CN112779156A - Nanosecond pulse laser perforation system and method based on spatial light modulation technology - Google Patents
Nanosecond pulse laser perforation system and method based on spatial light modulation technology Download PDFInfo
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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 flow 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 the high-speed micro-jet flow.
Description
Technical Field
The invention relates to the field of liquid photoinduced breakdown and cell photo-perforation, in particular to a nanosecond pulse laser perforation system and method based on a spatial light modulation technology.
Background
The cell membrane micro-operation has great scientific significance for deeply analyzing the life process and the pathogenesis of diseases, for example, exogenous biological macromolecules such as protein, DNA, RNA, siRNA and the like are introduced into living cells to intervene in the functional expression of the protein and even kill or reverse diseased cells in a targeted manner in the micro field. In this process, how to achieve recoverable cell perforation without affecting cell viability is a critical step.
In the process of introducing exogenous substances into cells, the ability of active introduction is the key to improve the introduction efficiency, the success rate and the controllable dosage, and meanwhile, the realization of the targeting and high-flux operation of the cells is also a very important requirement. Traditional cell membrane microsurgical methods include capillary microinjection, electroporation, sonoporation, and viral or chemical delivery, among others. In addition, with the development of laser technology, a method of photo-perforation with non-invasive, non-contact and targeting operation is applied to the field of cell membrane microsurgery. On the one hand, targeted perforation of individual cells can be achieved with tightly focused laser light, and on the other hand, high-throughput perforation of cell populations can be achieved by means of strong absorption of laser light by some metal nanomaterials. However, both methods utilize passive diffusion of exogenous substances into cells after perforation, and thus have the disadvantages of uncontrollable introduced dose and poor reproducibility. In view of the above, a method for performing targeted perforation of single cells by using micro-jet formed by multi-point breakdown induced by a double-pulse laser in water is proposed. The high-speed movement process of the micro jet drives the surrounding medium to move, so that the application potential of actively targeted introduced energy and controllable dosage exists. However, the two pulse lasers for inducing jet formation to realize cell photo-perforation have the defects of high manufacturing cost, complex operation and the like.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a nanosecond pulse laser perforation system and a nanosecond pulse laser perforation method based on a spatial light modulation technology, which utilize single-pulse induced controllable jet flow to realize cell targeting and high-flux perforation in a microfluidic chip, and have the advantages of simple system, easy operation and low manufacturing cost.
The invention is realized by the following technical scheme:
a nanosecond pulse laser perforation system based on a spatial light modulation technology comprises a pulse laser, a spatial light modulator, a white light source, an energy measurement module and a system control storage module;
pulse laser emitted by a pulse laser is incident on a spatial light modulator, and a wired polaroid and a first convex lens are sequentially arranged in the propagation direction of the pulse laser reflected by the spatial light modulator;
the micro-fluidic chip, the objective lens, the dichroic mirror and the high-speed imager are sequentially arranged in the propagation direction of continuous light emitted by the white light source; the pulse laser transmitted by the first convex lens enters the objective lens after being reflected by the dichroic mirror;
the micro-fluidic chip is provided with a cell flow channel and a reagent flow channel, wherein a breakdown cavity is arranged in the reagent flow channel and is communicated with the cell flow channel through a jet orifice; the pulse laser focused by the objective lens is incident into the breakdown cavity;
the energy measuring module is used for measuring the pulse laser energy emitted by the pulse laser;
the system control storage module is electrically connected with the pulse laser, the spatial light modulator and the high-speed imager; the device is used for controlling the time sequence and triggering among the pulse laser, the spatial light modulator and the high-speed imager, controlling the pulse laser energy of the pulse laser and the phase hologram loading of the spatial light modulator, and receiving and storing the image data of the high-speed imager.
Preferably, the energy measurement module comprises a beam splitter prism and an energy meter; the beam splitter prism is arranged in the pulse laser transmission direction of the pulse laser, the energy meter is arranged in the pulse laser transmission direction reflected by the beam splitter prism, and the energy meter is electrically connected with the system control storage module.
