CN112779156B - Nanosecond pulse laser perforation system and method based on spatial light modulation technology - Google Patents

Abstract

The invention provides a nanosecond pulse laser perforation system and a method based on a spatial light modulation technology, wherein a spatial light modulator is used for modulating a spatial light field of single pulse laser; the modulated pulse laser forms controllable micro-jet in the micro-fluidic chip after being focused by the objective lens, and the perforation of cells and the introduction of exogenous substances are realized by utilizing high-speed micro-jet.

Description

A nanosecond pulsed laser perforation system and method based on spatial light modulation technology

technical field

The present invention relates to the field of photo-induced breakdown of liquid and cell photo-perforation, in particular to a nanosecond pulsed laser perforation system and method based on spatial light modulation technology.

Background technique

Cell membrane microsurgery is of great scientific significance for our in-depth analysis of life processes and the pathogenesis of diseases. For example, the introduction of some exogenous biological macromolecules such as proteins, DNA, RNA and siRNA into living cells can interfere with the expression of protein functions. Even targeted killing or reversal of diseased cells in the microscopic field. In this process, how to achieve recoverable perforation of cells without affecting cell viability as much as possible is a key step.

In the process of introducing foreign substances into cells, the ability to actively introduce is the key to improving the efficiency, success rate and dose control of the introduction. At the same time, it is also a very important requirement to achieve targeted and high-throughput operations on cells. Traditional cell membrane microsurgery methods include capillary microinjection, electroporation, sonoporation, and viral or chemical delivery. In addition, with the development of laser technology, a non-invasive, non-contact and target-oriented photoperforation method has been applied to the field of cell membrane microsurgery. On the one hand, a tightly focused laser can be used to achieve targeted perforation of single cells, and on the other hand, high-throughput perforation of cell populations can be achieved by means of the strong absorption of laser light by some metal nanomaterials. However, these two methods use passive diffusion of exogenous substances to enter cells after perforation, so there are disadvantages of uncontrollable introduction dose and poor repeatability. In view of this, a method for targeted perforation of single cells using microfluidics formed by inducing multi-point perforation in water by a double-pulse laser is proposed. The high-speed motion of the microfluidic fluid will drive the movement of the surrounding medium. Therefore, it has the potential to control the energy and dose of active targeted introduction. However, using two pulsed lasers to induce jet formation to realize cell photoperforation has disadvantages such as high cost and complicated operation.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a nanosecond pulse laser perforation system and method based on spatial light modulation technology, which utilizes a single-pulse-induced controllable jet to achieve cell puncture in a microfluidic chip. Targeted and high-throughput perforation, and the system is simple, easy to operate, and low cost.

The present invention is achieved through the following technical solutions:

A nanosecond pulsed laser perforation system based on spatial light modulation technology, comprising a pulsed laser, a spatial light modulator, a white light source, an energy measurement module and a system control storage module;

The pulsed laser light emitted by the pulsed laser is incident on the spatial light modulator, and the linear polarizer and the first convex lens are sequentially arranged in the propagation direction of the pulsed laser light reflected by the spatial light modulator;

A microfluidic chip, an objective lens, a dichroic mirror and a high-speed imager are arranged in sequence in the direction of continuous light propagation from the white light source; the pulsed laser light transmitted from the first convex lens is reflected by the dichroic mirror and then enters the objective lens;

The microfluidic chip is provided with a cell flow channel and a reagent flow channel, a breakdown cavity exists in the reagent flow channel, and the breakdown cavity communicates with the cell flow channel through a jet port; the pulsed laser focused by the objective lens is incident in the breakdown cavity;

The energy measurement module is used to measure the pulsed laser energy emitted by the pulsed laser;

The system control storage module is electrically connected to the pulsed laser, the spatial light modulator and the high-speed imager; it is used to control the timing and triggering between the pulsed laser, the spatial light modulator and the high-speed imager, and to control the pulsed laser energy and spatial light of the pulsed laser The phase hologram of the modulator loads, receives and stores image data from the high-speed imager.

