CN114112864A - Counting, sorting and speed measuring system and method for biological samples and storage medium - Google Patents
Counting, sorting and speed measuring system and method for biological samples and storage medium Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- G01N15/1023—
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01P—MEASURING LINEAR OR ANGULAR SPEED, ACCELERATION, DECELERATION, OR SHOCK; INDICATING PRESENCE, ABSENCE, OR DIRECTION, OF MOVEMENT
- G01P5/00—Measuring speed of fluids, e.g. of air stream; Measuring speed of bodies relative to fluids, e.g. of ship, of aircraft
- G01P5/26—Measuring speed of fluids, e.g. of air stream; Measuring speed of bodies relative to fluids, e.g. of ship, of aircraft by measuring the direct influence of the streaming fluid on the properties of a detecting optical wave
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
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Abstract
The invention discloses a system, a method and a storage medium for counting, sorting and measuring speed of biological samples, which relate to the technical field of optics, and the system comprises: the device comprises a laser, a light beam modulation module, an inverted fluorescence module, a micro-fluidic chip, a second objective, a multimode optical fiber, an optical detector, a control box and a computer; the beam modulation module modulates the laser beam emitted by the laser and converges the beam into the channel of the microfluidic chip through the inverted fluorescence module to generate a plurality of parallel ultrathin polished sections; after being excited by the ultrathin light sheet, the biological sample dyed with fluorescence is converged to a multimode optical fiber through an inverted fluorescence module and a second objective lens, and is received by an optical detector; the control box controls the micro-fluidic chip to sort the biological samples based on the analysis and calculation results, and stores and displays the biological samples through a computer. The light sheets in the focusing area of the first objective lens can be independently controlled to generate multiple light sheets, the number of the intervals of the multiple light sheets is adjustable, and high-flux counting, sorting and speed measurement of biological samples can be realized.
Description
Technical Field
The invention relates to the technical field of optics, in particular to a system, a method and a storage medium for counting, sorting and measuring speed of biological samples.
Background
The detection of the amount of biological samples and identification of the type of sample is a common medical identification method in medicine and is widely used in disease detection. The flow cytometer is an advanced cell counting technology at present, different cells are differently stained with fluorescence, and different cells are counted through the difference of fluorescence signals among the cells. However, the prior art can only identify different cell types through the difference of fluorescence signals, and only aims at cell types with more consistent sizes. However, different biological samples, such as various normal cells and cancer cells and their respective exosomes, usually have different sizes and morphologies, and are mixed together, so that it is difficult to measure the biological samples with larger size difference simultaneously in the prior art, and it is impossible to provide other information besides the type of the biological sample, such as the size of the cells or the biological sample and the flowing behavior thereof.
Therefore, how to count different biological samples, analyze the size and motion information of the biological samples, and realize sorting of cells and biological samples is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of this, the invention provides a system, a method and a storage medium for counting, sorting and measuring the speed of a biological sample, which can perform quick and high-throughput counting, sorting and measuring the speed of the biological sample and are applied to the fields of biological and medical detection, flow field detection and the like.
In order to achieve the above purpose, the invention provides the following technical scheme:
a system for counting, sorting and measuring speed of biological samples, comprising: the device comprises a laser, a light beam modulation module, an inverted fluorescence module, a micro-fluidic chip, a second objective, a multimode optical fiber, an optical detector, a control box and a computer;
the laser emits laser beams and enters the beam modulation module; the light beam modulation module modulates the incident laser light beam and irradiates the laser light beam to the inverted fluorescence module; the inverted fluorescence module focuses an incident light beam and converges the light beam into a channel of the microfluidic chip to generate a plurality of parallel ultrathin polished sections; the micro-fluidic chip controls the flow of the biological sample dyed with fluorescence through the control box; the biological sample dyed with fluorescence is excited by the ultrathin optical sheet, and the excited fluorescence is converged to the multimode optical fiber through the inverted fluorescence module and the second objective lens and is received by the optical detector; the optical detector monitors the change of the fluorescence signal and sends the fluorescence signal to the control box; the control box distinguishes the size and the type of the biological sample based on the fluorescence signal, calculates the speed and the number of the sample, and sorts the biological sample through the microfluidic chip; and the control box sends the counting and sorting results to a computer for storage and display.
