CN114433262A - Multi-particle rapid capturing system and operation method thereof - Google Patents

Multi-particle rapid capturing system and operation method thereof Download PDF

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CN114433262A
CN114433262A CN202210090799.2A CN202210090799A CN114433262A CN 114433262 A CN114433262 A CN 114433262A CN 202210090799 A CN202210090799 A CN 202210090799A CN 114433262 A CN114433262 A CN 114433262A
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target object
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image
array
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CN114433262B (en
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张晨初
赵强强
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Hefei University of Technology
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Hefei University of Technology
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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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Abstract

The invention belongs to the field of cell biology, and particularly relates to a multi-particle rapid capture system and an operation method thereof. The system takes micron-scale particles or cells as a target object and captures the target object; the capture process is performed within a microfluidic chip. The system comprises a liquid supply assembly, a femtosecond laser processing assembly, a micro-motion platform, an image acquisition assembly, a manual input module and a central control module. Wherein, the liquid supply subassembly includes first liquid reserve tank, second liquid reserve tank, miniflow pump, flow divider, pipe. The femtosecond laser processing component comprises a femtosecond laser, an attenuating mirror, a spatial light modulator and a 4f lens. The micro-moving platform is a micron-sized biaxial moving platform. The image acquisition assembly includes a focusing objective, a camera, and a dichroic mirror. The central control module is electrically connected with the liquid supply assembly, the spatial light modulator, the micro-moving platform, the image acquisition assembly and the manual input module. The invention solves the problems of low capture efficiency of particles or cells, easy adhesion or damage among particles and the like in the prior method.

Description

Multi-particle rapid capturing system and operation method thereof
Technical Field
The invention belongs to the field of cell biology, and particularly relates to a multi-particle rapid capture system and an operation method thereof.
Background
In order to study the properties of some specific particles or cells, it is first necessary to capture specific cells or particles, and the existing particle or cell capture task is mainly performed by micro-capture technology based on microfluidic chips. The micro-capture technology based on the micro-fluidic chip has the advantages of low cost, less reagent consumption, convenience for cell operation and the like.
At present, there are some methods for capturing a large amount of suspended particles by integrating a trap in a microfluidic chip. For example, particles or cells are trapped using acoustic traps, microwells, dielectrophoresis, and hydrodynamic traps formed by micro-structured arrays. Compared to the above-mentioned methods, the hydrodynamic array capture method is the most commonly used method in microfluidic systems, which has the advantage of simplicity of operation. The specific capture process is as follows: firstly, a micro trap array corresponding to the size of a target particle is manufactured in advance, then, the liquid mixed with the particles or cells is injected into a microfluidic chip, and finally, the micro trap array captures the particles or the cells. The principle of the method is that a 'trap' is preset in a fluid channel, and the trap is equivalent to a 'fence'. The particles are blocked when passing through the trap and can normally pass through the trap. Therefore, in this method, it is necessary to maintain a constant unidirectional external pressure in order to firmly trap the particles or cells. This results in a complicated and cumbersome operation of capturing the target.
Also in the existing capture method, since the hydraulic resistance of the micro-channel is smaller than that of the trap array, most of the particles bypass the traps, resulting in relatively low capture efficiency (10%). In addition, the hydrodynamic trap in the existing microchannel is usually only suitable for capturing targets with specific sizes, and cannot be effectively and adaptively designed for particles or cells with different structures and sizes. This not only results in a low trap trapping rate, but also easily causes adhesion between objects or damages to the trapped objects.
Disclosure of Invention
The invention provides a multi-particle rapid capturing system and an operating method thereof, aiming at solving the problems that the capturing efficiency of particles or cells is low, the particles are easy to adhere or damage and the like in the existing method.
The invention is realized by adopting the following technical scheme:
a multi-particle rapid capture system for capturing objects with micron-scale particles or cells as the objects. The capture process is carried out in a micro-fluidic chip which is transparent and contains a fluid channel for the circulation of a target object; the multi-particle rapid capture system comprises: the device comprises a liquid supply assembly, a femtosecond laser processing assembly, a micro-moving platform, an image acquisition assembly, a manual input module and a central control module.
Wherein, the liquid supply subassembly includes first liquid reserve tank, second liquid reserve tank, miniflow pump, flow divider valve to and pipe. The first liquid storage tank is used for containing a first working solution containing a target object and a light curing agent. The second liquid storage tank is used for containing second working liquid which is convenient for target storage. The flow dividing valve is a two-in one-out electromagnetic valve, is connected among the first liquid storage tank, the second liquid storage tank and the micro-flow pump and is used for controlling working liquid in the first liquid storage tank or the second liquid storage tank to enter the micro-flow pump. The micro-flow pump is used for pumping the working fluid into the fluid channel of the micro-fluidic chip through the conduit.
The femtosecond laser processing assembly is used for generating exciting light curing agent for curing, and further generating a plurality of processing light beams parallel to the microcolumns. The femtosecond laser processing assembly comprises a femtosecond laser, an attenuation mirror, a spatial light modulator and a 4f lens according to the direction of an optical path. Among them, the femtosecond laser is used to generate the outgoing laser. The attenuation mirror is used for modulating the laser energy and the polarization direction of the emergent laser. The spatial light modulator is used for modulating the emergent laser light passing through the attenuator into a plurality of parallel laser beams of which the projection focuses meet the arrangement relation of a target array. The 4f lens is used for shrinking the modulated laser beam and filtering out unmodulated 0-level light. The laser beam after passing through the 4f lens is a processing beam meeting the working array, and the processing beam is projected on the micro-fluidic chip below the processing beam.
Wherein, the target array that each focus corresponds in the processing beam that femto second laser processing subassembly produced satisfies: the connecting line between adjacent focuses forms a polygon, the inscribed circle corresponding to the polygon is larger than the maximum section circle of the target object, and the distance between any two adjacent focuses in the multi-focus array is smaller than the particle size of the target object.
The micro-moving platform is a micron-sized biaxial moving platform and is used for fixing the micro-fluidic chip to be processed and accurately adjusting the position of the micro-fluidic chip according to the requirements of the processing process.
The image acquisition assembly includes a focusing objective, a camera, and a dichroic mirror. The focusing objective lens is movably arranged right above the displacement platform and used for amplifying the image of the micro-fluidic chip below the focusing objective lens, so that a camera can conveniently acquire a local image of a target object in the fluid channel. The dichroic mirror is capable of fully transmitting natural light and fully reflecting laser light of the processing light beam. The incident direction of the working light beam passing through the 4f lens in the femtosecond laser processing assembly forms a 45-degree included angle with the reflecting plane of the dichroic mirror, and the laser light reflected by the dichroic mirror is projected onto the micro-moving platform. The camera is positioned on the reverse extension line of the laser reflection light in the dichroic mirror, and the camera is positioned on the other side of the dichroic mirror relative to the focusing objective lens. The camera is used for acquiring a real-time amplified image of the microfluidic chip below the focusing objective lens.
The manual input module is used for inputting characteristic information of the target object to be captured and other manual instructions. The characteristic information includes specification information of the target object and a characteristic image of the target object acquired in advance.
