, and calculating the flow rate of each cell or particle according to the pulse number, wherein the flow rate is inversely related to the pulse number. The invention can effectively overcome the defects of large sorting error and the like caused by adopting an average delay value in the prior art, realizes the normal form conversion from 'group average' to 'individual precision', and improves the sorting precision, the system stability and the repeatability of the flow cytometer on the premise of not increasing the hardware cost.

Inventors

  • ZHONG JINFENG
  • SONG FEIFEI
  • CHEN JIANSHENG
  • WANG CE
  • MA YUTING
  • CHEN ZHONGXIANG
  • CHEN MENGLI
  • YAN XINTAO
  • PEI ZHIGUO
  • WANG YAO
  • HE SHUAI

Assignees

  • 中国科学院苏州生物医学工程技术研究所

Dates

Publication Date
20260512
Application Date
20260211

Claims (10)

  1. 1. A method of determining flow rate, time delay of a flow cytometer, the method comprising the steps of: step 1, numbering the detected cells or particles, and measuring the pulse number of each cell or particle passing through the same laser beam; step2, calculating corresponding liquid drop delay according to the following formula according to the pulse number of each cell or particle: ; In the formula, Representing the corresponding drop delay of the nth cell or particle, Indicating the number of pulses corresponding to the nth cell or particle, Represents a custom set drop delay reference value, Representing a self-defined set pulse number reference value; step3, calculating the flow rate of each cell or particle according to the pulse number of the cell or particle, wherein the flow rate is inversely related to the pulse number; wherein step 2 and step 3 are performed synchronously or sequentially; In the step 3, the flow rate is calculated according to the pulse number, and the calculation formula is as follows: ; In the formula, Indicating the flow rate of the nth cell or particle, Represents the number of standard pulses corresponding to the nth cell or particle, Represents a custom set drop delay reference value, Representing a custom set pulse number reference value, Associated with droplets A corresponding physical distance parameter, which parameter is only relevant for the device.
  2. 2. The method for determining flow rate and time delay of a flow cytometer according to claim 1, wherein the droplet time delay reference value Pulse number reference value The acquisition mode of (a) is as follows: Collecting pulse number of a section of particles in a step 1 mode, calculating pulse number distribution, and extracting pulse number at peak value as pulse number reference value The corresponding liquid drop delay is a delay value measured by a flow cytometer, and the delay value is used as a liquid drop delay reference value 。
  3. 3. The method for determining flow rate and time delay of a flow cytometer according to claim 2, wherein the droplet time delay reference value The calculation formula of (2) is as follows: ; In the formula, For the physical distance parameter associated with droplet sorting, Is the pulse width of the standard particle, Is the flow rate of the standard particles.
  4. 4. The method for determining flow rate and time delay of flow cytometer according to claim 3, wherein the flow rate of the standard particle The calculation formula of (2) is as follows: ; In the formula, The pulse width and flow rate of standard particles under certain specific conditions, respectively.
  5. 5. The method for determining flow rate and time delay of flow cytometer according to claim 1, wherein the flow rate of each cell or particle in step 3 can be obtained by laser time delay, and the calculation formula is: ; In the formula, For the distance between any two lasers, Delay the laser between the two lasers; The drop delay reference value The calculation formula of (2) is as follows: ; In the formula, Physical distance parameters associated with sorting droplets.
  6. 6. The method according to claim 1, wherein the laser beam in step 1 is any one of the laser beams in the flow cytometer or is output by a specific laser, and the spot size is much larger than the cell or particle size.
  7. 7. The method of determining flow rate, time delay of a flow cytometer of claim 1 further comprising performing after step 1, prior to step 2: the number of pulses is corrected so that the number of pulses accurately reflects the time it takes to pass the spot.
  8. 8. The method of determining flow rate and time delay of a flow cytometer of claim 7 wherein the number of pulses is modified by an optimal trigger extension.
  9. 9. A system for determining flow rate, time delay of a flow cytometer based on the method of any of claims 1 to 8, the system comprising: a first module for numbering the detected cells or particles and measuring the number of pulses of each cell or particle passing through the same laser beam; A second module for realizing that, for each cell or particle, according to the pulse number, the corresponding drop delay is calculated according to the following formula: ; In the formula, Representing the corresponding drop delay of the nth cell or particle, Indicating the number of pulses corresponding to the nth cell or particle, Represents a custom set drop delay reference value, Representing a self-defined set pulse number reference value; A third module for realizing that, for each cell or particle, its flow rate is calculated from its number of pulses, which flow rate is inversely related to the number of pulses.
  10. 10. A computer device comprising a memory, a processor and a computer program stored on the memory and executable on the processor, wherein the processor implements the method of any one of claims 1 to 8 when the computer program is executed by the processor.