Optionally, the optical beam modulation module includes: the device comprises a spatial light filter, a first aperture diaphragm, a single lens, a polarizer, a beam splitter, a second aperture diaphragm, a spatial light modulator and a quarter wave plate;
the spatial light filter, the first small aperture diaphragm and the single lens sequentially perform filtering, beam expanding and collimating operations on a laser beam emitted by the laser, modulate the laser beam into a linearly polarized light beam with a fundamental transverse mode, and irradiate the linearly polarized light beam to the polarizer;
the polarizer modulates the polarization direction of the linearly polarized light beam to be consistent with the working direction of a liquid crystal plate of the spatial light modulator and transmits the linearly polarized light beam to the beam splitter;
the beam splitter divides the linearly polarized light beam passing through the polarizer into two parts, and after the linearly polarized light beam passes through the second small aperture diaphragm, the linearly polarized light beam is vertically incident on a liquid crystal plate of the spatial light modulator and is subjected to phase modulation to form a plurality of parallel flaky Airy type light spots;
the quarter-wave plate converts the plurality of parallel sheet Airy type light spots into circularly polarized light and emits the circularly polarized light to the inverted fluorescence module.
Optionally, the inverted fluorescence module comprises: the device comprises a first reflecting mirror, a dichroic mirror, a first objective lens, a second reflecting mirror and a filter plate;
the laser beam modulated by the beam modulation module enters the entrance pupil plane of the first objective lens after passing through the first reflector and the dichroic mirror; the first objective lens focuses an incident light beam and converges the light beam into a channel of the microfluidic chip to generate a plurality of parallel ultrathin polished sections; and a fluorescence signal generated by exciting the biological sample dyed with fluorescence by the ultrathin optical sheet is filtered and extracted by the dichroic mirror and the filter plate and is emitted out by the second reflecting mirror.
Optionally, the microfluidic chip comprises a liquid inlet area K1, a light sheet detection area K2, and a sorting channel K3;
wherein the liquid inlet region K1 includes: the liquid inlet area K1 controls a biological sample to enter the channel;
the light sheet detection area K2 includes: a rectangular cross-section channel S3 for exciting the biological sample to generate a fluorescence signal;
the sorting channel K3 includes: and the control valve S4, the control valve S5, the control valve S6, the control valve S7, the liquid outlet S8, the liquid outlet S9, the liquid outlet S10 and the liquid outlet S11 output different detected biological samples.
A method for counting, sorting and measuring speed of biological samples comprises the following steps:
the laser emits a laser beam and is incident to the beam modulation module;
the light beam modulation module modulates the incident laser light beam and irradiates the laser light beam to the inverted fluorescence module;
the inverted fluorescence module focuses the incident light beam and converges the light beam into a channel of the microfluidic chip to generate a plurality of parallel ultrathin polished sections;
the micro-fluidic chip controls the flow of the biological sample dyed with fluorescence through the control box; the biological sample dyed with fluorescence is excited by the ultrathin light sheet, and the excited fluorescence is converged to the multimode optical fiber through the inverted fluorescence module and the second objective lens and is received by the optical detector;
the optical detector monitors the change of the fluorescence signal and sends the fluorescence signal to the control box;
based on the received fluorescence signals, the control box distinguishes the sizes and the types of the biological samples, calculates the speed and the number of the samples, and controls the micro-fluidic chip to sort the biological samples; and the control box sends the counting and sorting results to a computer for storage and display.
Optionally, modulating the incident laser beam specifically includes the following steps:
the spatial light filter, the first small aperture diaphragm and the single lens sequentially carry out filtering, beam expanding and collimation operations on a laser beam emitted by the laser, the laser beam is modulated into a linearly polarized light beam with a base transverse mode, and the linearly polarized light beam is incident to the polarizer;
the polarizer modulates the polarization direction of the linearly polarized light beam to be consistent with the working direction of a liquid crystal plate of the spatial light modulator and transmits the linearly polarized light beam to the beam splitter;
the beam splitter divides the linearly polarized light beam passing through the polarizer into two parts, and after the linearly polarized light beam passes through the second small aperture diaphragm, the linearly polarized light beam is vertically incident on a liquid crystal plate of the spatial light modulator to be subjected to phase modulation into a plurality of parallel flaky Airy type light spots;
the quarter-wave plate converts the plurality of parallel plate-shaped Airy type light spots into circularly polarized light, and the circularly polarized light is incident to the inverted fluorescence module.
Optionally, generating a plurality of parallel ultrathin optical sheets in a channel of the microfluidic chip specifically includes the following steps:
dividing a TONG-in plane of a first objective lens in the inverted fluorescence module into S strip-shaped areas by the diameter R, and changing the uniformity of light spots by adjusting the size of S;
each strip-shaped area is further divided into SX small strip-shaped areas;
the Airy beams and the multifocal pure phase distribution are sequentially filled into the small strip-shaped area, the number of the Airy beams is changed by adjusting the SX size, and the phase distribution is as follows:
wherein:
as an expression for the phase of the Airy beam,
is the phase expression of the multifocal facula, alpha is the attenuation factor of Airy beam, lambda is the outgoing wavelength of the laser, NA is the numerical aperture of the objective lens, ntIn terms of the refractive index of the objective lens, deltax and deltay are respectively the positions of the light spot focuses in the focusing area of the objective lens, R is the radius of the TONG plane of the objective lens, x 'and y' are respectively rectangular coordinates on the TONG plane of the objective lens, and kx、kyThe spatial frequency distribution of x 'and y' respectively, i is an imaginary unit;
increasing a rotation matrix to enable the Airy light beam to rotate into a sheet Airy type light spot, wherein the phase function expression of the rotated sheet Airy type light spot is as follows:
wherein: the wave vector after rotation of the rotation matrix is:
combining the phase function of the sheet Airy type light spot with the phase function of the multi-focus light spot and filling the phase function into each small strip-shaped area to modulate the multi-light sheet; by designing Δ x and Δ y, the position of the light sheet is controlled and a phase map of the gray scale transformation from 0 to 2 π is plotted.