Preferably, the pulse laser has a half-wave plate, a concave lens, and a second convex lens arranged in this order in the pulse laser propagation direction, and the pulse laser transmitted through the second convex lens is incident on the spatial light modulator.
Preferably, the pulse laser has a first reflecting mirror disposed in a pulse laser propagation direction, a second reflecting mirror disposed in a pulse light propagation direction after being reflected by the first reflecting mirror, and the spatial light modulator is disposed in the pulse light propagation direction after being reflected by the second reflecting mirror.
Furthermore, the included angle between the first reflector and the pulse laser propagation direction of the pulse laser is 45 degrees anticlockwise, and the included angle between the second reflector and the pulse laser propagation direction reflected by the first reflector is 85 degrees anticlockwise; the spatial light modulator mirror is parallel to the second mirror.
Preferably, a convex lens is arranged between the white light source and the microfluidic chip.
Preferably, a wave trap plate is arranged between the dichroic mirror and the high-speed imager.
Preferably, the microfluidic chip further comprises a pressure pump, wherein the pressure pump is used for pumping the suspension cell solution and the reagent into the cell flow channel and the reagent flow channel of the microfluidic chip respectively; the pressure pump is electrically connected with the system control storage module.
A nanosecond pulse laser perforation method based on a spatial light modulation technology comprises the following steps:
s1, debugging the system: establishing a relation between the strength and the direction of the micro-jet flow, the pulse laser energy emitted by a pulse laser and the phase hologram loaded in the spatial light modulator;
s2, introducing a suspension cell solution and a reagent solution in the microfluidic chip: injecting a suspension cell solution into the cell flow channel, and injecting an exogenous reagent solution into the reagent flow channel;
s3, cell perforation and introduction of exogenous substances: the high-speed imager is used for dynamically monitoring the cells in the cell flow channel of the microfluidic chip in real time; when the situation that a cell is positioned at a perforation position is monitored, the system loads a corresponding phase hologram into the spatial light modulator according to the position of the cell and the relationship between the strength and the direction of the micro-jet obtained by S1 and the energy of the pulse laser emitted by the pulse laser and the phase hologram loaded into the spatial light modulator, sends a trigger signal to the pulse laser in proper time delay, controls the pulse laser to emit pulse laser with corresponding energy and forms micro-jet in a breakdown cavity of the microfluidic chip, realizes targeted reversible perforation of a single cell, and injects a reagent solution in a reagent flow channel into the perforation cell by using directional flow caused by the micro-jet.
Preferably, the specific implementation steps of S1 are as follows:
(1) respectively injecting a suspension cell solution and a reagent solution into a cell flow channel and a reagent flow channel of the microfluidic chip;
(2) adjusting the pulse laser energy, loading different phase holograms in different pulse laser energy ranges, and controlling the number, relative position and size of breakdown points in the breakdown cavity; real-time imaging is carried out on the evolution process of the cavitation bubbles and the micro jet flow by using a high-speed imager;
(3) recording the direction of the micro-jet flow by using an image shot by a high-speed imager, and calculating the strength information of the micro-jet flow; and establishing the relationship between the pulse laser energy and the phase hologram and the strength and direction of the micro-jet.
Compared with the prior art, the invention has the following beneficial technical effects:
the spatial light modulator is used for carrying out light field modulation on the single-pulse laser, so that multi-point breakdown of the single-pulse laser with controllable size, quantity and position on a focused focal plane is realized; the interaction of the multi-point breakdown vacuole in the oscillation process can form directional microjet, the microjet is finally utilized to realize the targeted perforation of single cells, exogenous substances are introduced into the cells through the perforation by means of the directional flow caused by the asymmetric oscillation of the vacuole, the controllability of the introduction dosage is realized, the whole perforation process is arranged in a microfluidic channel, and the perforation speed and the perforation flux can be obviously increased. The high-speed imager can be used for imaging the formation process of the micro-jet with clear and high spatial and temporal resolution, and meanwhile, the position and the flow of the cells can be monitored in real time in the perforation process.
