CN-121989573-A - Particle printing method, system and device based on fluorescence field and bright field time sequence imaging

CN121989573ACN 121989573 ACN121989573 ACN 121989573ACN-121989573-A

Abstract

The invention discloses a particle printing method, a system and a device based on fluorescence field and bright field time sequence imaging, wherein the method comprises the steps of monitoring a micro-fluidic chip sorting area under the condition that a multicolor fluorescence field is continuously illuminated, and collecting fluorescence images of the micro-fluidic chip sorting area in real time; when the target fluorescent particles are identified in the fluorescent image, the field illumination is automatically touched, the bright field image is acquired, fusion analysis processing is carried out on the fluorescent image and the bright field image, multidimensional characteristic parameters are extracted, whether the multidimensional characteristic parameters meet printing conditions or not is judged according to a preset multi-mode judging rule, when the multidimensional characteristic parameters meet the printing conditions, a printing instruction is sent, and the printing channel of the microfluidic chip is driven by the control pressure and motion control unit to print the target fluorescent particles to the appointed hole site of the microporous plate in response to the printing instruction. The invention combines the advantages of fluorescence imaging and bright field imaging to realize comprehensive feature analysis and high-precision sorting of particles.

Inventors

CHEN HUAYING
GUO SITONG
LIU XIAOYING
LI KANG
ZHAO WENTAO

Assignees

珠海大略科技有限公司

Dates

Publication Date: 20260508
Application Date: 20260106

Claims (10)

1. The particle printing method based on fluorescence field and bright field time sequence imaging is characterized by comprising the following steps of: Monitoring a micro-fluidic chip sorting area under the condition that a multicolor fluorescence field continuously illuminates, and collecting fluorescence images of the micro-fluidic chip sorting area in real time; When the target fluorescent particles are identified in the fluorescent image, automatically triggering field illumination, and collecting a bright field image; Carrying out fusion analysis processing on the fluorescent image and the bright field image, and extracting multidimensional characteristic parameters; Judging whether the multidimensional characteristic parameters meet printing conditions according to a preset multimodal judging rule; when the multidimensional characteristic parameters meet printing conditions, sending a printing instruction; And responding to the printing instruction, controlling the pressure and motion control unit to drive the printing channel of the microfluidic chip to print the target fluorescent particles to the appointed hole site of the micro-pore plate.
2. The method for printing particles based on fluorescence field and bright field time sequence imaging according to claim 1, wherein the performing fusion analysis processing on the fluorescence image and the bright field image, and extracting the multidimensional feature parameter comprises: Analyzing and processing the fluorescent image through an image processing unit to obtain target characteristic parameters of the fluorescent image, wherein the target characteristic parameters comprise the fluorescent intensity of the target fluorescent particles and the fluorescent color of the target fluorescent particles; And analyzing and processing the bright field image through an image processing unit to obtain target morphology parameters of the bright field image, wherein the target morphology parameters comprise the size of the target fluorescent particles and the roundness of the target fluorescent particles.
3. The method for printing particles based on fluorescence field and bright field time sequence imaging according to claim 2, wherein the determining whether the multi-dimensional feature parameter satisfies a printing condition according to a preset multi-modal determination rule comprises: When the fluorescence intensity of the target fluorescent particles is within a preset range and the fluorescence color of the target fluorescent particles is consistent with the set color, determining that the target characteristic parameters of the fluorescent image meet the preset multi-mode judgment rule; When the size of the target fluorescent particles is in a preset range and the roundness of the target fluorescent particles is in the preset range, determining that the target morphological parameters of the bright field image meet the preset multi-mode judging rule; And when the target feature parameters of the fluorescent image and the target morphology parameters of the bright field image simultaneously meet the preset multi-mode judging rule, determining whether the multi-dimensional feature parameters meet printing conditions.
4. The fluorescence field and bright field timing imaging-based particulate printing method of claim 1, further comprising: Uploading the fluorescence image to an image processing unit; Processing the fluorescent image in real time by the image processing unit based on an identification algorithm to identify a potential target; judging whether the potential target accords with a preset verification condition or not; And when the potential target meets a preset verification condition, determining the potential target as a target fluorescent particle.
5. The method for printing particles based on fluorescence field and bright field time sequence imaging according to claim 1, wherein the multicolor fluorescence field adopts a multicolor LED array and optical filter wheel scheme, and the intelligent switching of multicolor fluorescence is realized through time sequence control and spectrum matching; the multi-color LED array comprises a three-color LED array consisting of ultraviolet, blue and green, and the filter wheel is driven by a stepping motor to complete filter switching within preset time so as to uniformly acquire fluorescent signals under multi-wavelength excitation.
6. The fluorescence field and bright field timing imaging-based particulate printing method of claim 1, further comprising: and when the multidimensional characteristic parameters do not meet the printing conditions, immediately turning off bright field illumination, recovering to a multi-color fluorescent field monitoring state, and waiting for the next potential target to appear.
7. The fluorescence and bright field timing imaging based particulate printing method of claim 3, further comprising: After determining whether the multi-dimensional characteristic parameter meets a printing condition, judging whether the printing quantity of the target fluorescent particles meets a set threshold value; If the printing quantity of the target fluorescent particles does not meet the set threshold, closing bright field illumination, and recovering to a multi-color fluorescent field monitoring state to wait for the next potential target to appear; And if the printing quantity of the target fluorescent particles meets a set threshold value, ending the printing operation.
8. The fluorescence field and bright field timing imaging-based particulate printing method of claim 1, further comprising: and when the printing operation is finished, the bright field illumination is turned off, the multi-color fluorescent field monitoring state is restored, and the next potential target is waited for.
9. A fluorescence field and brightfield time sequential imaging-based particle printing system for performing a fluorescence field and brightfield time sequential imaging-based particle printing method according to any one of claims 1 to 8, comprising: the microfluidic chip comprises a sorting area and a printing channel, wherein the sorting area is used for carrying a particle sample to be sorted, and the printing channel is used for providing a flow channel for target fluorescent particles for performing printing operation; The optical imaging unit comprises a multi-wavelength LED light source and a camera, wherein the multi-wavelength LED light source is used for providing multicolor fluorescent field illumination, and the camera is used for monitoring the sorting area of the microfluidic chip and collecting fluorescent images of the sorting area of the microfluidic chip in real time under the condition that the multicolor fluorescent field is continuously illuminated; The image processing unit is used for carrying out fusion analysis processing on the fluorescent image and the bright field image, extracting multi-dimensional characteristic parameters, judging whether the multi-dimensional characteristic parameters meet printing conditions according to a preset multi-mode judging rule, and sending a printing instruction when the multi-dimensional characteristic parameters meet the printing conditions; And the pressure and motion control unit is used for responding to the printing instruction and driving the printing channel of the microfluidic chip to print the target fluorescent particles to the appointed hole sites of the micro-pore plate.
10. A computer device comprising a memory and a processor, wherein the processor implements the method of any of claims 1 to 8 when executing a computer program stored in the memory.