Preferably, the energy measurement module includes a beam splitter prism and an energy meter; the beam splitter prism is arranged in the pulse laser propagation direction of the pulsed laser, the energy meter is arranged in the pulse laser propagation direction reflected by the beam splitter prism, and the energy meter and the system control storage module with electrical connection.

Preferably, a half-wave plate, a concave lens and a second convex lens are arranged in sequence in the pulse laser propagation direction of the pulse laser, and the pulse laser transmitted from the second convex lens is incident on the spatial light modulator.

Preferably, the pulse laser is provided with a first reflection mirror in the direction of propagation of the pulsed laser light, a second reflection mirror is arranged in the transmission direction of the pulsed light reflected by the first reflection mirror, and the spatial light modulator is placed on the second reflection mirror. in the direction of propagation of the reflected pulsed light.

Further, the included angle between the first reflection mirror and the propagation direction of the pulsed laser light of the pulsed laser is 45° counterclockwise, and the included angle between the second reflection mirror and the propagation direction of the pulsed light reflected by the first reflection mirror is 85° counterclockwise; spatial light modulation The mirror surface of the reflector is parallel to the mirror surface of the second reflector.

Preferably, a convex lens is arranged between the white light source and the microfluidic chip.

Preferably, a notch plate is arranged between the dichroic mirror and the high-speed imager.

Preferably, it also includes a pressure pump, which is used for pumping suspended cell solution and reagents into the cell flow channel and reagent flow channel of the microfluidic chip respectively; the pressure pump is electrically connected to the system control storage module.

A nanosecond pulse laser perforation method based on spatial light modulation technology, based on the nanosecond pulse laser perforation system based on spatial light modulation technology, comprising:

S1. System debugging: establish the relationship between the strength and direction of the micro-jet, the pulsed laser energy emitted by the pulsed laser and the phase hologram loaded into the spatial light modulator;

S2. Introduction of suspended cell solution and reagent solution in the microfluidic chip: inject the suspended cell solution into the cell flow channel, and inject the exogenous reagent solution into the reagent flow channel;

S3. Cell perforation and introduction of exogenous substances: use a high-speed imager to dynamically monitor the cells in the cell flow channel of the microfluidic chip in real time; when a cell is detected in the perforation position, the system obtains the cell position according to the position of the cell and S1 The relationship between the intensity and direction of the micro-jet and the pulsed laser energy emitted by the pulsed laser and the phase hologram loaded into the spatial light modulator, the corresponding phase hologram is loaded into the spatial light modulator, and with a suitable delay Send a trigger signal to the pulsed laser, control the pulsed laser to emit a pulsed laser of corresponding energy and form a microjet in the breakdown cavity of the microfluidic chip, realize the targeted reversible perforation of a single cell, and use the directional flow caused by the microjet to The reagent solution in the reagent flow channel is injected into the perforated cells.

Preferably, the specific implementation steps of the S1 are:

(1) Inject the suspended cell solution and the reagent solution into the cell flow channel and the reagent flow channel of the microfluidic chip respectively;

(2) Adjust the pulsed laser energy, load different phase holograms in different pulsed laser energy ranges, and control the number, relative position and size of the breakdown points in the breakdown cavity; use a high-speed imager to detect the cavitation and Real-time imaging of the evolution of microfluidics;

(3) Using the image captured by the high-speed imager, record the direction of the microjet, and calculate the intensity information of the microjet; establish the relationship between the pulsed laser energy and phase hologram and the intensity and direction of the microjet.

Compared with the prior art, the present invention has the following beneficial technical effects:

The invention uses the spatial light modulator to modulate the light field of the single-pulse laser, so as to realize the single-pulse laser on the focused focal plane to form multiple tap penetrations with controllable size, quantity and position; The action will form a directional microfluid, and finally use the microfluid to realize the targeted perforation of single cells, and use the directional flow caused by the asymmetric oscillation of the vacuoles to introduce the exogenous substances into the cells through the perforation to achieve the introduction. The dose is controllable, and the entire perforation process is placed in a microfluidic channel, which can significantly increase the perforation speed and throughput. By setting up a high-speed imager, the formation process of the microfluidic fluid can be imaged clearly and with high spatial and temporal resolution, and the position and flow of cells can be monitored in real time during the perforation process.