Furthermore, the half wave plate is used for adjusting the polarization direction of the incident laser, and the concave lens and the first convex lens can realize beam expansion and collimation of the pulse laser.
Further, a first reflecting mirror and a second reflecting mirror are provided for adjusting an incident angle of the pulse laser to the spatial light modulator.
Further, a second convex lens is arranged between the white light source and the micro-fluidic chip and used for collimating the white light source.
Furthermore, a wave trap plate is arranged between the dichroic mirror and the high-speed imager and used for preventing the pulse laser from entering the high-speed imager and playing a role in protecting the high-speed imager.
The invention introduces a simple and feasible micro-jet cell perforation way, provides a new technical scheme for the cell membrane micro-operation, and has the following advantages: (1) the forming mode and the controllability scheme of the micro-jet are simple, and the micro-jet can be realized only by loading different phase holograms and adjusting proper pulse laser energy input; (2) the targeted perforation of the microjet has active introduction capability, can more accurately control the introduction dosage of exogenous substances, has smaller microjet size and causes more controllable cell damage; (3) the whole perforation process is carried out in the microfluidic chip, and the perforated cells are monitored in real time, so that the perforation success rate and the perforation efficiency can be obviously improved; (4) the requirement on the operation skill of an operator is low.
Drawings
Fig. 1 is a schematic structural diagram of a nanosecond pulse light perforation system based on a spatial light modulation technology.
Fig. 2 is a schematic structural diagram of a microfluidic chip used in the present invention.
Fig. 3 is a flow chart of a nanosecond pulse light perforation method based on a spatial light modulation technology.
Wherein: the device comprises a pulse laser 1, a beam splitting prism 2, an energy meter 3, a half-wave plate 4, a concave lens 5, a second convex lens 6, a first reflecting mirror 7, a second reflecting mirror 8, a spatial light modulator 9, a linear polarizer 10, a second convex lens 11, a white light source 12, a third convex lens 13, a microfluidic chip 14, an objective lens 15, a dichroic mirror 16, a notch film 17, a high-speed camera 18, a system control and storage module 19 and a pressure pump 20.
Detailed Description
The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the 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.
The pulse laser 1 is sequentially provided with a beam splitter prism 2, a half-wave plate 4, a concave lens 5, a second convex lens 6 and a first reflector 7 in the pulse laser propagation direction; the transmission-reflection splitting ratio of the beam splitter prism 2 is 9: 1; the image focus of the concave lens 5 coincides with the object focus of the second convex lens 6; the included angle between the first reflector 7 and the incident pulse light is 45 degrees anticlockwise; the energy meter 3 is placed in the direction of the reflected light of the beam splitting prism 2.
The pulse laser 1, the beam splitting prism 2, the half wave plate 4, the concave lens 5, the second convex lens 6 and the first reflector 7 are positioned on the same horizontal plane and meet the coaxial condition.
The third convex lens 13, the micro-fluidic chip 14, the objective lens 15, the dichroic mirror 16, the notch film 17 and the high-speed imager 18 are sequentially arranged in the propagation direction of the continuous light emitted by the white light source 12.
A second reflecting mirror 8 is arranged in the propagation direction of the pulse light reflected by the first reflecting mirror 7, and the included angle between the second reflecting mirror 8 and the incident pulse light is 85 degrees anticlockwise.
The spatial light modulator 9 is disposed on a propagation path of the pulse light reflected by the second mirror 8, which is mirror-parallel to the second mirror 8.
The linear polarizer 10 and the first convex lens 11 are disposed in the propagation direction of the pulse laser light reflected by the spatial light modulator 9, wherein the linear polarizer is used for suppressing higher order diffraction parts in the modulated pulse laser light, and the convex lens 11 is used for collimating the modulated pulse laser light.
The dichroic mirror 16 is arranged in the direction of the pulse laser reflected by the spatial light modulator 9, and forms an included angle of 45 degrees with the direction of the pulse laser anticlockwise; the dichroic mirror 16 is used to reflect the pulsed laser light 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; dichroic mirror 16 is a long-wave pass dichroic mirror with a cut-off wavelength of 567 nm.
