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CN-122016615-A - Flow type sorting liquid drop delay measuring method, system and flow type cell sorter

CN122016615ACN 122016615 ACN122016615 ACN 122016615ACN-122016615-A

Abstract

The invention provides a flow separation liquid drop delay measuring method, a system and a flow cell sorter, wherein the method comprises the steps of enabling calibration particles to pass through a detection area and be identified by a laser detector to generate a trigger signal; the method comprises the steps of acquiring a first sensing signal and a second sensing signal, acquiring a first time difference based on the trigger signal and the first sensing signal, acquiring a second time difference based on the first sensing signal and the second sensing signal, acquiring an image of a liquid column between the position of the second sensor and a liquid drop breaking point, determining the cycle number of a liquid drop forming waveform between the second sensor and the liquid drop breaking point based on the image, acquiring total liquid drop delay time, decomposing the total delay into a time difference which can be directly measured with high precision and a waveform cycle which can be directly counted, solving the problems of low efficiency, dependence on experience and insufficient precision of the traditional trial and error method, and realizing quick, direct, accurate and automatic calibration of the liquid drop delay time.

Inventors

  • MIAO RUIFENG
  • LI JINDONG
  • LIU ZHENG

Assignees

  • 深圳市特博赛科技有限公司

Dates

Publication Date
20260512
Application Date
20260227

Claims (10)

  1. 1. The method for measuring the delay time of the flow sorting liquid drops is characterized by comprising the following steps of: Passing the calibration particles through a detection zone of the flow sorter and being identified by a laser detector to generate a trigger signal; Acquiring a first sensing signal and a second sensing signal generated when the calibration particles sequentially pass through a first sensor and a second sensor arranged on a downstream liquid flow path of the nozzle; Acquiring a first time difference based on the trigger signal and the first sensing signal; obtaining a second time difference based on the first sensing signal and the second sensing signal; acquiring an image of a liquid column between the position of the second sensor and a droplet breaking point, and determining the number of periods of a droplet forming waveform between the second sensor and the droplet breaking point based on the image; and obtaining the total drop delay time according to the first time difference, the second time difference, the cycle number and the drop oscillation frequency.
  2. 2. The method of claim 1, wherein the step of determining the position of the substrate comprises, the first sensor and the second sensor are inductive sensors or capacitive sensors.
  3. 3. The method of claim 1, wherein the calibration particles are magnetic particles or conductive coated particles.
  4. 4. The method of claim 1, wherein the step of determining the number of cycles comprises: identifying a periodic waveform of drop formation in the image; positioning a corresponding position of the second sensor in the image; From this location, the number of complete waveforms between the drop break-up points is counted in the direction of the flow, the number of periods being an integer or a half-integer.
  5. 5. The method of claim 1, further comprising a system stability verification step: measuring the second time difference a plurality of times at a steady sheath hydraulic pressure and oscillation frequency; and if the fluctuation range of the second time difference measured for a plurality of times exceeds a preset threshold, judging that the liquid flow system is unstable and giving out a warning.
  6. 6. The method of claim 1, further comprising the step of accuracy verification: Calculating an average velocity of the calibration particles based on the second time difference and a known distance between the first sensor and the second sensor; calculating a theoretical flight time from the second sensor to the droplet break-off point according to the known distance between the second sensor and the droplet break-off point and the average speed; and comparing the visual time calculated based on the cycle number with the theoretical flight time, and correcting the cycle number or the image analysis process according to a comparison result.
  7. 7. The method of claim 1, further comprising a final calibration step: repeatedly executing the measuring method for a plurality of times to obtain a plurality of total droplet delay times; And after eliminating the abnormal value, calculating the average value or the median of a plurality of total droplet delay times to be used as the final calibrated droplet delay time.
  8. 8. A flow sort drop delay measurement system, comprising: The laser detection module is used for detecting the calibration particles and generating a trigger signal; a sensing module comprising a first sensor and a second sensor disposed along the flow path for generating a sensing signal when the calibration particles pass; The image acquisition module is used for acquiring a liquid column image from the second sensor to the liquid drop breaking point; a process control module, in communication with the laser detection module, the sensing module, and the image acquisition module, configured to perform the method of any one of claims 1 to 7.
  9. 9. The system of claim 8, wherein the process control module comprises: The timing unit is used for recording the time stamps of the trigger signal and the induction signal with high precision; an image analysis unit for processing the liquid column image to identify waveforms and counting cycles; A calculating unit for calculating the first time difference, the second time difference and the total droplet delay time; and the calibration output unit is used for outputting the final calibrated drop delay time to the sorting control system.
  10. 10. A flow cytometer integrated with a flow cytometer drop delay measurement system as described in any of claims 8-9.