Optionally, the operation process of the microfluidic chip includes:
introducing solutions carrying different biological samples into the microfluidic chip through a liquid inlet S1, adjusting the flow of the liquid inlet S1 to enable the multiple biological samples to enter the microfluidic chip one by one, and opening a control valve S2, a control valve S4, a control valve S5, a control valve S6 and a control valve S7;
when the solution carrying different biological samples passes through the plurality of parallel ultrathin light sheets in the channel S3 with the rectangular cross section, a fluorescence signal is generated and received by the optical detector;
and based on the analysis and calculation results of the control box on the fluorescence signals, the switching states of the control valve S4, the control valve S5, the control valve S6 and the control valve S7 are controlled, so that different biological samples are output through the corresponding liquid outlet S8, liquid outlet S9, liquid outlet S10 and liquid outlet S11, and the biological samples are sorted.
Optionally, sorting the biological sample specifically includes the following steps:
when a biological sample A passes through a plurality of parallel ultrathin light sheets, a generated fluorescence signal is received by an optical detector, and the speed of the biological sample A is obtained based on the time interval of the fluorescence signal and the interval of the ultrathin light sheets;
acquiring the size of the A biological sample based on the time length of the fluorescence signal pulse and the speed of the A biological sample;
calculating the time when the A biological sample reaches the control valve S4 based on the distance between the ultrathin optical sheet and the control valve S4 and the speed of the A biological sample; at this time, the control box opens the control valve S4, closes the control valve S5, the control valve S6 and the control valve S7, and outputs the biological sample a through the liquid outlet S8;
repeating the steps, and distinguishing, classifying and counting different biological samples based on the sizes of the biological samples; and (3) opening the corresponding control valve and closing other control valves by obtaining the time when different biological samples reach the control valve, so that different biological samples are output through the liquid outlets corresponding to the control valves until the set running stopping condition is reached, closing the control valve S2, and stopping counting and sorting.
A computer-readable medium, on which a computer program is stored, which, when being executed by a processor, carries out the steps of the above method of counting, sorting and measuring speed of biological samples.
According to the technical scheme, compared with the prior art, the invention discloses a system, a method and a storage medium for counting, sorting and speed measuring of biological samples, parallel flaky Airy type light spots are introduced through a spatial light modulation technology, and rapid and high-flux counting, sorting and speed measuring are carried out on the biological samples such as cells continuously passing through a flaky light in a channel by combining a micro-fluidic chip technology, so that the system, the method and the storage medium can be applied to the fields of biological and medical detection, flow field detection and the like. In addition, when the counting, sorting and speed measuring functions are realized, the number of the parallel sheet-shaped light spots can be an integer larger than or equal to 2, and when only the counting and sorting functions are needed, a single light sheet can be used.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic diagram of a system for counting, sorting and measuring speed of biological samples in example 1;
FIG. 2 is a schematic structural diagram of a microfluidic chip;
FIG. 3 is a schematic diagram of a multi-focal spot modulation method;
FIG. 4 is a phase diagram of a single slab of spots generated when loaded onto a spatial light modulator according to the present invention;
FIG. 5 is a graph showing the results of an experiment to generate a single sheet-like spot;
FIG. 6 is a phase diagram of three sheet-like spots of light generated by loading the spatial light modulator of the present invention;
FIG. 7 is a graph showing the results of an experiment for generating three sheet-like spots;
FIG. 8 is a graph of time-series simulated signals of four different sizes of biological samples through three light sheets;
FIG. 9 is a diagram of time-series analog signals of four biological samples of different sizes and flow rates counted and tested by a three-light sheet;
reference numerals: the system comprises a laser 1, a spatial light filter 2, a first aperture diaphragm 3, a single lens 4, a polarizer 5, a beam splitter 6, a second aperture diaphragm 7, a spatial light modulator 8, a quarter wave plate 9, a first reflector 10, a dichroic mirror 11, a first objective 12, a microfluidic chip 13, a second reflector 14, a filter 15, a second objective 16, a multimode optical fiber 17, an optical detector 18, a control box 19, a computer 20, an M beam modulation module, an M1 inverted fluorescence module, a K1 liquid inlet region, a K2 light detection region, a K3 differential detection channel, an S1 liquid inlet, an S2 control valve, an S3 rectangular section channel, an S4 control valve, an S5 control valve, an S6 control valve, an S7 control valve, an S8 liquid outlet, an S9 liquid outlet, an S10 liquid outlet and an S11 liquid outlet.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment of the invention discloses a counting, sorting and speed measuring system for biological samples, which comprises the following components as shown in figure 1: the device comprises a laser 1, a light beam modulation module M, an inverted fluorescence module M1, a microfluidic chip 13, a second objective lens 16, a multimode optical fiber 17, an optical detector 18, a control box 19 and a computer 20;