Further, the half-wave plate is used to adjust the polarization direction of the incident laser light, and the concave lens and the first convex lens can realize beam expansion and collimation of the pulsed laser light.

Further, a first reflecting mirror and a second reflecting mirror are provided for adjusting the incident angle of the pulsed laser light incident on the spatial light modulator.

Further, a second convex lens is arranged between the white light source and the microfluidic chip for collimating the white light source.

Further, a notch plate is arranged between the dichroic mirror and the high-speed imager to prevent the pulsed laser light from entering the high-speed imager and to protect the high-speed imager.

The present invention introduces a simple and feasible microfluidic cell perforation method, which provides a new technical solution for cell membrane microsurgery. Specifically, it has the following advantages: (1) The microfluidic formation method and controllability scheme are simple, and only need to load Different phase holograms can be achieved by adjusting the appropriate pulsed laser energy input; (2) the targeted perforation of the microjet has the ability to actively introduce, which can more accurately control the introduction dose of exogenous substances, and the size of the microjet is smaller. (3) The entire perforation process is carried out in a microfluidic chip, and the perforated cells are monitored in real time, which can significantly improve the perforation success rate and perforation efficiency; (4) The operation of the operator Proficiency requirements are low.

Description of drawings

FIG. 1 is a schematic structural diagram of a system of nanosecond pulse optical perforation based on spatial light modulation technology according to the present invention.

FIG. 2 is a schematic structural diagram of the microfluidic chip used in the present invention.

FIG. 3 is a flow chart of the nanosecond pulse optical perforation method based on the spatial light modulation technology of the present invention.

Among them: pulse laser 1, beam splitting prism 2, energy meter 3, half wave plate 4, concave lens 5, second convex lens 6, first mirror 7, second mirror 8, spatial light modulator 9, line Polarizer 10, first convex lens 11, white light source 12, third convex lens 13, microfluidic chip 14, objective lens 15, dichroic mirror 16, notch plate 17, high-speed imager 18, system control and storage module 19, pressure pump 20.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, which are to explain rather than limit the present invention.

As shown in FIG. 1 , a nanosecond pulsed laser perforation system based on spatial light modulation technology includes a pulsed laser 1 , a spatial light modulator 9 , a white light source 12 and a system control storage module 19 .

A beam splitter prism 2, a half-wave plate 4, a concave lens 5, a second convex lens 6 and a first reflector 7 are sequentially arranged in the pulse laser propagation direction of the pulse laser 1; the beam splitter prism 2 has a transmission reflection splitting ratio of 9 : 1; the image focus of the concave lens 5 coincides with the object focus of the second convex lens 6; the angle between the first mirror 7 and the incident pulse light is 45° counterclockwise; the energy meter 3 is placed in the direction of the reflected light of the beam splitter prism 2.

The pulsed laser 1 , the beam splitting prism 2 , the half-wave plate 4 , the concave lens 5 , the second convex lens 6 and the first reflecting mirror 7 are on the same horizontal plane and satisfy the coaxial condition.

A third convex lens 13 , a microfluidic chip 14 , an objective lens 15 , a dichroic mirror 16 , a notch plate 17 and a high-speed imager 18 are sequentially arranged in the direction of continuous light propagation from the white light source 12 .

A second reflection mirror 8 is arranged in the propagation direction of the pulsed light reflected by the first reflection mirror 7 , and the included angle between the second reflection mirror 8 and the incident pulsed light is 85° counterclockwise.

The spatial light modulator 9 is placed on the propagation path of the pulsed light reflected by the second reflecting mirror 8 , which is kept parallel to the mirror surface of the second reflecting mirror 8 .