The device also comprises a pressure pump 20, and the pressure pump 20 is connected with the cell flow channel and the reagent flow channel of the microfluidic chip 14.
The output wavelength of the pulse laser 1 is 532nm, the pulse width is 6ns, and the maximum single pulse energy can reach 200 mJ.
The cut-off wavelength of the notch plate 17 is 532nm, and the notch plate is used for preventing pulse laser from entering the high-speed imager 18.
The system control storage module 19 comprises a processing storage part and a timing control part, and is electrically connected with the pulse laser 1, the energy meter 3, the spatial light modulator 9, the high-speed imager 18 and the pressure pump 20, and is used for timing control among devices on one hand and data recording and storage on the other hand.
The numerical aperture of the objective lens 15 is 0.65, and the magnification is 40 times; the micro-fluidic chip is used for focusing pulse laser to form controllable micro-jet flow in the micro-fluidic chip to perforate cells; on the other hand, the micro-fluidic chip is used as an imaging magnifying objective lens for carrying out high-speed imaging on the micro-fluidic process in the channel and carrying out real-time monitoring on the cell flow.
The highest frame frequency of the high-speed imager 18 can reach 50 ten thousand frames, and the high-speed imager can be used for imaging with clear high space-time resolution in the process of forming the micro jet flow in the debugging process; meanwhile, the position and the flow of the cells can be monitored in real time during the perforation process.
The schematic structural diagram of the microfluidic chip is shown in fig. 2, and the microfluidic chip comprises 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 is provided with a breakdown cavity, and the diameter of the breakdown cavity is 100 mu m; the reagent flow channel is communicated with the cell flow channel at the jet orifice of the puncture cavity, and the diameter of the jet orifice is 10 mu m; the multipoint breakdown controllable jet flow which is formed in the breakdown cavity and points to the cell flow channel realizes the targeted perforation of the cells passing through the jet orifice;
as shown in fig. 3, the present invention provides a method for nanosecond pulsed laser perforation based on spatial light modulation technology, which firstly modulates the incident pulsed laser by using a spatial light modulator; the modulated pulse laser is focused in the micro-fluidic chip by using the objective lens to form controllable micro-jet, the micro-jet is used for realizing the targeted perforation of cells and the controllable targeted introduction of exogenous substances, and the fluidity of the cells in the micro-fluidic chip is used for realizing the high-flux operation of the micro-surgery; the control of the micro-jets is achieved by means of a spatial light modulator. The method comprises the following specific implementation steps:
and S1, debugging the system. Establishing the relationship between the strength and the direction of the micro-jet flow, the pulse laser energy and the phase hologram loaded in the spatial light modulator;
s2, preparing a suspension cell solution: digesting the cultured cells in a culture dish for two minutes by using pancreatin, removing the pancreatin, adding a proper amount of culture medium, blowing and beating the cells to remove the walls of the cells, and enabling the cell concentration to be less than 8000 cells/ml;
s3, introducing a suspension cell solution and a reagent solution in the microfluidic chip: injecting the suspended cell solution after the wall is removed into a cell flow channel in the microfluidic chip, simultaneously injecting an exogenous substance solution to be introduced into a reagent flow channel in the microfluidic chip, and debugging a pressure pump to ensure that the solution in the cell flow channel and the solution in the reagent flow channel can be driven by the pressure pump;
s4, collection of the perforated suspension cell solution and waste liquid: the cell flow channel outlet of the microfluidic chip is connected with the culture dish and used for collecting the perforated cells; the outlet of the reagent flow channel is connected with a waste liquid tank;
s5, fixing the position of the microfluidic chip: ensuring that breakdown occurs within the breakdown cavity; opening the pressure pump and controlling the flow rates of the cell flow channel and the reagent flow channel according to the cell concentration of the suspension cell solution;
s6, cell perforation and introduction of exogenous substances: and (3) carrying out real-time dynamic monitoring on cells in the cell flow channel of the microfluidic chip by using a high-speed imager. When cells are monitored to be positioned at the perforating position of the microfluidic chip, the system loads a specific phase hologram into the spatial light modulator according to the position of the cells, sends a trigger signal to the pulse laser 1 in proper time delay, controls the pulse laser to send pulse laser and forms a micro-jet in a breakdown cavity of the microfluidic chip, realizes targeted reversible perforation of single cells, and injects dye reagent solution in a reagent flow channel into the perforated cells by utilizing directional flow caused by the micro-jet;
s7, processing after punching: after all cells are treated, safely treating the used microfluidic chip, and replacing the used microfluidic chip with a new microfluidic chip for the next use; the collected punch suspension cell solution was aliquoted, cultured and used for subsequent studies.