The central control module is electrically connected with the liquid supply assembly, the spatial light modulator, the micro-moving platform, the image acquisition assembly and the manual input module. The central control module is used for: (1) and acquiring the characteristic information of the target object input by the manual input module. (2) And controlling the type, flow speed and fluid movement direction of the working fluid pumped by the micro-flow pump in the liquid supply system. (3) And receiving the real-time image of the microfluidic chip acquired by the image acquisition assembly, identifying the target object contained in the channel of the microfluidic chip from the real-time image according to the characteristic image of the target object, and calculating the position of the target object. (3) And adjusting the spatial light modulator according to the received specification information of the target object, so that the focal array of the working beam generated by the femtosecond laser processing assembly meets the requirement of the target array corresponding to the target object. (4) And adjusting the position of the micro-motion platform according to the distinguished position of the target object, so that the processing light beams generated by the femtosecond laser processing assembly are sequentially irradiated around each identified target object.
As a further improvement of the invention, the central control module comprises a target identification unit, a target positioning unit, a pumping control subunit, a displacement control subunit and a laser control subunit.
The target identification unit is used for acquiring a characteristic image of a target object serving as a reference and a real-time image acquired by the camera, and then extracting all target objects contained in the real-time image by using the target in the characteristic image as the reference through characteristic identification.
The target positioning unit is used for calculating the coordinate information of each target object on the micro-moving platform according to the pixel position of each target object identified by the target identification unit in the real-time image.
The pumping control subunit is used for controlling the on-off state of the diverter valve at different processing stages and controlling the fluid direction and flow rate of the pumping process of the micro-flow pump.
The displacement control subunit is used for acquiring coordinate information of each target object, and then adjusting the micro-displacement platform according to the coordinate information in the capturing stage of each target object, so that the target object in each micro-fluidic chip is exactly positioned in the center of the processing light beam.
The laser control subunit is used for acquiring the specification information of the target object, then inquiring a preset target array matching table according to the specification information of the target object to obtain a hologram of the target array meeting the conditions, and loading the hologram into the spatial light modulator, so that the femtosecond laser processing assembly generates a processing beam meeting the requirements. The target array matching table is a pre-stored expert experience table, and the one-to-one mapping relation between the specification information of different particles and the grating multiplexing holograms of different target arrays is stored in the expert experience table.
As a further improvement of the present invention, the target array match table is a data set that allows for manual editing. Before each particle or cell is captured for the first time, determining a hologram corresponding to the current target object in a manual setting mode, and adding the corresponding hologram and a mapping relation into a data set; when the current target object is captured again, the corresponding hologram is obtained through a table look-up method.
As a further improvement of the present invention, each hologram is provided corresponding to a multifocal array having a specific number and satisfying a specific pitch; after the spatial light modulator loads a specific hologram, the focus and the light intensity of a processing beam generated by the femtosecond laser processing assembly meet the corresponding multi-focus array; and the number of the focuses of the processing beams of the parameters of the femtosecond laser processing component, the positions of the focuses and the light intensity of light emitted from each focus are adjusted by adjusting different holograms.
As a further improvement of the invention, the microfluidic chip is an assembly which is automatically selected according to the particle size of the target to be captured, and comprises a detachable cover plate and a detachable bottom plate, wherein the bottom plate is provided with grooves for forming all fluid channels. The cover plate and the bottom plate of the microfluidic chip are made of transparent glass-based or organic resin materials.
As a further improvement of the invention, the spatial light modulator generates a multi-focus array satisfying a target array by loading a Dammann grating and a blazed grating multiplexed hologram, and then modulates the laser generated by the femtosecond laser into a laser beam including a plurality of parallel lasers. In the process of constructing the target array, the number of the focuses is 2N (N belongs to N, N is more than or equal to 2), and N is the sum of the used Dammann grating and the blazed grating; the number of the used gratings is adjusted to reach the number of focuses in a preset multi-focus array; adjusting the distance between each focus in the multi-focus array by adjusting the period of the Dammann grating; one or more Dammann gratings are replaced by blazed gratings, and the phase depth of the blazed gratings is adjusted to further change the light intensity of the focus, so that the light intensity of each focus in the multi-focus array is uniformly distributed.
As a further improvement of the invention, the multi-particle rapid capture system further comprises a display module, and the display module is electrically connected with the central control module. The display module is used for displaying the real-time image acquired by the camera or the image processed by the central control module.
And/or the central control module also comprises a characteristic extraction module which is used for confirming the characteristic information of the target object to be captured. The feature extraction module acquires the real-time image acquired by the image acquisition assembly, frames the target object according to the manual instruction input by the manual input module, and then segments the frame part in the real-time image as the feature image of the target object. And calculating the contour and the particle size of the particles in the frame selection part as the specification information of the target object.
The invention also comprises an operation method of the multi-particle rapid capture system, which comprises a preliminary preparation phase, an equipment initialization phase and a system automatic capture phase. Specifically, the method comprises the following steps:
the first stage of preparation comprises the following contents:
(1) dispersing a substance containing a target object to be captured into a transparent liquid photocuring agent to obtain a first working solution; selecting liquid suitable for storing the target object as second working liquid; and adding the first working fluid and the second working fluid to the liquid supply assembly, respectively.
(2) Selecting a corresponding micro-fluidic chip according to the particle size of a target object to be captured, and mounting the micro-fluidic chip on a micro-moving platform; in the selected microfluidic chip, the height of the fluid channel is greater than the particle size of the target object and less than 2 times of the particle size of the target object; and injecting the first working solution into the microfluidic chip.
Secondly, the equipment initialization phase comprises the following contents:
(1) judging whether the target object to be captured is captured for the first time by the system: if so, acquiring a real-time image of the target object, determining the target object through manual frame selection, further calculating the actual specification information of the target, and extracting a characteristic image of the target object. Otherwise, the specification information and the characteristic image of the current target object are directly determined through manual input.
(2) When the target object is captured for the first time, determining a target array of the target object according to the specification information of the target object, and further designing a hologram which meets the multiplexing requirements of a blazed grating and a Dammann grating for responding to the target array; the hologram is loaded into the spatial light modulator. And when the target object is not captured for the first time, querying a target array matching table according to the specification information, and further obtaining a hologram applicable to the current target object and loading the hologram into the spatial light modulator.
(3) And taking the characteristic image of the target object, which is acquired in real time or determined manually, as a reference image for identifying the target object in the automatic capturing stage of the system.
Thirdly, the automatic capture stage of the system comprises the following contents:
(1) and adjusting the flow divider valve to be communicated with the first liquid storage tank, quantitatively injecting the first working solution into a fluid channel of the microfluidic chip, and then closing the microfluidic pump.
(2) And a camera is used for acquiring a local image of the microfluidic chip above the displacement platform, which is amplified by the focusing objective lens, and the micro-displacement platform is moved to acquire a local image of the whole area of the microfluidic chip.