A linear polarizer 10 and a first convex lens 11 are placed in the propagation direction of the pulsed laser reflected by the spatial light modulator 9, wherein the linear polarizer is used to suppress the high-order diffraction part of the modulated pulsed laser, and the convex lens 11 is used to collimate the modulated pulsed laser. pulsed laser.

The dichroic mirror 16 is arranged in the direction of the pulsed laser reflected by the spatial light modulator 9, and the included angle with the pulsed laser is 45 degrees counterclockwise; the dichroic mirror 16 is used to reflect the pulsed laser into the objective lens 15 for focusing.

The microfluidic chip 14, the objective lens 15 and the dichroic mirror 16 are in the same vertical plane; the dichroic mirror 16 is a long-pass dichroic mirror with a cut-off wavelength of 567 nm.

A pressure pump 20 is also included, and the pressure pump 20 is fluidly connected to the cell flow channel and the reagent flow channel of the microfluidic chip 14 .

The output wavelength of the pulsed laser 1 is 532 nm, the pulse width is 6 ns, and the maximum single pulse energy can reach 200 mJ.

The cut-off wavelength of the notch plate 17 is 532 nm, which is used to prevent the pulsed laser light from entering the high-speed imager 18 .

The system control storage module 19 includes a processing storage part and a timing control part, and is electrically connected to the pulsed laser 1, the energy meter 3, the spatial light modulator 9, the high-speed imager 18, and the pressure pump 20, and is used between devices on the one hand. The timing control, on the other hand, is used for data recording and storage.

The numerical aperture of the objective lens 15 is 0.65 and the magnification is 40 times; on the one hand, it is used to focus the pulsed laser to form a controllable micro-jet in the microfluidic chip to perforate the cells; on the other hand, it is used as a magnifying objective lens for imaging For high-speed imaging of microfluidic processes within channels and real-time monitoring of cell flow.

The highest frame rate of the high-speed imager 18 can reach 500,000 frames, which can be used to image the formation process of the micro-fluid clearly and with high spatial and temporal resolution during the debugging process; at the same time, the position and flow of the cells can be monitored in real time during the perforation process. monitor.

The schematic diagram of the structure of the microfluidic chip is shown in Figure 2, which includes a cell flow channel and a reagent flow channel; the diameter of the cell flow channel is 40 μm, and the diameter of the reagent flow channel is 30 μm; the reagent flow channel has a breakdown cavity, The diameter of the breakdown cavity is 100 μm; the reagent flow channel and the cell flow channel are connected at the jet opening of the breakdown cavity, and the diameter of the jet opening is 10 μm; the multi-point puncture controllable jet pairs formed in the breakdown cavity and pointing to the cell flow channel pass through the jet opening The cells achieve targeted perforation;

As shown in FIG. 3 , the present invention provides a method for nanosecond pulse laser perforation based on spatial light modulation technology. The method firstly uses a spatial light modulator to modulate the incident pulsed laser light; Focusing in the microfluidic chip forms a controllable microfluidic flow, using the microfluidic flow to achieve targeted perforation of cells and controllable targeted introduction of exogenous substances, and use the fluidity of the cells in the microfluidic chip to achieve High-throughput operation of microsurgery; control of microfluidics is achieved through spatial light modulators. The specific implementation steps of the method are:

S1, system debugging. Establish the relationship between the intensity and direction of the microjet and the energy of the pulsed laser and the phase hologram loaded into the spatial light modulator;

S2. Preparation of suspension cell solution: digest the cells cultured in the petri dish with trypsin for two minutes, remove the trypsin, add an appropriate amount of medium by pipetting to make the cells detached and make the cell concentration less than 8000 cells/ml;

S3. Introduction of suspended cell solution and reagent solution in the microfluidic chip: inject the detached suspended cell solution into the cell flow channel in the microfluidic chip, and at the same time inject the exogenous substance solution that needs to be introduced into the microfluidic chip The reagent flow channel in the device, adjust the pressure pump to ensure that the solution in the cell flow channel and the reagent flow channel can be driven by the pressure pump;