The specific implementation steps of S1 are as follows:
(1) the position and the angle of each device of the system are adjusted to ensure that the pulse laser can be incident to the modulation target surface of the spatial light modulator at an incidence angle of about 5 degrees and ensure that the modulated laser can be correctly incident to the objective lens;
(2) adjusting the position of the objective lens and the position of the micro-fluidic chip to ensure that the pulse laser energy can be correctly focused into a breakdown cavity of the micro-fluidic chip;
(3) respectively injecting a suspension cell solution and a reagent solution into a cell flow channel and a reagent flow channel of the microfluidic chip;
(4) adjusting the pulse laser energy, and loading different phase holograms in different pulse laser energy ranges so as to control the number, relative positions and sizes of breakdown points in the breakdown cavity; imaging the evolution process of cavitation and jet flow in real time by using a high-speed imager 18;
(5) recording the direction, strength and other information of the micro-jet flow by utilizing a cavitation dynamic evolution image shot by the high-speed imager 18; and establishes a relationship between the pulsed laser energy, the particular phase hologram, and the microfluidic stream.
The principle of the invention is as follows: the pulse laser 1, the beam splitter prism 2, the half wave plate 4, the concave lens 5 and the second convex lens 6 form a pulse laser pumping module which is used for outputting the energy-adjustable single pulse laser; the first reflector 7, the second reflector 8, the spatial light modulator 9, the linear polarizer 10 and the second convex lens 11 form a spatial light modulation module for performing spatial light field modulation on the pulse laser; the modulated pulse laser enters an objective lens 15 after being reflected by a dichroic mirror 16, and is focused in a breakdown cavity of a microfluidic chip 14 to form controllable multi-point micro-jet; when the target cell flows to the jet orifice in the cell flow channel of the microfluidic chip 14, the system sends out an instruction according to the position and the flow speed of the cell, so as to induce the formation of the microjet pointing to the cell and perforate the cell, thereby realizing the controllable introduction of the exogenous substance.
The white light source 12, the third convex lens 13, the objective lens 15, the dichroic mirror 16, the notch film 17 and the high-speed imager 18 form an imaging module for imaging with high time-space resolution in the microfluidic chip microfluidic forming process and monitoring the cell flow in the perforation process in real time; the third convex lens 13 is used for collimating the white light source; the objective lens 15 is used for magnification of imaging; the wave trap 17 is used to prevent the reflected pulse laser from entering the high-speed imager 18, and plays a role in protecting the high-speed imager 18.
The system utilizes a spatial light modulator to perform spatial light field modulation on single-pulse laser, so that microjet with controllable strength and direction and capable of reaching submicron level is formed in a microfluidic chip, and a high-speed imager is utilized to image the microjet in the process, so that the relationship between the characteristics of the microjet and the energy of pulse laser and a holographic phase diagram loaded into the spatial light modulator can be established; on the basis, cell membrane micro-surgery and introduction of exogenous substances are carried out on cells in the micro-fluidic chip by using controllable micro-jet, so that the micro-fluidic chip has good perforation efficiency and perforation speed, and simultaneously provides a new scheme for controllability of introduction dosage, has great application potential for application of a photo-perforation technology in the field of biomedicine, and has great application value.