(3) And synthesizing a full image according to all the local images, then performing feature recognition on the full image according to the received feature image of the target object to be captured, extracting all the target objects contained in the full image, and calculating the coordinate information of the target object.
(4) Sorting the capture sequence of all selected targets, and then sequentially executing the following capture processes:
and moving the micro-moving platform to move the target object to be captured to a preset working beam irradiation area.
And ii, opening an optical shutter switch of the femtosecond laser, and irradiating the modulated working beam meeting the target array around the target object through the microfluidic chip.
And iii, closing the optical gate switch after the preset irradiation duration is reached, in the process, exciting and curing the light curing agent at the working beam to form a microcolumn, and forming a fence in the fluid channel at a plurality of positions to realize directional capture of the current target object.
And iv, completing the capture tasks of all the targets by circulating the steps i-iii.
(5) And after all the targets are captured, adjusting the shunt valve to be communicated with the second liquid storage tank, and quantitatively injecting the second working solution into the fluid channel of the microfluidic chip to replace the original first working solution.
As a further improvement of the invention, after the action of first particle capture is completed for a specific type of target object, the target object is moved to a position below a focusing objective lens, and the first working solution is controlled by a micro-flow pump to repeatedly flow forward and backward, so that the captured target object repeatedly collides with the microcolumn; then, observing whether each microcolumn is broken or not through a display module; when a certain microcolumn is broken, marking the serial number of the focus corresponding to the microcolumn, then readjusting the Darman grating and the blazed grating to increase the laser intensity at the focus corresponding to the broken microcolumn, and generating a new hologram and loading the new hologram into the spatial light modulator.
As a further improvement of the invention, after the capture task of each target object is completed, the fluid in the microfluidic chip is controlled to flow back and forth through the microfluidic pump, and whether each target object is adhered to the microcolumn or not is observed in a sampling mode through the display module: if yes, the current target object is failed to be captured, otherwise, the current target object is successfully captured.
As a further improvement of the present invention, the method for preparing the first working fluid is as follows: selecting a transparent liquid light curing agent; adding a substance containing a target object into a selected light curing agent for uniform dispersion; centrifuging the dispersion system to remove the impurity-containing part and only retaining the target-containing part; and (3) continuously supplementing a light curing agent into the dispersion system after the centrifugal impurity removal according to the standard that the particle solubility of the target object is not higher than 30000/mL, and uniformly dispersing again to obtain the required first working solution.
The technical scheme provided by the invention has the following beneficial effects:
the multi-particle rapid capturing system provided by the invention can directionally capture target particles or cells in the microfluidic chip, has high capturing precision, does not damage the particles or the cells, and simultaneously overcomes the problem that the particles or the cells are easy to adhere under the capturing condition due to reasons. The system provided by the invention can capture not only inorganic matter or organic matter particles, but also living cells, and cannot cause great influence on the life activity of the cells in the capturing process.
The system provided by the invention has high automation degree, can capture a plurality of targets in one capturing task at the same time, and greatly improves the capturing efficiency compared with the prior equipment and method. The system is low in operation cost, has a good capturing effect on particles with different specifications and scales, and is high in universality.
Drawings
Fig. 1 is a schematic system architecture diagram of a multi-particle fast capture system according to an embodiment of the present invention.
Fig. 2 is a schematic diagram of capturing a single particle in a microfluidic chip according to an embodiment of the present invention.
FIG. 3 is a schematic illustration of the results of a liquid supply assembly in an embodiment of the invention.
Fig. 4 is a schematic structural diagram of a femtosecond laser processing assembly in an embodiment of the invention.
Fig. 5 is a system deployment diagram of a femtosecond laser processing assembly and an image acquisition assembly in an embodiment of the invention.
Fig. 6 is a schematic structural diagram of an image capturing assembly with an additional optical filter in an embodiment of the present invention.
FIG. 7 is a block diagram of a central control module connected to other components according to an embodiment of the present invention.
FIG. 8 is a schematic diagram of a four focus array building process.
FIG. 9 is a schematic diagram of a hexafocal array construction process.
Fig. 10 is a flow of the verification stage after the target capture, which is composed of an electron microscope image.
Labeled as:
1. a liquid supply assembly; 2. femtosecond laser processing the component; 3. a micro-moving platform; 4. an image acquisition component; 5. a manual input module; 6. a microfluidic chip; 7. a display module; 11. a first liquid storage tank; 12. a second liquid storage tank; 13. a flow divider valve; 14. a microflow pump; 15. a conduit; 21. a femtosecond laser; 22. an attenuating mirror; 23. a spatial light modulator; 24. a 4f lens; 41. a camera; 42. a dichroic mirror; 43. a focusing objective lens; 44. an optical filter; 100. a central control module; 101. an object recognition unit; 102. a target positioning unit; 103. a displacement control subunit; 104. a laser control subunit; 105. a feature extraction module; 106. a pumping control subunit.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The present embodiment provides a multi-particle rapid capture system for capturing a target object with a micron-scale particle or cell as the target object. The capture process is carried out in the microfluidic chip 6, the microfluidic chip 6 is transparent and contains a fluid channel for the circulation of a target object; as shown in fig. 1, the multi-particle rapid capturing system includes: the device comprises a liquid supply component 1, a femtosecond laser processing component 2, a micro-motion platform 3, an image acquisition component 4, a manual input module 5 and a central control module 100. In this embodiment, the central control module 100 is electrically connected to the liquid supply assembly 1, the femtosecond laser processing assembly 2, the micro-motion platform 3, the image acquisition assembly 4, and the manual input module 5. The central control module 100 is a data processing center and a state control center of the whole system. The hardware form of the central control module 100 is an upper computer, and the devices connected with the upper computer, such as a mouse, a keyboard, a rocker and the like, are the corresponding manual input modules 5. The manual input module 5 is used for inputting characteristic information of the target object to be captured and other manual instructions. The characteristic information includes specification information of the target object and a characteristic image of the target object acquired in advance.
In addition, according to needs, the upper computer can also be connected with a display, and the display can show the data of gathering or each item processing result etc.. Such as an image of the material of the image capturing assembly 4, or an image processed by the central control module 100, etc.
Specifically, with reference to fig. 2, the working principle and mode of the multi-particle fast capturing system provided by this embodiment are as follows:
dispersing the target liquid to be captured into a light curing agent, and introducing the light curing agent into a fluid channel of the microfluidic chip 6; then all the targets in the microfluidic chip 6 are determined by collecting images and identifying the images, and the positions of the targets are calculated. And then sequentially starting the femtosecond laser processing assembly 2, adjusting the position of the micro-moving platform 3, irradiating the light curing agent around the target object by using a plurality of parallel beams generated by the femtosecond laser 21, and curing the light curing agent into a micro-column under the excitation of laser, thereby realizing the capture of the target object. Because the system can continuously switch on and off the laser and adjust the micro-moving platform 3 after one-time identification, the quantitative capture of a plurality of target objects can be realized in one-time operation, and the capture efficiency is high. And the captured target objects are in a single dispersed state, and adhesion or damage cannot occur.