S4. Collection of perforated suspension cell solution and waste liquid: the outlet of the cell flow channel of the microfluidic chip is connected to the culture dish, which is used to collect the perforated cells; the outlet of the reagent flow channel is connected to the waste liquid tank;

S5. Fixing the position of the microfluidic chip: ensure that the breakdown occurs in the breakdown cavity; turn on the pressure pump, and control the flow rate of the cell flow channel and the reagent flow channel according to the cell concentration of the suspended cell solution;

S6. Cell perforation and introduction of exogenous substances: real-time dynamic monitoring of cells in the cell flow channel of the microfluidic chip using a high-speed imager. When a cell is detected at the perforation position of the microfluidic chip, the system loads a specific phase hologram into the spatial light modulator according to the position of the cell, and sends a trigger signal to the pulsed laser 1 with an appropriate delay to control the pulse The laser emits a pulsed laser and forms a microjet in the breakdown cavity of the microfluidic chip to achieve targeted reversible perforation of a single cell, and the directional flow caused by the microjet is used to inject the dye reagent solution in the reagent flow channel into the perforated cells;

S7. Post-processing after perforation: After all cells are processed, the used microfluidic chip is safely processed, and a new microfluidic chip is replaced for next use; and used for follow-up research.

The specific implementation steps of the S1 are:

(1) Adjust the position and angle of each device of the system to ensure that the pulsed laser can be incident on the modulation target surface of the spatial light modulator at an incident angle of about 5°, and at the same time ensure that the modulated laser can be correctly incident on the objective lens;

(2) Adjust the position of the objective lens and the position of the microfluidic chip so that the pulsed laser can be correctly focused into the breakdown cavity of the microfluidic chip;

(3) injecting the suspended cell solution and the reagent solution into the cell flow channel and the reagent flow channel of the microfluidic chip respectively;

(4) Adjust the pulsed laser energy, and load different phase holograms in different pulsed laser energy ranges, so as to control the number, relative position and size of the breakdown points in the breakdown cavity; Real-time imaging of the evolution of bubbles and jets;

(5) Use the image of the dynamic evolution of the cavitation captured by the high-speed imager 18 to record information such as the direction and strength of the microjet; and establish the relationship between the pulsed laser energy, a specific phase hologram and the microjet.

The principle of the present invention is as follows: a pulsed laser 1, a beam splitter prism 2, a half-wave plate 4, a concave lens 5 and a second convex lens 6 form a pulsed laser pumping module, which is used for the output of a single-pulse laser with adjustable energy; A reflecting mirror 7, a second reflecting mirror 8, a spatial light modulator 9, a linear polarizer 10 and a first convex lens 11 form a spatial light modulation module, which is used to modulate the light field of the pulsed laser in space; the modulated pulsed laser is After being reflected by the dichroic mirror 16, it enters the objective lens 15 and is focused into the breakdown cavity of the microfluidic chip 14 to form a controllable multi-point microjet; when the target cells flow through the cell flow channel of the microfluidic chip 14 to the jet port At the same time, the system sends out instructions according to the position and flow speed of the cells, inducing the formation of micro-fluids directed at the cells and perforating the cells, so as to realize the controllable introduction of exogenous substances.

The white light source 12 , the third convex lens 13 , the objective lens 15 , the dichroic mirror 16 , the notch plate 17 and the high-speed imager 18 constitute an imaging module, which is used to perform high temporal and spatial resolution analysis of the microfluidic formation process in the microfluidic chip. Imaging, and real-time monitoring of the flow of cells during the perforation process; the third convex lens 13 is used for collimating the white light source; the objective lens 15 is used for imaging magnification; the notch plate 17 is used to prevent the reflected pulsed laser from entering high-speed imaging The camera 18 plays the role of protecting the high-speed imager 18 .