Claims (10)
1. A nanosecond pulse laser perforation system based on a spatial light modulation technology is characterized by comprising a pulse laser (1), a spatial light modulator (9), a white light source (12), an energy measurement module and a system control storage module (19);
pulse laser emitted by the pulse laser (1) is incident on the spatial light modulator (9), and a linear polaroid (10) and a first convex lens (11) are sequentially arranged in the propagation direction of the pulse laser reflected by the spatial light modulator (9);
a micro-fluidic chip (14), an objective lens (15), a dichroic mirror (16) and a high-speed imager (18) are sequentially arranged in the propagation direction of continuous light emitted by the white light source (12); the pulse laser transmitted by the first convex lens (11) is reflected by a dichroic mirror (16) and enters an objective lens (15);
the micro-fluidic chip (14) is provided with a cell flow channel and a reagent flow channel, a breakdown cavity is arranged in the reagent flow channel, and the breakdown cavity is communicated with the cell flow channel through a jet orifice; the pulse laser focused by the objective lens (15) is incident into the breakdown cavity;
the energy measuring module is used for measuring the pulse laser energy emitted by the pulse laser (1);
the system control storage module (19) is electrically connected with the pulse laser (1), the spatial light modulator (9) and the high-speed imager (18); the device is used for controlling the timing and triggering among the pulse laser (1), the spatial light modulator (9) and the high-speed imager (18), controlling the pulse laser energy of the pulse laser (1) and the phase hologram loading of the spatial light modulator (9), and receiving and storing the image data of the high-speed imager (18).
2. The nanosecond pulsed laser perforation system based on spatial light modulation technique according to claim 1, wherein the energy measurement module comprises a beam splitting prism (2) and an energy meter (3); the beam splitting prism (2) is arranged in the pulse laser propagation direction of the pulse laser (1), the energy meter (3) is arranged in the pulse laser propagation direction reflected by the beam splitting prism (2), and the energy meter (3) is electrically connected with the system control storage module (19).
3. The nanosecond pulsed laser perforation system based on spatial light modulation technology according to claim 1, wherein a half wave plate (4), a concave lens (5) and a second convex lens (6) are sequentially arranged in a pulse laser propagation direction of the pulse laser (1), and the pulse laser transmitted from the second convex lens (6) is incident on the spatial light modulator (9).
4. The nanosecond pulsed laser perforation system based on spatial light modulation technology according to claim 1, wherein a first mirror (7) is disposed in a pulse laser propagation direction of the pulsed laser (1), a second mirror (8) is disposed in the pulse light propagation direction after being reflected by the first mirror (7), and the spatial light modulator (9) is disposed in the pulse light propagation direction after being reflected by the second mirror (8).
5. The nanosecond pulsed laser perforation system based on the spatial light modulation technology according to claim 4, wherein the angle between the first mirror (7) and the propagation direction of the pulsed laser (1) is 45 ° counterclockwise, and the angle between the second mirror (8) and the propagation direction of the pulsed light reflected by the first mirror (7) is 85 ° counterclockwise; the mirror surface of the spatial light modulator (9) is parallel to the mirror surface of the second reflector (8).
6. The nanosecond pulsed laser perforation system based on spatial light modulation technology according to claim 1, wherein a convex lens is arranged between the white light source (12) and the microfluidic chip (14).
7. Nanosecond pulsed laser perforation system based on spatial light modulation technique according to claim 1, characterized in that a wave trap plate (17) is arranged between the dichroic mirror (16) and the high-speed imager (18).
8. The nanosecond pulsed laser perforation system based on spatial light modulation technology according to claim 1, further comprising a pressure pump (20), wherein the pressure pump (20) is used for pumping suspended cell solution and reagent into the cell flow channel and the reagent flow channel of the microfluidic chip (14), respectively; the pressure pump (20) is electrically connected with the system control storage module (19).