Specifically, as shown in fig. 3, the liquid supply assembly 1 includes a first tank 11, a second tank 12, a micro-flow pump 14, a flow dividing valve 13, and a conduit 15. The first liquid storage tank 11 is used for containing a first working fluid containing a target object and a light curing agent. The second liquid storage tank 12 is used for containing a second working fluid which is convenient for storing the target object. The flow dividing valve 13 is a two-in one-out electromagnetic valve, is connected between the first liquid storage tank 11, the second liquid storage tank 12 and the micro-flow pump 14, and is used for controlling the working fluid in the first liquid storage tank 11 or the second liquid storage tank 12 to enter the micro-flow pump 14. The micro-fluid pump 14 in this embodiment is a combined positive and negative pressure micro-fluid control pressure pump. The microfluidic pump 14 is used for pumping the working fluid into the fluid channel of the microfluidic chip 6 through the conduit 15.
The femtosecond laser processing assembly 2 is used for generating exciting light curing agent for curing, and further generating a plurality of processing light beams of parallel microcolumns. As shown in fig. 4, the femtosecond laser processing assembly 2 includes a femtosecond laser 21, an attenuating mirror 22, a spatial light modulator 23, and a 4f lens 24 in terms of an optical path direction. Among them, the femtosecond laser is used to generate the outgoing laser. The attenuator 22 is used to modulate the laser energy and polarization direction of the exiting laser light. The spatial light modulator 23 is configured to modulate the emitted laser light having passed through the attenuator 22 into a plurality of parallel laser beams whose projection focuses satisfy an arrangement relationship of a target array. The 4f lens 24 is used to demagnify the modulated laser beam and filter out the unmodulated 0 th order light. The laser beam after passing through the 4f lens 24 is a processing beam satisfying the working array, and the processing beam is projected on the microfluidic chip 6 below.
In the femtosecond laser processing component 2 applied in this embodiment, the model of the femtosecond laser 21 is Coherent, Chamelon Vision-S; the femtosecond laser 21 firstly emits femtosecond laser, the wavelength of a laser light source generated by the femtosecond laser 21 is 800nm, the frequency is 80MHz, the pulse width is 75fs, and the output power is 2.2W. The laser energy and polarization direction are adjusted by the attenuator 22 consisting of a half-wave plate (P0) and a glan-taylor prism (H0), and for best modulation, the polarization direction of the incident laser light is the same as the direction of the liquid crystal molecules.
The diameter of the laser spot passing through the attenuator 22 is about 10mm, and is slightly larger than the panel of the spatial light modulator 23(SLM), so that the femtosecond laser can completely cover the panel of the spatial light modulator 23, and the pixel points on the spatial light modulator 23 are fully utilized. The spatial light modulator 23 used in the present embodiment is Holoeye Pluto NIR-2 of Holoeye corporation, germany, the resolution of the spatial light modulator 23 is 1920 × 1080, and 1080 × 1080 pixels (the size of a single pixel is 8 μm) at the center are used in actual processing; after the expanded laser irradiates the blazed grating and Dammann grating multiplexing hologram spatial light modulator 23 which is loaded and modulated, the incident light is modulated successfully.
Then, the laser beam modulated by the spatial light modulator 23 reaches the 4f Lens 24 system constituted by the Lens one (Lens1), the stop (P), and the Lens two (Lens 2). In the present embodiment, considering that the spatial light modulator 23 is also a diffractive optical element, a plurality of diffraction orders occur in the modulated femtosecond laser light; the beam is filtered using a 4f lens 24. In the 4f lens 24, the laser beam reaches a diaphragm after being focused by the first lens, and the diaphragm filters out unmodulated 0-level light; the rest of the modulated laser is processed by a lens II confocal with the lens I and then is changed into a contracted parallel laser beam. The parallel laser beam is a processing beam meeting the requirement, and each focus of the processing beam forms a target array capable of surrounding and capturing a target object.
In this embodiment, the target array corresponding to each focus in the processing beam generated by the femtosecond laser processing assembly 2 satisfies: the connecting line between adjacent focuses forms a polygon, the inscribed circle corresponding to the polygon is larger than the maximum section circle of the target object, and the distance between any two adjacent focuses in the multi-focus array is smaller than the particle size of the target object.
The micro-moving platform 3 used in the embodiment is a micron-sized biaxial moving platform, and the micro-moving platform 3 is used for fixing the micro-fluidic chip 6 to be processed and accurately adjusting the position of the micro-fluidic chip 6 according to the requirements of the processing process. The equipment can adopt various UVW precision alignment platforms sold in the existing market, and the equipment can apply corresponding adjusting instructions to the platforms to enable the platforms to realize micron-level controllable displacement. The performance of the current commercial products can be accurately controlled on the order of 2-3 μm. In this embodiment, the micro-fluidic chip 6 mounted on the micro-moving platform 3 can be accurately moved, so that the processing light beam generated by the femtosecond laser processing assembly 2 is accurately irradiated to the periphery of the target, which is the center of the processing light beam.
The image acquisition assembly 4 comprises a focusing objective 43, a camera 41 and a dichroic mirror 42. Fig. 5 shows a position schematic diagram of the femtosecond laser processing assembly 2 and the image acquisition assembly 4 when the assemblies are installed and deployed.
In this embodiment, a focusing objective 43 is movably mounted right above the displacement stage, considering that a clean image of a micrometer-scale object in the microfluidic chip 6 cannot be completely obtained by the conventional high-resolution industrial-scale CCD camera 41. The focusing objective 43 corresponds to a microscope for magnifying the image of the underlying microfluidic chip 6. The focusing objective 43 allows the camera 41 to acquire a partial image of the object in the fluid channel. After the local images of all the areas of the microfluidic chip 6 are obtained, the whole image of all the areas of the microfluidic chip 6 on the micro-moving platform 3 can be synthesized by image splicing.
Considering that the viewing area of the camera 41 and the projection direction of the working beam during the curing process completely overlap; this embodiment mounts a dichroic mirror 42 above the fine moving stage 3. Dichroic mirror 42 is characterized by: the light of a specific wavelength is totally reflected and the light of the remaining wavelengths is totally transmitted, and the dichroic mirror 42 that is totally transmitted by natural light and totally reflected by the laser light of the processing light beam is selected in the present embodiment. Specifically, the incidence direction of the working light beam passing through the 4f lens 24 in the femtosecond laser processing assembly 2 is the horizontal direction, the incident light beam and the reflection plane of the dichroic mirror 42 form an included angle of 45 degrees, and the laser light beam of the incident light beam reflected by the dichroic mirror 42 is in the vertical direction and is projected onto the micro-moving platform 3. The camera 41 is located on the reverse extension of the laser reflected light in the dichroic mirror 42, and the camera 41 is located on the other side of the dichroic mirror with respect to the focusing objective 43. The camera 41 is used to acquire a real-time magnified image of the microfluidic chip 6 under the focusing objective 43.
In the present embodiment, the dichroic mirror 42 is provided to achieve the coincidence of the optical paths of framing and laser irradiation, but does not interfere with each other. In addition, as shown in fig. 6, a filter 44 or a polarizing plate may be added between the camera 41 and the dichroic mirror 42 to filter the interference of stray light on the sharpness of the image viewed by the camera 41.