the laser 1 emits laser beams with corresponding wavelengths and enters the beam modulation module M; the light beam modulation module M modulates the incident laser light beam and irradiates the laser light beam to the inverted fluorescence module M1; the inverted fluorescence module M1 focuses the incident light beam and converges the light beam into the channel of the microfluidic chip 13 to generate a plurality of parallel ultrathin light sheets; the micro-fluidic chip 13 controls the flow of biological samples such as cells stained with fluorescence and the like through the control box 19; the biological sample dyed with fluorescence is excited by the ultrathin light sheet, and the excited fluorescence is converged to the multimode optical fiber 17 through the inverted fluorescence module M1 and the second objective lens 16 and is received by the optical detector 18; the optical detector 18 monitors the change of the fluorescence signal and transmits the fluorescence signal to the control box 19; the control box 19 distinguishes the size and the type of the biological samples based on the fluorescence signals, calculates the speed and the quantity of the biological samples, and sorts the biological samples through the microfluidic chip 13; the control box 19 sends the counting and sorting results to the computer 20 for storage and display.
Further, the beam modulation module M includes: the device comprises a spatial light filter 2, a first aperture diaphragm 3, a single lens 4, a polarizer 5, a beam splitter 6, a second aperture diaphragm 7, a spatial light modulator 8 and a quarter wave plate 9;
the spatial light filter 2, the first small aperture diaphragm 3 and the single lens 4 sequentially filter, expand and collimate laser beams emitted by the laser 1, modulate the laser beams into linearly polarized light beams with a fundamental transverse mode, and enter the polarizer 5; the polarizer 5 modulates the polarization direction of the linearly polarized light beam to be consistent with the working direction of a liquid crystal panel of the spatial light modulator 8 and transmits the linearly polarized light beam to the beam splitter 6; the beam splitter 6 divides the linearly polarized light beam passing through the polarizer 5 into two parts, and after the linearly polarized light beam passes through the second small aperture diaphragm 7, the linearly polarized light beam is vertically incident on a liquid crystal plate of the spatial light modulator 8 to be phase-modulated into a plurality of parallel flaky Airy type light spots; the quarter-wave plate 9 converts the plurality of parallel plate-like Airy-type spots into circularly polarized light and is incident to the inverted fluorescence module M1.
Further, the inverted fluorescence module M1 includes: a first reflector 10, a dichroic mirror 11, a first objective 12, a second reflector 14, and a filter 15;
the laser beam modulated by the beam modulation module M passes through the first reflecting mirror 10 and the dichroic mirror 11, and then enters the entrance pupil plane of the first objective lens 12; the first objective lens 12 focuses the incident light beam, and converges the light beam into a channel of the microfluidic chip 13 to generate a plurality of parallel ultrathin polished sections; the fluorescence signal generated by the fluorescence-stained biological sample excited by the ultrathin optical sheet is filtered and extracted by the dichroic mirror 11 and the filter 15, and is emitted out by the second reflecting mirror 14.
Further, referring to fig. 2, the microfluidic chip 13 includes a liquid inlet region K1, a light sheet detection region K2, and a sorting channel K3;
wherein, the regional K1 of feed liquor includes: the liquid inlet S1 and the control valve S2 are used for controlling the biological sample to enter the channel; the light sheet detection area K2 includes: the long and straight rectangular cross-section channel S3 is used for exciting a biological sample to generate a fluorescence signal; the sorting passage K3 includes: and the control valve S4, the control valve S5, the control valve S6, the control valve S7, the liquid outlet S8, the liquid outlet S9, the liquid outlet S10 and the liquid outlet S11 output different detected biological samples.
Example 2
The embodiment of the invention discloses a method for counting, sorting and measuring speed of biological samples, which comprises the following steps:
the laser emits laser beams with corresponding wavelengths, and the laser beams are incident to the beam modulation module;
the light beam modulation module modulates the incident laser light beam and irradiates the laser light beam to the inverted fluorescence module;
the inverted fluorescence module focuses the incident light beam and converges the light beam into a channel of the microfluidic chip to generate three parallel ultrathin polished sections;
the micro-fluidic chip controls the flow of the biological sample dyed with fluorescence through the control box; the biological sample dyed with fluorescence is excited by the ultrathin light sheet, and the excited fluorescence is converged to the multimode optical fiber through the inverted fluorescence module and the second objective lens and is received by the optical detector;
the optical detector monitors the change of the fluorescence signal and sends the fluorescence signal to the control box;
based on the fluorescence signal, the control box distinguishes the size and the type of the biological sample, calculates the speed and the number of the sample, and controls the micro-fluidic chip to sort the biological sample; and the control box sends the counting and sorting results to a computer for storage and display.