9. A nanosecond pulsed laser perforation method based on spatial light modulation technology, characterized in that, the nanosecond pulsed laser perforation system based on spatial light modulation technology according to any claim 1-8, comprises:
s1, debugging the system: establishing a relation between the strength and the direction of the micro-jet flow and the energy of the pulse laser emitted by the pulse laser (1) and the phase hologram loaded in the spatial light modulator;
s2, introducing a suspension cell solution and a reagent solution in the microfluidic chip: injecting a suspension cell solution into the cell flow channel, and injecting an exogenous reagent solution into the reagent flow channel;
s3, cell perforation and introduction of exogenous substances: the high-speed imager is used for dynamically monitoring the cells in the cell flow channel of the microfluidic chip in real time; when the situation that a cell is positioned at a perforation position is monitored, the system loads a corresponding phase hologram into the spatial light modulator according to the position of the cell and the relationship between the strength and the direction of the micro-jet obtained by S1 and the energy of the pulse laser emitted by the pulse laser (1) and the phase hologram loaded into the spatial light modulator, sends a trigger signal to the pulse laser in proper time delay, controls the pulse laser to emit pulse laser with corresponding energy and forms micro-jet in a breakdown cavity of the microfluidic chip, realizes targeted reversible perforation of a single cell, and injects a reagent solution in a reagent flow channel into the perforated cell by using directional flow caused by the micro-jet.
10. The nanosecond pulsed laser perforation method based on spatial light modulation technology according to claim 9, wherein the specific implementation steps of S1 are as follows:
(1) respectively injecting a suspension cell solution and a reagent solution into a cell flow channel and a reagent flow channel of the microfluidic chip;
(2) adjusting the pulse laser energy, loading different phase holograms in different pulse laser energy ranges, and controlling the number, relative position and size of breakdown points in the breakdown cavity; imaging the evolution process of cavitation bubbles and micro-jet in real time by using a high-speed imager (18);
(3) recording the direction of the micro-jet flow by using an image shot by a high-speed imager, and calculating the strength information of the micro-jet flow; and establishing the relationship between the pulse laser energy and the phase hologram and the strength and direction of the micro-jet.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114371621A (en) * | 2021-12-28 | 2022-04-19 | 复旦大学 | Automatic control device and method for light-operated microfluidic platform |
| CN114486687A (en) * | 2022-01-27 | 2022-05-13 | 北京理工大学 | Multi-scale continuous observation feedback method and device for femtosecond laser processing cells |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102021212505A1 (en) | 2021-11-08 | 2023-05-11 | Robert Bosch Gesellschaft mit beschränkter Haftung | Optical device for exciting a sample, analyzer and method for exciting a sample |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6083583A (en) * | 1983-10-13 | 1985-05-11 | Rikagaku Kenkyusho | Perforation apparatus of live cell with laser |
| CN1653185A (en) * | 2002-05-23 | 2005-08-10 | 卡斯滕·柯尼希 | Method for transferring molecules in vital cells by means of laser beams and arrangement for carrying out said method |
| US20140073027A1 (en) * | 2011-06-21 | 2014-03-13 | University Court Of The University Of St Andrews | Microfluidic photoporation |
| US20140256047A1 (en) * | 2010-07-06 | 2014-09-11 | The Ohio State University | Dose and location controlled drug/gene/particle delivery to individual cells by nanoelectroporation |
| DE102015101838A1 (en) * | 2015-02-09 | 2016-08-11 | Jenlab Gmbh | Method and device for reprogramming living cells |