The central control module 100 of the present invention performs the following operations in the actual application process, respectively: (1) and acquiring the characteristic information of the target object input by the manual input module 5. (2) And controlling the type, flow speed and fluid movement direction of the working fluid pumped by the micro-flow pump 14 in the liquid supply system. (3) And receiving the real-time image of the microfluidic chip 6 acquired by the image acquisition assembly 4, identifying the target object contained in the channel of the microfluidic chip 6 from the real-time image according to the characteristic image of the target object, and calculating the position of the target object. (3) The spatial light modulator 23 is adjusted according to the received specification information of the target object, so that the focal array of the working beam generated by the femtosecond laser processing assembly 2 meets the requirement of the target array corresponding to the target object. (4) And adjusting the position of the micro-moving platform 3 according to the distinguished position of the target object, so that the processing beams generated by the femtosecond laser processing assembly 2 are sequentially irradiated around each identified target object.
Specifically, as shown in fig. 7, the central control module 100 includes an object recognition unit 101, an object positioning unit 102, a pumping control subunit 106, a displacement control subunit 103, and a laser control subunit 104. The above subunits are different microprocessors for processing data in the upper computer and different microcontrollers for issuing instructions.
The target recognition unit 101 is a dedicated microprocessor, and the microprocessor is configured to acquire a feature image of a target object as a reference and a real-time image acquired by the camera 41, and then extract all target objects included in the real-time image by feature recognition with a target in the feature image as a reference. This part of the process can be completed by adopting various existing image recognition algorithms based on the neural recognition network.
The target positioning unit 102 is a dedicated microprocessor, and the microprocessor is configured to calculate coordinate information of each target on the micro-moving platform 3 according to the pixel position of each target in the real-time image, which is identified by the target identifying unit 101.
The pumping control subunit 106 is a microcontroller for controlling the switching state of the diverter valve 13 at different stages of the process, as well as controlling the direction and flow rate of the fluid pumped by the micropump 14.
The displacement control subunit 103 is a processing module including a microprocessor and a microcontroller, and is configured to acquire coordinate information of each target, and then adjust the micro-displacement platform 3 according to the coordinate information in the capturing stage of each target, so that the target in each microfluidic chip 6 is exactly located at the center of the processing beam.
The laser control subunit 104 is a processing module including a microprocessor and a microcontroller, and is configured to acquire specification information of the target object, query a preset target array matching table according to the specification information of the target object to obtain a hologram of the target array that meets the condition, and load the hologram into the spatial light modulator 23, so that the femtosecond laser processing assembly 2 generates a processing beam that meets the requirement. The target array matching table is a pre-stored expert experience table, and the one-to-one mapping relation between the specification information of different particles and the grating multiplexing holograms of different target arrays is stored in the expert experience table.
It is considered that when capturing a completely new particle or cell, the system cannot accurately identify the corresponding target. Therefore, in the system of the present embodiment, the central control module 100 further includes a feature extraction module 105, which is used to confirm feature information of the target object to be captured. The feature extraction module 105 obtains the real-time image acquired by the image acquisition assembly 4, and performs framing on the target object according to the manual instruction input by the manual input module 5, and then divides the framed part in the real-time image as the feature image of the target object. And calculating the contour and the particle size of the particles in the frame selection part as the specification information of the target object.
The working content of the feature extraction module 105 can be understood as: before each particle is captured for the first time, it is necessary to acquire an image in the microfluidic chip 6 and then manually frame the portion containing the target particle. The feature extraction module 105 of the system then takes the object included in the selected part as the target object in the current round of capturing. Meanwhile, the accurate specification information such as the particle size of the target object is calculated according to the selected image, and the specification information can be used as a basis for designing a corresponding target array at a later stage and further used for adjusting working parameters of the spatial light modulator 23, so that a working beam generated by the femtosecond laser 21 meets the requirement of the constructed target array.
Consider that the system provided by the present embodiment performs a self-learning process every time a new particle is identified and captured. The target array matching table in this embodiment is a data set that allows for manual editing. Before each particle or cell is captured for the first time, determining a hologram corresponding to the current target object in a manual setting mode, and adding the corresponding hologram and a mapping relation into a data set; when the current target object is captured again, the corresponding hologram is obtained through a table look-up method. Thus, the workload of system operators can be greatly reduced; the intelligence of the system is improved.
In the present embodiment, each hologram provided in the spatial light modulator 23 corresponds to a multifocal array having a specific number and satisfying a specific pitch; after the spatial light modulator 23 loads a specific hologram, the focus and the light intensity of the processing beam generated by the femtosecond laser processing assembly 2 meet the corresponding multi-focus array; the number of the focus points, the position of the focus points and the light intensity of the light emitted from each focus point of the processing light beam of the parameters of the femtosecond laser processing component 2 are adjusted by adjusting different holograms.
The spatial light modulator 23 generates a multifocal array satisfying a target array by loading a hologram multiplexed by a dammann grating and a blazed grating, and modulates the laser beam generated by the femtosecond laser 21 into a laser beam including a plurality of parallel laser beams. In the process of constructing the target array, the number of the focuses is 2N (N belongs to N, N is more than or equal to 2), and N is the sum of the used Dammann grating and the blazed grating; the number of the used gratings is adjusted to reach the number of focuses in a preset multi-focus array; adjusting the distance between each focus in the multi-focus array by adjusting the period of the Dammann grating; one or more Dammann gratings are replaced by blazed gratings, and the phase depth of the blazed gratings is adjusted to further change the light intensity of the focus, so that the light intensity of each focus in the multi-focus array is uniformly distributed.
In the process of adjusting the focal array, firstly, 2 uniformly distributed points are generated by using a Dammann grating; then, a blazed grating with adjustable phase depth is added on the Dammann grating, and the blazed grating can also generate 2 two focuses which are uniformly distributed. And then, a quadrilateral focal array with proper distance and light intensity is formed by adjusting the grating period of the Dammann grating and the phase of the blazed grating. Finally, the modulated dammann grating and blazed grating multiplexed hologram are loaded into the spatial light modulator 23.
For conventional micron-sized particles or cells, 4-focal and 6-focal arrays have generally been able to achieve the capture requirements. When a multi-focus array with more focuses and uniform intensity is required to be modulated, a larger number of gratings are used and the adjustment is performed according to the method. For example, when one-dimensional dammann grating is used with a rotation angle of 0 degrees and the other one-dimensional dammann grating is used with a rotation angle of 90 °, the hologram-generating array pattern of the multiplexed two-dimensional dammann grating is square as shown in fig. 8. When three one-dimensional dammann gratings are used and the rotation angles are set to 0 °, 60 °, and 120 °, respectively, the array pattern generated by the holograms of the multiplexed three-dimensional dammann gratings is a regular hexagon as shown in fig. 9. The distance between each focus in the multi-focus array can be changed by changing the period of the Dammann grating to adjust the focus.