Further, modulating the incident laser beam specifically comprises the following steps:
the spatial light filter, the first small aperture diaphragm and the single lens sequentially carry out filtering, beam expanding and collimation operations on a laser beam emitted by the laser, the laser beam is modulated into a linearly polarized light beam with a base transverse mode, and the linearly polarized light beam is incident to the polarizer;
the polarizer modulates the polarization direction of the linearly polarized light beam to be consistent with the working direction of a liquid crystal plate of the spatial light modulator and transmits the linearly polarized light beam to the beam splitter;
the beam splitter divides the linearly polarized light beam passing through the polarizer into two parts, and after the linearly polarized light beam passes through the second aperture diaphragm, the linearly polarized light beam is vertically incident on a liquid crystal plate of the spatial light modulator to be subjected to phase modulation into three parallel flaky Airy type light spots (one of Airy light beam families);
the quarter-wave plate converts the plurality of parallel plate-shaped Airy type light spots into circularly polarized light, and the circularly polarized light is incident to the inverted fluorescence module.
Fig. 3 is a schematic diagram of a method for modulating multiple focal spots, where S ═ 10 denotes dividing the tone-in plane of the first objective lens into 10 main regions, SX ═ 3 denotes dividing the main region into 3 sub-regions, S is used to adjust the uniformity of multiple focal spots, and SX is used to adjust the number of multiple focal spots.
Further, a plurality of parallel ultrathin optical sheets are generated in the channel of the microfluidic chip, and the method specifically comprises the following steps:
dividing a tone-in plane of a first objective lens in the inverted fluorescence module into S strip-shaped areas by the diameter R, changing the uniformity of light spots by adjusting the size of S, and taking S as 70 in the embodiment in order to ensure that the generated 3 light sheets have better uniformity of multi-focus light spots;
each strip-shaped area is further divided into SX small strip-shaped areas, the number of light spots is changed by adjusting the size of SX, and 3 light sheets are generated, wherein SX is 3;
the Airy beams and the multifocal pure phase distribution are sequentially filled into the small strip-shaped area, the number of the Airy beams is changed by adjusting the SX size, and the phase distribution is as follows:
wherein:
as an expression for the phase of the Airy beam,
is the phase expression of the multifocal facula, alpha is the attenuation factor of Airy beam, lambda is the outgoing wavelength of the laser, NA is the numerical aperture of the objective lens, ntIs a fold of an objective lensRefractive index, Deltax and Delay are respectively the position of a light spot focus in a focusing area of the objective lens, R is the radius of the TONG plane of the objective lens, x 'and y' are respectively rectangular coordinates on the TONG plane of the objective lens, and kx、kyThe spatial frequency distribution of x 'and y' respectively, i is an imaginary unit;
the Airy light beam is rotated into a sheet Airy type light spot by adding a rotation matrix, and the phase function expression of the rotated sheet Airy type light spot is as follows:
wherein: the wave vector after rotation of the rotation matrix is:
combining the phase function of the sheet Airy type light spot with the phase function of the multi-focus light spot and filling the phase function into each small strip-shaped area to modulate the multi-light sheet; by designing Δ x and Δ y, the positions of the light sheets are controlled, and a phase map of the gray scale transformation from 0 to 2 pi is plotted, for example, 3 light sheets Δ x ═ 3,0,3.5, and Δ y ═ 0,0,0 are generated.
Further, the operation process of the microfluidic chip comprises the following steps:
introducing a solution loaded with different biological samples such as cells into the microfluidic chip through the liquid inlet S1, adjusting the flow rate of the liquid inlet S1 to enable the biological samples such as cells to enter the microfluidic chip one by one, and opening the control valve S2, the control valve S4, the control valve S5, the control valve S6 and the control valve S7;
the solution carrying different biological samples passes through a long straight rectangular cross section channel S3, generates fluorescence signals when passing through three parallel ultrathin light sheets and is received by an optical detector;
based on the analysis and calculation results of the control box on the fluorescence signals, the switching states of the control valve S4, the control valve S5, the control valve S6 and the control valve S7 are controlled, so that different biological samples are correspondingly output from the liquid outlet S8, the liquid outlet S9, the liquid outlet S10 and the liquid outlet S11, and the biological samples are sorted.