Description

Particle printing method, system and device based on fluorescence field and bright field time sequence imaging Technical Field The invention relates to the technical field of biological medicine and microorganism research, in particular to a particle printing method, a system and a device based on fluorescence field and bright field time sequence imaging. Background The fluorescence sample sorting and printing technology has important application value in the fields of biological medicine, microbiological research and the like. However, the current fluorescent particle printing method generally only depends on the fluorescent signal generated by laser excitation as the sorting and printing criterion, and cannot identify and exclude non-fluorescent particles, and cannot identify and distinguish fluorescent particles and non-fluorescent particles at the same time. The single signal discrimination mechanism is difficult to realize accurate screening and printing of the mixed sample, and severely limits the application of the single signal discrimination mechanism in complex biological samples. In addition, the current fluorescent particle printing method only acquires fluorescent signals, can not provide bright field images, and cannot realize unified imaging and synchronous analysis of bright field and multicolor fluorescence, so that morphology and target characteristics of particles or cells cannot be analyzed, multidimensional identification and quality judgment of target fluorescent particles cannot be realized due to lack of morphology and target characteristic information of fluorescent particles, non-target components such as fragments and aggregates cannot be removed, and accurate identification and quality control are difficult to carry out. In addition, the existing fluorescent particle detection method realizes the acquisition of multicolor fluorescent signals by configuring a plurality of sets of photomultiplier tubes or photodiodes, but each detection channel only outputs light intensity values instead of space imaging information, signals among different channels are independent, space alignment and morphology analysis cannot be realized, and rapid switching and combined acquisition of fluorescence and bright field illumination cannot be realized in a high-speed flowing sample, so that the comprehensive judgment cannot be carried out by utilizing the fluorescent information and the particle morphology features at the same time, and the existing methods have obvious defects in multicolor marked sample sorting, morphology verification and complex sample identification. Disclosure of Invention The invention provides a particle printing method, a system and a device based on fluorescence field and bright field time sequence imaging, which aim to at least solve one of the technical problems in the prior art. The technical scheme of the invention is a particle printing method based on fluorescence field and bright field time sequence imaging, which comprises the following steps: Monitoring a micro-fluidic chip sorting area under the condition that a multicolor fluorescence field continuously illuminates, and collecting fluorescence images of the micro-fluidic chip sorting area in real time; When the target fluorescent particles are identified in the fluorescent image, automatically triggering field illumination, and collecting a bright field image; Carrying out fusion analysis processing on the fluorescent image and the bright field image, and extracting multidimensional characteristic parameters; Judging whether the multidimensional characteristic parameters meet printing conditions according to a preset multimodal judging rule; when the multidimensional characteristic parameters meet printing conditions, sending a printing instruction; And responding to the printing instruction, controlling the pressure and motion control unit to drive the printing channel of the microfluidic chip to print the target fluorescent particles to the appointed hole site of the micro-pore plate. According to some embodiments of the invention, the performing fusion analysis processing on the fluorescent image and the bright field image, and extracting the multidimensional feature parameter includes: Analyzing and processing the fluorescent image through an image processing unit to obtain target characteristic parameters of the fluorescent image, wherein the target characteristic parameters comprise the fluorescent intensity of the target fluorescent particles and the fluorescent color of the target fluorescent particles; And analyzing and processing the bright field image through an image processing unit to obtain target morphology parameters of the bright field image, wherein the target morphology parameters comprise the size of the target fluorescent particles and the roundness of the target fluorescent particles. According to some embodiments of the invention, the determining whether the multi-dimensional feature parame