When the light intensity of the focus is adjusted, 2 uniformly distributed points are generated by using the Dammann grating. And then adding a blazed grating with adjustable phase depth on the Dammann grating, wherein the blazed grating and the Dammann grating can jointly generate 4 focuses. And adjusting the grating period of the Dammann grating and the phase of the blazed grating to adjust the intensity distribution and the position distribution of the four focuses.
The foregoing embodiments provide a method for operating a multi-particle rapid capture system, which generally includes a preliminary preparation phase, an equipment initialization phase, and an automatic capture phase. The early preparation stage is mainly to complete the preparation of materials, the equipment initialization stage is mainly to complete the parameter setting of the system, and the automatic capture stage is to automatically capture each target particle or cell in the microfluidic chip 6 by the system. The working contents of the three parts are as follows:
first, early preparation stage
(1) Dispersing a substance containing a target object to be captured into a transparent liquid light curing agent to obtain a first working solution; selecting liquid suitable for storing the target object as second working liquid; and the first working fluid and the second working fluid are separately added to the liquid supply assembly 1.
The first working liquid in this example is obtained by sufficiently dispersing a substance containing an object in a dispersion liquid having a photo-setting characteristic. During the preparation process of the first working solution, the concentration of the target is adjusted according to experience, so that the target is abundant in the dispersion system and is convenient to search and capture at a later stage. Meanwhile, the concentration of the target is not high enough, otherwise the target agglomeration can be caused to influence the capture of the single target.
In this embodiment, the photo-curing agent used in the first working solution preparation process is a liquid photoresist IPL manufactured by Nanoscibe corporation. The type of the photoresist is not limited to the type, and in fact, the type selection process of the photoresist used in this embodiment includes at least the following three criteria:
A. the photoresist is liquid, has good fluidity, and can form a stable solid (target object) liquid (finger dispersion glue) dispersion system.
B. The photoresist should be transparent or sufficiently light in color to at least facilitate effective and accurate resolution of the target and non-removable impurities contained therein.
C. The photoresist should maintain stable properties under a normal state and not undergo a physicochemical reaction with a target or impurities, in addition to having a photocuring property.
Any photoresist that satisfies the above criteria may be used as the photoresist used in the present embodiment. In addition, in order to keep the properties of the first working fluid stable, a processing aid such as an anticoagulant and an antioxidant may be added thereto in an appropriate amount without affecting the final application effect.
After the target is uniformly dispersed in the light curing agent to obtain the first working solution, the target needs to be subjected to preliminary impurity removal, the purpose of the impurity removal is to remove large-particle-size impurities contained in the target, and the impurities may cause the blockage of a fluid channel in the later period or be mistakenly captured as the target. Therefore, in capturing a particular particle, the material should be selected as pure as possible and then dispersed in the light curing agent. The impurities already present can be removed by filtration, centrifugation, etc. In the dispersing process of the target and the light curing agent, the modes of stirring, ultrasonic dispersion treatment and the like can be adopted to improve the uniformity of the target in a dispersing system as much as possible; avoiding the agglomeration of the target.
Specifically, the configuration process of the second working fluid in this embodiment is as follows: selecting a transparent liquid light curing agent; adding a substance containing a target object into a selected light curing agent for uniform dispersion; centrifuging the dispersion system to remove the impurity-containing part and only retaining the target-containing part; and (3) continuously supplementing a light curing agent into the dispersion system after the centrifugal impurity removal according to the standard that the particle solubility of the target object is not higher than 30000/mL, and uniformly dispersing again to obtain the required first working solution.
The second working fluid in this embodiment needs to be adaptively selected for different targets. For example, for active cells, it should be suitable for cell survival, stable and appropriate in properties, and not harmful to active cells, buffers, etc. In contrast, in the case of inorganic particles which are easily oxidized, it is necessary to use a site containing an antioxidant as the second working fluid.
(2) Selecting a corresponding micro-fluidic chip 6 according to the particle size of a target object to be captured and installing the micro-fluidic chip on the micro-moving platform 3; in the selected microfluidic chip 6, the height of the fluid channel is greater than the particle size of the target object and less than 2 times of the particle size of the target object; the first working liquid is injected into the microfluidic chip 6.
In this embodiment, the microfluidic chip 6 is an assembly that is automatically selected according to the particle size of the target object to be captured, and the microfluidic chip 6 includes a detachable cover plate and a bottom plate, and the bottom plate includes grooves that form the fluid channels. The cover plate and the bottom plate of the microfluidic chip 6 are made of transparent glass-based or organic resin materials.
The reason why the microfluidic chip 6 is designed as a detachable split structure in this embodiment is to facilitate the target to be extracted from the microfluidic chip 6 after the target is captured.
Second, device initialization phase
The early preparation stage is to set corresponding parameters and provide related data for the automatic capture of the system, so that the work content of the part is different between the target object for the primary capture and the target object for the non-primary capture, and specifically, the method comprises the following steps:
(1) judging whether the target object to be captured is captured for the first time by the system: if so, acquiring a real-time image of the target object, determining the target object through manual frame selection, further calculating the actual specification information of the target, and extracting a characteristic image of the target object. Otherwise, the specification information and the characteristic image of the current target object are directly determined through manual input.
(2) When the target object is captured for the first time, determining a target array of the target object according to the specification information of the target object, and further designing a hologram which meets the multiplexing requirements of a blazed grating and a Dammann grating for responding to the target array; the hologram is loaded into the spatial light modulator 23. When the target object is not captured for the first time, a target array matching table is queried according to the specification information, and a hologram suitable for the current target object is obtained and loaded into the spatial light modulator 23.
(3) And taking the characteristic image of the target object, which is acquired in real time or determined manually, as a reference image for identifying the target object in the automatic capturing stage of the system.
Third, the automatic capture stage of the system
The automatic capture stage of the system is automatically executed by a multi-particle rapid capture system, so as to simultaneously capture a plurality of targets contained in the microfluidic chip 6, and the automatic capture stage comprises the following steps:
(1) and adjusting the flow dividing valve 13 to be communicated with the first liquid storage tank 11, quantitatively injecting the first working solution into a fluid channel of the microfluidic chip 6, and then closing the microfluidic pump 14.
(2) The camera 41 is used for acquiring a local image of the microfluidic chip 6 above the displacement platform, which is amplified by the focusing objective 43, and the micro-displacement platform 3 is moved to acquire a local image of the whole area of the microfluidic chip 6.
(3) And synthesizing a full image according to all the local images, then performing feature recognition on the full image according to the received feature image of the target object to be captured, extracting all the target objects contained in the full image, and calculating the coordinate information of the target object.
(4) Sorting the capture sequence of all selected targets, and then sequentially executing the following capture processes:
and i, moving the micro-moving platform 3 to move the target object to be captured to a preset working beam irradiation area.
And ii, turning on an optical shutter switch of the femtosecond laser 21, and irradiating the modulated working beam meeting the target array around the target object through the microfluidic chip 6.
And iii, closing the optical gate switch after the preset irradiation duration is reached, in the process, exciting and curing the light curing agent at the working beam to form a microcolumn, and forming a fence in the fluid channel at a plurality of positions to realize directional capture of the current target object.