Further, sorting the biological sample specifically comprises the following steps:
when a biological sample A passes through a plurality of parallel ultrathin light sheets, a generated fluorescence signal is received by an optical detector, and the speed of the biological sample A is obtained based on the time interval of the fluorescence signal and the interval of the ultrathin light sheets;
acquiring the size of the A biological sample based on the time length of the fluorescence signal pulse and the speed of the A biological sample;
calculating the time when the A biological sample reaches the control valve S4 based on the distance between the ultrathin optical sheet and the control valve S4 and the speed of the A biological sample; at this time, the control box opens the control valve S4, closes the control valve S5, the control valve S6 and the control valve S7, and outputs the biological sample a through the liquid outlet S8;
repeating the steps, and distinguishing, classifying and counting different biological samples based on the sizes of the biological samples; and (3) opening the corresponding control valve and closing other control valves by obtaining the time when different biological samples reach the control valve, so that different biological samples are output through the liquid outlets corresponding to the control valves until the set running stopping condition is reached, closing the control valve S2, and stopping counting and sorting.
The invention also discloses a computer-storable medium on which a computer program is stored, which computer program, when being executed by a processor, realizes the steps of the above method for counting, sorting and measuring the speed of biological samples.
As shown in fig. 4, which is a phase map loaded onto a spatial light modulator to produce a single light sheet, the phase map is plotted into a 1080 x 1080 pixel PNG format.
FIG. 5 shows a single optical sheet generated experimentally in the focusing region of an oil immersion objective (63X/NA1.4), the thickness of the generated optical sheet is 0.8 μm, the width is 37 μm, and the height is 3.5 μm (the height is obtained from the simulation result).
As shown in fig. 6, which is a phase diagram loaded onto a spatial light modulator to produce three light sheets, the phase diagram is plotted into a 1080 x 1080 pixel PNG format.
As shown in FIG. 7Shows that three optical sheets are generated in the focusing area of an oil immersion objective lens (63X/NA1.4) experimentally, the thickness of the generated optical sheet is 0.5 mu m, the width is 10 mu m, the height is 3.5 mu m (the height is obtained according to a simulation result), and the distance between the optical sheets is l1＝3μm，l23.5 μm. It is emphasized that the number of light sheets can be arbitrarily adjusted within the field of view of the objective lens.
FIG. 8 is a graph of fluorescence time series signals for counting and sorting four different sizes of biological samples using a single light sheet, in which four different sizes of cells (A, B, C and D, respectively) are pulsed at different widths (full width at half maximum τ) respectivelyA～τD) And different biological samples are distinguished according to different pulse widths, and then the cells are sorted by respectively controlling different control valve switches through the control box. Of course, the present embodiment is also applicable to sorting of biological samples such as multicolor cells by changing the wavelength of the laser light and the biological samples such as fluorescent-stained cells.
FIG. 9 is a fluorescence time-series signal diagram of counting, measuring and sorting biological samples of different sizes by using a three-light-sheet method, wherein A, B, C and D represent biological samples of different sizes, and A, B, C and D represent four biological samples passing through a three-light-sheet interval l1(first and second light sheet) and2(the second and third light sheets) are each at a time Δ t1A、Δt2A、Δt1B、Δt2B、Δt1C、Δt2C、Δt1DAnd Δ t2DThen A, B, C and D are the speeds of passing the four biological samples respectively
The speed measurement of biological samples such as cells can be realized by the method. At the same time, by the pulse width τA、τB、τC、τDTo distinguish and count the biological samples of different sizes, i.e. the total number of pulses of different widths1/3 in quantity. Finally, by vA、vB、vC、vDAnd the distances from the light sheet to the control valve S4, the control valve S5, the control valve S6 and the control valve S7, respectively calculate the arrival time of each biological sample, further control the switches of the control valve S4, the control valve S5, the control valve S6 and the control valve S7, respectively guide different biological samples into different channels, and realize the sorting of the different biological samples.
The fluorescent dye used in the experiment was CY5 dye with an excitation peak of 651nm and an emission peak of 670 nm.