| CN108531396A (en) * | 2018-03-30 | 2018-09-14 | 东南大学 | A kind of micro-fluidic chip for cell transfecting |
| US20190017071A1 (en) * | 2015-12-30 | 2019-01-17 | Fondazione Istituto Italiano Di Tecnologia | Method for the optoinjection of exogenous material into a biological cell |
| US20190071695A1 (en) * | 2017-08-28 | 2019-03-07 | Cellino Biotech, Inc. | Microfluidic laser-activated intracellular delivery systems and methods |
| CN110536634A (en) * | 2017-02-19 | 2019-12-03 | 索里顿有限责任公司 | Selective laser induced optical breakdown in Biomedia |
| CN110618133A (en) * | 2019-10-17 | 2019-12-27 | 江苏卓易信息科技股份有限公司 | Device and method for detecting dynamics of symmetric vacuoles in transparent liquid environment |
| CN110736749A (en) * | 2019-10-17 | 2020-01-31 | 淮阴工学院 | Multipoint detection device and method for millimeter-scale vacuoles on fixed wall surface |
| CN112649595A (en) * | 2020-11-11 | 2021-04-13 | 西安交通大学 | System and method based on single-pulse laser-induced photoinduced breakdown controllable jet flow |
-
2020
- 2020-12-29 CN CN202011612112.4A patent/CN112779156B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6083583A (en) * | 1983-10-13 | 1985-05-11 | Rikagaku Kenkyusho | Perforation apparatus of live cell with laser |
| CN1653185A (en) * | 2002-05-23 | 2005-08-10 | 卡斯滕·柯尼希 | Method for transferring molecules in vital cells by means of laser beams and arrangement for carrying out said method |
| US20140256047A1 (en) * | 2010-07-06 | 2014-09-11 | The Ohio State University | Dose and location controlled drug/gene/particle delivery to individual cells by nanoelectroporation |
| US20140073027A1 (en) * | 2011-06-21 | 2014-03-13 | University Court Of The University Of St Andrews | Microfluidic photoporation |
| DE102015101838A1 (en) * | 2015-02-09 | 2016-08-11 | Jenlab Gmbh | Method and device for reprogramming living cells |
| US20190017071A1 (en) * | 2015-12-30 | 2019-01-17 | Fondazione Istituto Italiano Di Tecnologia | Method for the optoinjection of exogenous material into a biological cell |
| CN110536634A (en) * | 2017-02-19 | 2019-12-03 | 索里顿有限责任公司 | Selective laser induced optical breakdown in Biomedia |
| US20190071695A1 (en) * | 2017-08-28 | 2019-03-07 | Cellino Biotech, Inc. | Microfluidic laser-activated intracellular delivery systems and methods |
| CN108531396A (en) * | 2018-03-30 | 2018-09-14 | 东南大学 | A kind of micro-fluidic chip for cell transfecting |
| CN110618133A (en) * | 2019-10-17 | 2019-12-27 | 江苏卓易信息科技股份有限公司 | Device and method for detecting dynamics of symmetric vacuoles in transparent liquid environment |
| CN110736749A (en) * | 2019-10-17 | 2020-01-31 | 淮阴工学院 | Multipoint detection device and method for millimeter-scale vacuoles on fixed wall surface |
| CN112649595A (en) * | 2020-11-11 | 2021-04-13 | 西安交通大学 | System and method based on single-pulse laser-induced photoinduced breakdown controllable jet flow |
Non-Patent Citations (4)
| Title |
|---|
| ING-HSIANGWU等: "Pulsed laser triggered high speed microfluidic fluorescence activated cell sorter", 《LAB ON A CHIP》 * |
| KANG YUAN LIM等: "Nonspherical laser-induced cavitation bubbles", 《PHYSICAL REVIEW E》 * |
| SIQI WANG等: "Quantitative Evaluation and Optimization of Photothermal Bubble Generation around Overheated Nanoparticles Excited by Pulsed Lasers", 《THE JOURNAL OF PHYSICAL CHEMISTRY C》 * |
| 付磊: "纳秒脉冲激光诱导的水中双空泡振荡研究", 《中国激光》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114371621A (en) * | 2021-12-28 | 2022-04-19 | 复旦大学 | Automatic control device and method for light-operated microfluidic platform |
| CN114486687A (en) * | 2022-01-27 | 2022-05-13 | 北京理工大学 | Multi-scale continuous observation feedback method and device for femtosecond laser processing cells |
| CN114486687B (en) * | 2022-01-27 | 2023-09-15 | 北京理工大学 | Multi-scale continuous observation feedback method and device for femtosecond laser processing cells |
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