And iv, completing the capture tasks of all the targets by circulating the steps i-iii.
(5) After all the targets are captured, the shunt valve 13 is adjusted to be communicated with the second liquid storage tank 12, and the second working solution is quantitatively injected into the fluid channel of the microfluidic chip 6 to replace the original first working solution.
In particular, after the first particle capturing action is completed for a specific type of target object by using the present system, it should be verified whether the laser intensity of the working beam of the femtosecond laser processing assembly 2 set in the system initialization setting process is reliable or not by manual operation.
The specific verification process is as follows: as shown in fig. 10, the target object is moved to a position below the focusing objective 43, and the micro-flow pump 14 controls the first working fluid to repeatedly flow forward and backward, so that the captured target object repeatedly collides with the microcolumn; then, whether each microcolumn is broken or not is observed through a display module 7; when a microcolumn is broken, the serial number of the focus corresponding to the microcolumn is marked, and then the Dammann grating and the blazed grating are readjusted to increase the laser intensity at the focus corresponding to the broken microcolumn, so as to generate a new hologram and load the new hologram into the spatial light modulator 23.
In particular, after the system is used to complete the capturing task of each target object, each captured target object should be subjected to sampling verification or full verification. And judging whether the solidified microcolumns are adhered to the target object before. The verification process, like fig. 10, includes the following steps: firstly, the fluid in the microfluidic chip 6 is controlled to flow back and forth by the microfluidic pump 14, and whether each target object is adhered to the microcolumn or not is sampled and observed by the display module 7: if yes, the current target object is failed to be captured, otherwise, the current target object is successfully captured.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (10)

1. A multi-particle rapid capture system, which is used for capturing a target object by using particles or cells with micron scale as the target object; the capture process is carried out in a microfluidic chip which is transparent and contains a fluid channel for the circulation of the target object; the multi-particle rapid capture system comprises:
the liquid supply assembly comprises a first liquid storage tank, a second liquid storage tank, a micro-flow pump, a flow divider valve and a guide pipe; the first liquid storage tank is used for containing a first working solution containing a target object and a light curing agent; the second liquid storage tank is used for containing second working liquid convenient for storing a target object; the flow dividing valve is a two-inlet one-outlet electromagnetic valve, is connected among the first liquid storage tank, the second liquid storage tank and the micro-flow pump and is used for controlling working liquid in the first liquid storage tank or the second liquid storage tank to enter the micro-flow pump; the micro-flow pump is used for pumping the working fluid into a fluid channel of the micro-fluidic chip through a conduit;
the femtosecond laser processing assembly is used for generating processing beams for exciting the curing of the curing agent and further generating a plurality of parallel microcolumns; according to the direction of a light path, the femtosecond laser processing assembly comprises a femtosecond laser, an attenuation mirror, a spatial light modulator and a 4f lens; the femtosecond laser is used for generating emergent laser; the attenuation mirror is used for modulating the laser energy and the polarization direction of the emergent laser; the spatial light modulator is used for modulating the emergent laser light after passing through the attenuator into a plurality of parallel laser beams of which the projection focuses meet the arrangement relation of a target array; the 4f lens is used for shrinking the modulated laser beam and filtering out unmodulated 0-level light; the laser beam passing through the 4f lens is a processing beam meeting the working array, and the processing beam is projected on the microfluidic chip below the working array; wherein, the target array corresponding to each focus of the processing light beam generated by the femtosecond laser processing assembly meets the following requirements: the connecting line between adjacent focuses forms a polygon, the inscribed circle corresponding to the polygon is larger than the maximum section circle of the target object, and the distance between any two adjacent focuses in the focus array is smaller than the particle size of the target object;
the micro-moving platform is a micron-sized biaxial moving platform and is used for fixing the micro-fluidic chip to be processed and accurately adjusting the position of the micro-fluidic chip according to the requirement of the processing process;
an image acquisition assembly comprising a focusing objective, a camera, and a dichroic mirror; the focusing objective lens is movably arranged right above the displacement platform and used for amplifying the image of the micro-fluidic chip below the focusing objective lens so as to facilitate the camera to acquire the local image of the target object in the fluid channel; the dichroic mirror can be completely transmitted by natural light and can be used for totally reflecting laser of a processing light beam; the incidence direction of the working light beam passing through the 4f lens in the femtosecond laser processing assembly forms an included angle of 45 degrees with the reflection plane of the dichroic mirror, and the laser light reflected by the dichroic mirror is projected onto the micro-moving platform; the camera is positioned on the reverse extension line of the laser reflection light in the dichroic mirror, and is positioned on the other side of the dichroic mirror relative to the focusing objective lens; the camera is used for acquiring a real-time amplified image of the microfluidic chip below the focusing objective lens;
the manual input module is used for inputting characteristic information of a target object to be captured and other manual instructions; the characteristic information comprises specification information of the target object and a characteristic image of the target object acquired in advance; and
the central control module is electrically connected with the liquid supply assembly, the spatial light modulator, the micro-moving platform, the image acquisition assembly and the manual input module; the central control module is used for: (1) acquiring characteristic information of the target object input by the manual input module; (2) controlling the type, flow rate and fluid movement direction of working fluid pumped by a micro-flow pump in the liquid supply system; (3) receiving a real-time image of the microfluidic chip acquired by the image acquisition assembly, identifying a target object contained in a channel of the microfluidic chip from the real-time image according to the characteristic image of the target object, and calculating the position of the target object; (3) adjusting the spatial light modulator according to the received specification information of the target object, so that a focal array of a working beam generated by the femtosecond laser processing assembly meets the requirement of a target array corresponding to the target object; (4) and adjusting the position of the micro-moving platform according to the distinguished position of the target object, so that the processing beams generated by the femtosecond laser processing assembly are sequentially irradiated around each identified target object.
2. The multiple particle rapid capture system of claim 1, wherein: the central control module comprises a target identification unit, a target positioning unit, a pumping control subunit, a displacement control subunit and a laser control subunit; the target identification unit is used for acquiring a characteristic image of a target object serving as a reference and a real-time image acquired by the camera, and then extracting all target objects contained in the real-time image by using a target in the characteristic image as a reference through characteristic identification; the target positioning unit is used for calculating the coordinate information of each target object on the micro-moving platform according to the pixel position of each target object in the real-time image, which is identified by the target identification unit; the pumping control subunit is used for controlling the switching state of the diverter valve at different processing stages and controlling the fluid direction and flow rate of the micro-flow pump in the pumping process; the displacement control subunit is used for acquiring coordinate information of each target object, and then adjusting the micro-displacement platform according to the coordinate information in the capturing stage of each target object, so that the target object in each micro-fluidic chip is exactly positioned at the midpoint of the processing light beam; the laser control subunit is used for acquiring the specification information of the target object, then inquiring a preset target array matching table according to the specification information of the target object to obtain a hologram of the target array meeting the conditions, and loading the hologram into the spatial light modulator so that the femtosecond laser processing assembly generates a processing beam meeting the requirements; the target array matching table is a pre-stored expert experience table, and the one-to-one mapping relation between the specification information of different particles and the grating multiplexing holograms of different target arrays is stored in the target array matching table.