In the prior art, different cell types can be identified only through the difference of fluorescence signals, and only for the cell types with relatively consistent sizes, a biological sample with relatively large size difference is difficult to measure simultaneously, and other information except the biological sample type cannot be provided. The parallel flaky Airy type light spots are introduced through the spatial light modulation technology, and the microfluidic chip technology is combined to perform rapid and high-flux counting, sorting and speed measurement on biological samples such as cells which continuously pass through the light sheets in a channel, and the method can be applied to the fields of biological and medical detection, flow field detection and the like. In addition, when the counting, sorting and speed measuring functions are realized, the number of the parallel sheet-shaped light spots can be an integer larger than or equal to 2, and when only the counting and sorting functions are needed, a single light sheet can be used.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A counting, sorting and speed measuring system for biological samples is characterized by comprising: the device comprises a laser, a light beam modulation module, an inverted fluorescence module, a micro-fluidic chip, a second objective, a multimode optical fiber, an optical detector, a control box and a computer;
the laser emits laser beams and enters the beam modulation module; the light beam modulation module modulates the incident laser light beam and irradiates the laser light beam to the inverted fluorescence module; the inverted fluorescence module focuses an incident light beam and converges the light beam into a channel of the microfluidic chip to generate a plurality of parallel ultrathin polished sections; the micro-fluidic chip controls the flow of the biological sample dyed with fluorescence through the control box; the biological sample dyed with fluorescence is excited by the ultrathin optical sheet, and the excited fluorescence is converged to the multimode optical fiber through the inverted fluorescence module and the second objective lens and is received by the optical detector; the optical detector monitors the change of the fluorescence signal and sends the fluorescence signal to the control box; the control box distinguishes the size and the type of the biological sample based on the fluorescence signal, calculates the speed and the number of the sample, and sorts the biological sample through the microfluidic chip; and the control box sends the counting and sorting results to a computer for storage and display.
2. The system for counting, sorting and velocimetry of biological samples according to claim 1, characterized in that the light beam modulation module comprises: the device comprises a spatial light filter, a first aperture diaphragm, a single lens, a polarizer, a beam splitter, a second aperture diaphragm, a spatial light modulator and a quarter wave plate;
the spatial light filter, the first small aperture diaphragm and the single lens sequentially perform filtering, beam expanding and collimating operations on a laser beam emitted by the laser, modulate the laser beam into a linearly polarized light beam with a fundamental transverse mode, and irradiate the linearly polarized light beam to the polarizer;
the polarizer modulates the polarization direction of the linearly polarized light beam to be consistent with the working direction of a liquid crystal plate of the spatial light modulator and transmits the linearly polarized light beam to the beam splitter;
the beam splitter divides the linearly polarized light beam passing through the polarizer into two parts, and after the linearly polarized light beam passes through the second small aperture diaphragm, the linearly polarized light beam is vertically incident on a liquid crystal plate of the spatial light modulator and is subjected to phase modulation to form a plurality of parallel flaky Airy type light spots;
the quarter-wave plate converts the plurality of parallel sheet Airy type light spots into circularly polarized light and emits the circularly polarized light to the inverted fluorescence module.
3. The system for counting, sorting and velocimetry of biological samples according to claim 1, characterized in that the inverted fluorescence module comprises: the device comprises a first reflecting mirror, a dichroic mirror, a first objective lens, a second reflecting mirror and a filter plate;
the laser beam modulated by the beam modulation module enters the entrance pupil plane of the first objective lens after passing through the first reflector and the dichroic mirror; the first objective lens focuses an incident light beam and converges the light beam into a channel of the microfluidic chip to generate a plurality of parallel ultrathin polished sections; and a fluorescence signal generated by exciting the biological sample dyed with fluorescence by the ultrathin optical sheet is filtered and extracted by the dichroic mirror and the filter plate and is emitted out by the second reflecting mirror.
4. The system for counting, sorting and speed measuring of biological samples according to claim 1, wherein the microfluidic chip comprises a liquid inlet area K1, a light sheet detection area K2, a sorting channel K3;
wherein the liquid inlet region K1 includes: the liquid inlet area K1 controls a biological sample to enter the channel;
the light sheet detection area K2 includes: a rectangular cross-section channel S3 for exciting the biological sample to generate a fluorescence signal;
the sorting channel K3 includes: and the control valve S4, the control valve S5, the control valve S6, the control valve S7, the liquid outlet S8, the liquid outlet S9, the liquid outlet S10 and the liquid outlet S11 output different detected biological samples.
5. A method for counting, sorting and measuring speed of biological samples is characterized by comprising the following steps:
the laser emits a laser beam and is incident to the beam modulation module;
the light beam modulation module modulates the incident laser light beam and irradiates the laser light beam to the inverted fluorescence module;
the inverted fluorescence module focuses the incident light beam and converges the light beam into a channel of the microfluidic chip to generate a plurality of parallel ultrathin polished sections;
the micro-fluidic chip controls the flow of the biological sample dyed with fluorescence through the control box; the biological sample dyed with fluorescence is excited by the ultrathin light sheet, and the excited fluorescence is converged to the multimode optical fiber through the inverted fluorescence module and the second objective lens and is received by the optical detector;
the optical detector monitors the change of the fluorescence signal and sends the fluorescence signal to the control box;
based on the received fluorescence signals, the control box distinguishes the sizes and the types of the biological samples, calculates the speed and the number of the samples, and controls the micro-fluidic chip to sort the biological samples; and the control box sends the counting and sorting results to a computer for storage and display.