Description

Flow type sorting liquid drop delay measuring method, system and flow type cell sorter Technical Field The invention relates to the technical field of biological detection and cell analysis, in particular to a flow type sorting liquid drop delay determination method, a flow type sorting liquid drop delay determination system and a flow type cell sorter. Background Flow cell sorting techniques enable high-speed, multi-parameter identification, analysis and sorting of specific cells in a mixed cell population. One of the core principles is to break the stream containing the target cells into tiny droplets and charge the droplets containing the target cells to deflect them in an electric field to be collected. In this process, the "drop delay" is a critical parameter, which refers to the time difference that a target cell experiences from being detected by the laser until it is wrapped around the very broken drop center, typically on the order of microseconds. Only if this delay time is accurately calibrated, the instrument can apply charge to the target droplet at the correct moment, achieving accurate sorting. Currently, a commonly used calibration method in the industry is the "calibration particle trial and error method". The specific operation is that before formal sorting, calibration particles with uniform size and optical characteristics are used for analog sorting. The operator presets a delay time according to experience, the instrument sorts the calibration particles to the target container under the setting, whether the preset delay time is accurate or not is judged by counting the difference between the number of the calibration particles which are actually sorted and the expected number, and the preset delay time is manually and iteratively adjusted until the sorting number is close to the expected number. The method has obvious defects that 1) the whole process needs repeated trial and error and takes long time, especially when the system state is unstable or the experience of an operator is insufficient, 2) the subjective dependence is strong, the selection of the adjustment step length and the judgment of the result are seriously dependent on the personal experience of an operator, standardization and automation are difficult to realize, and 3) the accuracy is limited, and the method is an indirect approximation method based on the statistical result and cannot directly and accurately measure the physical magnitude of the delay time. Therefore, there is an urgent need for a method of measuring drop delay that can be directly, accurately, rapidly and with a high degree of automation to improve the stability, accuracy and ease of use of flow sorting. Disclosure of Invention The invention provides a method and a system for measuring the delay of a flow separation liquid drop and a flow cell sorter, which are used for solving the defects of low efficiency, dependence on experience and low precision of a liquid drop delay calibration method in the prior art, and realizing the method for measuring the delay time of the liquid drop, which can be used for directly, accurately, rapidly and automatically measuring the delay time of the liquid drop with high degree of automation, so as to improve the stability, the accuracy and the usability of the flow separation. The invention provides a method for measuring delay time of flow separation liquid drops, which comprises the following steps. Passing the calibration particles through a detection zone of the flow sorter and being identified by a laser detector to generate a trigger signal; Acquiring a first sensing signal and a second sensing signal generated when the calibration particles sequentially pass through a first sensor and a second sensor arranged on a downstream liquid flow path of the nozzle; Acquiring a first time difference based on the trigger signal and the first sensing signal; obtaining a second time difference based on the first sensing signal and the second sensing signal; acquiring an image of a liquid column between the position of the second sensor and a droplet breaking point, and determining the number of periods of a droplet forming waveform between the second sensor and the droplet breaking point based on the image; and obtaining the total drop delay time according to the first time difference, the second time difference, the cycle number and the drop oscillation frequency. According to the method for measuring the delay time of the flow sorting liquid drops, the first sensor and the second sensor are inductive sensors or capacitive sensors. According to the method for measuring the delay time of the flow sorting liquid drops, the calibration particles are magnetic particles or conductive coating particles. According to the method for measuring the delay time of the flow sorting liquid drops provided by the invention, the step of determining the cycle number comprises the following steps: identifying a periodic waveform of drop fo