3. The multiple particle rapid capture system of claim 2, wherein: the target array matching table is a data set allowing manual editing; before each particle or cell is captured for the first time, determining a hologram corresponding to the current target object in a manual setting mode, and adding the corresponding hologram and a mapping relation into a data set; when the current target object is captured again, the corresponding hologram is obtained through a table look-up method.
4. The multiple particle rapid capture system of claim 2, wherein: each hologram is arranged corresponding to a multi-focus array with a specific number and satisfying a specific interval; after the spatial light modulator loads a specific hologram, the focus and the light intensity of a processing beam generated by the femtosecond laser processing assembly meet the corresponding multi-focus array; and the number of the focuses of the processing beams of the parameters of the femtosecond laser processing component, the positions of the focuses and the light intensity of light emitted from each focus are adjusted by adjusting different holograms.
5. The multiple particle rapid capture system of claim 1, wherein: the microfluidic chip is an assembly which is automatically selected according to the particle size of a target object to be captured, and comprises a detachable cover plate and a bottom plate, wherein the bottom plate is provided with grooves forming various fluid channels; the cover plate and the bottom plate of the microfluidic chip are made of transparent glass-based or organic resin materials.
6. The multiple particle rapid capture system of claim 1, wherein: the spatial light modulator generates a multi-focus array meeting a target array by loading a hologram multiplexed by a Dammann grating and a blazed grating, and then modulates laser generated by a femtosecond laser into a laser beam comprising a plurality of parallel lasers; in the process of constructing the target array, the number of the focuses is 2N (N belongs to N, N is more than or equal to 2), and N is the sum of the used Dammann grating and the blazed grating; the number of the used gratings is adjusted to reach the number of focuses in a preset multi-focus array; adjusting the distance between each focus in the multi-focus array by adjusting the period of the Dammann grating; one or more Dammann gratings are replaced by blazed gratings, and the phase depth of the blazed gratings is adjusted to further change the light intensity of the focus, so that the light intensity of each focus in the multi-focus array is uniformly distributed.
7. The multiple particle rapid capture system of claim 1, wherein: the display module is electrically connected with the central control module; the display module is used for displaying the real-time image acquired by the camera or the image processed by the central control module;
and/or
The central control module also comprises a feature extraction module which is used for confirming the feature information of the target object to be captured; the feature extraction module acquires a real-time image acquired by the image acquisition assembly, frames a target object according to a manual instruction input by the manual input module, and then segments a frame selection part in the real-time image to serve as a feature image of the target object; and calculating the contour and the particle size of the particles in the frame selection part as the specification information of the target object.
8. A method of operating a multiple particle rapid capture system according to claim 1, wherein: the operation method comprises a preliminary preparation stage, an equipment initialization stage and a system automatic acquisition stage;
the first stage of preparation comprises the following contents:
(1) dispersing a substance containing a target object to be captured into a transparent liquid light curing agent to obtain a first working solution; selecting liquid suitable for storing the target object as second working liquid; respectively adding the first working solution and the second working solution into the liquid supply assembly;
(2) selecting a corresponding micro-fluidic chip according to the particle size of a target object to be captured and installing the micro-fluidic chip on a micro-moving platform; in the selected microfluidic chip, the height of the fluid channel is greater than the particle size of the target object and less than 2 times of the particle size of the target object; injecting a first working solution into the microfluidic chip;
secondly, the equipment initialization phase comprises the following contents:
(1) judging whether the target object to be captured is captured for the first time by the system, if so, acquiring a real-time image of the target object, determining the target object through manual framing, further calculating the actual specification information of the target, and extracting a characteristic image of the target object: otherwise, the rule information and the characteristic image of the current target object are directly determined through manual input;
(2) when the target object is captured for the first time, determining a target array of the target object according to the specification information of the target object, and further designing a hologram which meets the multiplexing requirements of a blazed grating and a Dammann grating for responding to the target array; loading a hologram into a spatial light modulator; when the target object is not captured for the first time, a target array matching table is inquired according to the specification information, and a hologram suitable for the current target object is obtained and loaded into the spatial light modulator;
(3) taking the characteristic image of the target object which is acquired in real time or determined manually as a reference image for identifying the target object in the automatic capturing stage of the system;
thirdly, the automatic capture stage of the system comprises the following contents:
(1) adjusting the shunt valve to be communicated with the first liquid storage tank, quantitatively injecting the first working solution into a fluid channel of the microfluidic chip, and then closing the microfluidic pump;
(2) obtaining a local image of the microfluidic chip above the displacement platform, which is amplified by the focusing objective lens, by a camera, and moving the micro-displacement platform to obtain a local image of the whole area of the microfluidic chip;
(3) synthesizing a full image according to all the local images, then performing feature recognition on the full image according to the received feature images, extracting all target objects contained in the full image, and calculating coordinate information of the target objects;
(4) sorting the capture sequence of all selected targets, and then sequentially executing the following capture processes:
moving the micro-moving platform to move a target object to be captured to a preset working beam irradiation area;
opening an optical gate switch of the femtosecond laser, and irradiating the modulated working beam meeting the target array around the target object through the microfluidic chip;
closing the optical gate switch after the preset irradiation duration is reached, in the process, exciting and curing the light curing agent at the working beam to form a microcolumn, and forming a fence in the fluid channel at a plurality of positions to realize directional capture of the current target object;
iv, completing the capture tasks of all the targets by circulating the steps i-iii;
(5) and after all the targets are captured, adjusting the shunt valve to be communicated with the second liquid storage tank, and quantitatively injecting the second working solution into the fluid channel of the microfluidic chip to replace the original first working solution.
9. The method of claim 8, wherein: after the action of capturing the first particles/cells is finished aiming at a specific type of target object, the target object is moved to a position below a focusing objective lens, and the first working solution is controlled by a micro-fluid pump to repeatedly flow forwards and backwards so that the captured target object repeatedly collides with the microcolumn; then, observing whether each microcolumn is broken or not through a display module; when a certain microcolumn is broken, marking the serial number of the focus corresponding to the microcolumn, then readjusting the Darman grating and the blazed grating to increase the laser intensity at the focus corresponding to the broken microcolumn, and generating a new hologram and loading the new hologram into the spatial light modulator.
10. The method of claim 8, wherein: the preparation method of the first working solution comprises the following steps: selecting a transparent liquid light curing agent; adding a substance containing a target object into a selected light curing agent for uniform dispersion; centrifuging the dispersion system to remove the impurity-containing part and only retaining the target-containing part; and (3) continuously supplementing a light curing agent into the dispersion system after the centrifugal impurity removal according to the standard that the particle solubility of the target object is not higher than 30000/mL, and uniformly dispersing again to obtain the required first working solution.
CN202210090799.2A 2022-01-26 2022-01-26 Multi-particle rapid capturing system and operation method thereof Active CN114433262B (en)

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