Description

Method and system for determining flow velocity and time delay of flow cytometer Technical Field The invention belongs to the technical field of flow cytometry separation, in particular to the technical field of separation precision improvement in a high-flux sample analysis scene, and particularly relates to a method and a system for determining flow velocity and time delay of a flow cytometer. Background In a flow cell sorting system, the accuracy of a droplet delay directly determines the accuracy of a sorting result, including key indexes such as sorting purity, sorting yield and the like. In high-speed sorting systems, the spacing between particles is very small, and thus the delay accuracy is very high. Conventional methods for measuring drop delay mainly comprise a channel image method, an optical signal method, a trial and error method and the like, wherein the methods measure the sorted drop delay in a calibration flow, but the obtained value is based on the average value of integral particles. However, during the actual sorting process, there is a certain difference in the flow rate of each particle, which results in a difference in the corresponding drop delay. In addition, over time, the fluid flow system may fluctuate due to various factors such as sheath fluid pressure, fluid level, etc. If the uniform average delay value is adopted for sorting operation, error sorting is easy to occur, sorting purity and yield are reduced, and the requirement of high-precision sorting is difficult to meet. Disclosure of Invention The invention aims to overcome the defects of large sorting error and the like caused by adopting an average delay value in the prior art, and provides a system and a method for correcting sorting delay of each particle in real time, so that the model transition from 'group average' to 'individual precision' is realized, and the sorting precision, the system stability and the repeatability of a flow cytometer are improved on the premise of not increasing the hardware cost. The technical solution for realizing the purpose of the invention is that, on one hand, a method for determining the flow velocity and time delay of a flow cytometer is provided, and the method comprises the following steps: step 1, numbering the detected cells or particles, and measuring the pulse number of each cell or particle passing through the same laser beam; step2, calculating corresponding liquid drop delay according to the following formula according to the pulse number of each cell or particle: In the formula, Representing the corresponding drop delay of the nth cell or particle,Indicating the number of pulses corresponding to the nth cell or particle,Represents a custom set drop delay reference value,Representing a self-defined set pulse number reference value; step3, calculating the flow rate of each cell or particle according to the pulse number of the cell or particle, wherein the flow rate is inversely related to the pulse number; Wherein step 2 and step 3 are performed synchronously or sequentially. Further, in step 3, the flow rate is calculated according to the pulse number, and the calculation formula is as follows: In the formula, Indicating the flow rate of the nth cell or particle,Represents the number of standard pulses corresponding to the nth cell or particle,Represents a custom set drop delay reference value,Representing a custom set pulse number reference value,Associated with dropletsA corresponding physical distance parameter, which parameter is only relevant for the device. Further, the drop delay reference valuePulse number reference valueThe acquisition mode of (a) is as follows: Collecting pulse number of a section of particles in a step 1 mode, calculating pulse number distribution, and extracting pulse number at peak value as pulse number reference value The corresponding liquid drop delay is a delay value measured by a flow cytometer, and the delay value is used as a liquid drop delay reference value。 Further, the drop delay reference valueThe calculation formula of (2) is as follows: In the formula, For the physical distance parameter associated with droplet sorting,Is the pulse width of the standard particle,Is the flow rate of the standard particles. Further, the flow rate of the standard particlesThe calculation formula of (2) is as follows: In the formula, The pulse width and flow rate of standard particles under certain specific conditions, respectively. Further, the flow rate of each cell or particle in step 3 can be obtained by laser delay, and the calculation formula is: In the formula, For the distance between any two lasers,Delay the laser between the two lasers; The drop delay reference value The calculation formula of (2) is as follows: In the formula, Physical distance parameters associated with sorting droplets. Further, the laser beam in step 1 is any one of the laser beams in the flow cytometer, or is output by a specific laser, and the spot size of the laser be