The system uses a spatial light modulator to perform spatial light field modulation on a single-pulse laser, so as to form a microfluidic microfluidic with controllable strength and direction up to the sub-micron level in a microfluidic chip. The jet is imaged, so that the relationship between the characteristics of the microjet and the energy of the pulsed laser and the holographic phase map loaded into the spatial light modulator can be established; It has good perforation efficiency and perforation speed for cell membrane microsurgery and introduction of exogenous substances, and provides a new solution for the controllability of the introduced dose, which has great applications for the application of photoperforation technology in the field of biomedicine potential, with great application value.

Claims (2)

1. a nanosecond pulse laser perforation method based on spatial light modulation technology, it is characterized in that, based on the nanosecond pulse laser perforation system based on spatial light modulation technology, the nanosecond pulse laser perforation system of described spatial light modulation technology comprises pulse a laser (1), a spatial light modulator (9), a white light source (12), an energy measurement module and a system control storage module (19); The pulsed laser light emitted by the pulsed laser (1) is incident on the spatial light modulator (9), and a linear polarizer (10) and a first convex lens (11) are sequentially arranged in the propagation direction of the pulsed laser light reflected by the spatial light modulator (9) ; A microfluidic chip (14), an objective lens (15), a dichroic mirror (16) and a high-speed imager (18) are sequentially arranged in the direction of continuous light propagation from the white light source (12); from the first convex lens (11) The transmitted pulsed laser light enters the objective lens (15) after being reflected by the dichroic mirror (16); The microfluidic chip (14) is provided with a cell flow channel and a reagent flow channel, a breakdown cavity exists in the reagent flow channel, and the breakdown cavity communicates with the cell flow channel through a jet port; the pulsed laser focused by the objective lens (15) is incident on the breakdown cavity; The energy measurement module is used to measure the pulsed laser energy emitted by the pulsed laser (1); The system control storage module (19) is electrically connected with the pulsed laser (1), the spatial light modulator (9) and the high-speed imager (18); for controlling the pulsed laser (1), the spatial light modulator (9) and the high-speed imaging Timing between the instruments (18) and triggering and controlling the pulsed laser energy of the pulsed laser (1) and the phase hologram of the spatial light modulator (9) to load, receive and store the image data of the high-speed imager (18); Methods include: S1. System debugging: establish the relationship between the strength and direction of the microjet and the pulsed laser energy emitted by the pulsed laser (1) and the phase hologram loaded into the spatial light modulator; S2. Introduction of suspended cell solution and reagent solution in the microfluidic chip: inject the suspended cell solution into the cell flow channel, and inject the exogenous reagent solution into the reagent flow channel; S3. Cell perforation and introduction of exogenous substances: use a high-speed imager to dynamically monitor the cells in the cell flow channel of the microfluidic chip in real time; when a cell is detected in the perforation position, the system obtains the cell position according to the position of the cell and S1. The relationship between the intensity and direction of the micro-jet and the pulsed laser energy emitted by the pulsed laser (1) and the phase hologram loaded into the spatial light modulator, load the corresponding phase hologram into the spatial light modulator, and use the appropriate The time delay sends a trigger signal to the pulsed laser, controls the pulsed laser to emit a pulsed laser of corresponding energy and forms a microjet in the breakdown cavity of the microfluidic chip, realizes the targeted reversible perforation of a single cell, and uses the microfluidic induced perforation. Directed flow injects the reagent solution within the reagent flow channel into the perforated cells. 2. The nanosecond pulse laser perforation method based on spatial light modulation technology according to claim 1, wherein the specific implementation steps of the S1 are: (1) Inject the suspended cell solution and the reagent solution into the cell flow channel and the reagent flow channel of the microfluidic chip respectively; (2) Adjust the pulsed laser energy, load different phase holograms in different pulsed laser energy ranges, and control the number, relative position and size of the breakdown points in the breakdown cavity; use a high-speed imager (18) to Real-time imaging of the evolution of cavitation and microfluidics; (3) Using the image captured by the high-speed imager, record the direction of the microjet, and calculate the intensity information of the microjet; establish the relationship between the pulsed laser energy and phase hologram and the intensity and direction of the microjet.

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