6. The method for counting, sorting and speed measuring of biological samples according to claim 5, wherein the step of modulating the incident laser beam comprises the following steps:
the spatial light filter, the first small aperture diaphragm and the single lens sequentially carry out filtering, beam expanding and collimation operations on a laser beam emitted by the laser, the laser beam is modulated into a linearly polarized light beam with a base transverse mode, and the linearly polarized light beam is incident to the polarizer;
the polarizer modulates the polarization direction of the linearly polarized light beam to be consistent with the working direction of a liquid crystal plate of the spatial light modulator and transmits the linearly polarized light beam to the beam splitter;
the beam splitter divides the linearly polarized light beam passing through the polarizer into two parts, and after the linearly polarized light beam passes through the second small aperture diaphragm, the linearly polarized light beam is vertically incident on a liquid crystal plate of the spatial light modulator to be subjected to phase modulation into a plurality of parallel flaky Airy type light spots;
the quarter-wave plate converts the plurality of parallel plate-shaped Airy type light spots into circularly polarized light, and the circularly polarized light is incident to the inverted fluorescence module.
7. The method for counting, sorting and speed measuring of biological samples according to claim 5, wherein a plurality of parallel ultra-thin optical sheets are generated inside the channel of the microfluidic chip, comprising the following steps:
dividing a TONG-in plane of a first objective lens in the inverted fluorescence module into S strip-shaped areas by the diameter R, and changing the uniformity of light spots by adjusting the size of S;
each strip-shaped area is further divided into SX small strip-shaped areas;
the Airy beams and the multifocal pure phase distribution are sequentially filled into the small strip-shaped area, the number of the Airy beams is changed by adjusting the SX size, and the phase distribution is as follows:
wherein:
as an expression for the phase of the Airy beam,
is the phase expression of the multifocal facula, alpha is the attenuation factor of Airy beam, lambda is the outgoing wavelength of the laser, NA is the numerical aperture of the objective lens, ntIn terms of the refractive index of the objective lens, deltax and deltay are respectively the positions of the light spot focuses in the focusing area of the objective lens, R is the radius of the TONG plane of the objective lens, x 'and y' are respectively rectangular coordinates on the TONG plane of the objective lens, and kx、kyThe spatial frequency distribution of x 'and y' respectively, i is an imaginary unit;
increasing a rotation matrix to enable the Airy light beam to rotate into a sheet Airy type light spot, wherein the phase function expression of the rotated sheet Airy type light spot is as follows:
wherein: the wave vector after rotation of the rotation matrix is:
combining the phase function of the sheet Airy type light spot with the phase function of the multi-focus light spot and filling the phase function into each small strip-shaped area to modulate the multi-light sheet; by designing Δ x and Δ y, the position of the light sheet is controlled and a phase map of the gray scale transformation from 0 to 2 π is plotted.
8. The method for counting, sorting and measuring the speed of biological samples according to claim 5, wherein the operation process of the microfluidic chip comprises:
introducing solutions carrying different biological samples into the microfluidic chip through a liquid inlet S1, adjusting the flow of the liquid inlet S1 to enable the multiple biological samples to enter the microfluidic chip one by one, and opening a control valve S2, a control valve S4, a control valve S5, a control valve S6 and a control valve S7;
when the solution carrying different biological samples passes through the plurality of parallel ultrathin light sheets in the channel S3 with the rectangular cross section, a fluorescence signal is generated and received by the optical detector;
and based on the analysis and calculation results of the control box on the fluorescence signals, the switching states of the control valve S4, the control valve S5, the control valve S6 and the control valve S7 are controlled, so that different biological samples are output through the corresponding liquid outlet S8, liquid outlet S9, liquid outlet S10 and liquid outlet S11, and the biological samples are sorted.
9. The method for counting, sorting and speed measuring of biological samples according to claim 8, wherein the sorting of biological samples comprises the following steps:
when a biological sample A passes through a plurality of parallel ultrathin light sheets, a generated fluorescence signal is received by an optical detector, and the speed of the biological sample A is obtained based on the time interval of the fluorescence signal and the interval of the ultrathin light sheets;
acquiring the size of the A biological sample based on the time length of the fluorescence signal pulse and the speed of the A biological sample;
calculating the time when the A biological sample reaches the control valve S4 based on the distance between the ultrathin optical sheet and the control valve S4 and the speed of the A biological sample; at this time, the control box opens the control valve S4, closes the control valve S5, the control valve S6 and the control valve S7, and outputs the biological sample a through the liquid outlet S8;
repeating the steps, and distinguishing, classifying and counting different biological samples based on the sizes of the biological samples; and (3) opening the corresponding control valve and closing other control valves by obtaining the time when different biological samples reach the control valve, so that different biological samples are output through the liquid outlets corresponding to the control valves until the set running stopping condition is reached, closing the control valve S2, and stopping counting and sorting.
10. A computer-storable medium having stored thereon a computer program for performing the steps of the method for counting, sorting and measuring blood velocity of biological samples according to any of claims 5-9 when executed by a processor.
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