CN116497017A

CN116497017A - Acoustic perforation cell laser ablation purification method and device and culture imaging dish

Info

Publication number: CN116497017A
Application number: CN202310469408.2A
Authority: CN
Inventors: 胡亚欣; 张咪
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2023-04-24
Filing date: 2023-04-24
Publication date: 2023-07-28

Abstract

A method and a device for purifying sonoporation cells by laser ablation and a culture imaging dish, wherein the method comprises the following steps: carrying out ultrasonic treatment on the isolated cells in the microscopic field by adopting ultrasonic waves with preset parameters, wherein the isolated cells and physiological salt solution containing microbubbles are arranged in the microscopic field, so that the microbubbles are cavitated and the cells generate different degrees of acoustic perforation and micro damage; collecting unmarked microscopic images of the isolated cells, identifying effective sonoporated cells and ineffective sonoporated cells in the isolated cells according to the unmarked microscopic images and a machine learning algorithm, and outputting the position information of the ineffective sonoporated cells in a culture imaging dish; according to the position information, performing laser ablation on each ineffective sound-induced perforation cell; and re-collecting microscopic images of the isolated cells, determining that all the ineffective sonoporation cells are ablated, and completing the population purification of the effective sonoporation cells. The present application may enable purification of a population of effectively sonoporated cells.

Description

Acoustic perforation cell laser ablation purification method and device and culture imaging dish

Technical Field

The invention relates to the technical field of cell transfection, in particular to a laser ablation purification method and device for sound-induced perforation cells and a culture imaging dish.

Background

Microbubble-mediated sonoporation refers to micron-sized damage to nearby cell membranes caused by vibration and explosion of microbubbles under the drive of ultrasonic periodic positive and negative sound pressure, and the micro-damage to cells caused by the sonoporation can promote the transmission of gene drugs to cells. When ultrasound is applied to cells and microbubbles, three results are produced: no perforation, repairable sonoporation, and non-repairable sonoporation. Wherein repairable sonoporation can facilitate delivery of intracellular gene drugs without causing death of the cell, so called active gene drug delivery, such cells are defined as active sonoporated cells; while non-repairable sonoporation eventually leads to cell death due to the inability of the integrity of the cell membrane to recover, it is collectively referred to as ineffective gene drug delivery with no perforation, and these cells are defined as ineffective sonoporated cells.

Therefore, after transfection of microvesicle-type cells, population purification of the effective sonoporated cells is required, and the current purification mode is relatively lacking.

Disclosure of Invention

The invention mainly solves the technical problem that a method for purifying the effective sonoporation cells is lacked.

According to a first aspect, in one embodiment there is provided a method of sonoporation cell laser ablation purification, comprising:

acquiring microscopic images of the isolated cells in the culture imaging dish, identifying effective sound-induced perforated cells and ineffective sound-induced perforated cells in the isolated cells according to the microscopic images, and outputting position information of the ineffective sound-induced perforated cells in the culture imaging dish; in vitro cells and physiological salt solution containing microbubbles are added into a microscopic field of the culture imaging dish, and the culture imaging dish is subjected to ultrasonic treatment, so that the microbubbles are cavitated and the cells generate different degrees of acoustic perforation and micro damage;

according to the position information, performing laser ablation on each ineffective sound-induced perforation cell;

and re-collecting microscopic images of the isolated cells, determining that all the ineffective sonoporation cells are ablated, and completing the population purification of the effective sonoporation cells.

According to a second aspect, in one embodiment there is provided an sonoporation cell laser ablation purification apparatus comprising:

an ultrasound transmission system configured to transmit ultrasound waves to a preset ultrasound region;

the three-dimensional movement system is configured to load the culture imaging dish and drive the culture imaging dish to perform three-dimensional movement; the culture imaging dish is added with an isolated cell and a physiological saline solution containing microbubbles;

The image acquisition system is configured to acquire images of isolated cells of the culture imaging dish and output microscopic images;

a laser ablation system configured to emit laser light at a preset irradiation region;

the processing module is configured to control the ultrasonic emission system to carry out ultrasonic processing on the isolated cells in the culture imaging dish by adopting ultrasonic waves with preset parameters; controlling an image acquisition system to acquire images of the isolated cells; identifying effective sonoporation cells and ineffective sonoporation cells in the isolated cells according to the microscopic image, and outputting the position information of the ineffective sonoporation cells in the culture imaging dish; and controlling a laser ablation system to perform laser ablation on each ineffective sound-induced perforation cell according to the position information.

In a third aspect, an embodiment provides a culture imaging dish comprising:

the top cover is provided with a transducer placing hole which is used for installing and fixing the ultrasonic transducer;

the bottom box is matched with the top cover, and is provided with a culture chamber which is provided with a liquid injection hole;

the culture imaging dish is provided with a first use state, a second use state and a third use state;

in the first use state, the top cover is not assembled on the bottom box, the bottom box is arranged in a positive way, the culture chamber is injected with physiological saline solution containing isolated cells, and the isolated cells are adhered to the bottom of the culture chamber after being cultured for a preset period of time;

In the second use state, the top cover is not assembled on the bottom box, after the first use state, the culture chamber is injected with physiological saline solution containing microbubbles, and the bottom box is inverted, so that the microbubbles float to the bottom of the culture chamber and fully contact with the isolated cells;

in the third use state, after the first use state, the bottom box is arranged in the normal position, the top cover is assembled on the bottom box, the ultrasonic transducer is arranged on the transducer placing hole, and the acting area of the ultrasonic transducer covers the culture cavity.

According to the sonoporation cell laser ablation purification method and device and the culture imaging dish, the effective sonoporation cells and the ineffective sonoporation cells are classified through image recognition, corresponding position information of the ineffective sonoporation cells in the culture imaging dish is output, the ineffective sonoporation cells are precisely ablated through a laser ablation mode, and the population purification of the effective sonoporation cells is achieved.

Drawings

Fig. 1 is a schematic structural diagram (a) of a laser ablation and purification apparatus according to an embodiment of the present disclosure;

fig. 2 is a schematic structural diagram (ii) of a laser ablation and purification apparatus according to an embodiment of the present disclosure;

FIG. 3 is a schematic structural diagram of a laser ablation system and an image acquisition system according to an embodiment of the present disclosure;

Fig. 4 is a schematic structural diagram (ii) of a laser ablation system and an image acquisition system according to an embodiment of the present disclosure;

FIG. 5 is a schematic diagram of a processing module according to an embodiment of the present disclosure;

FIG. 6 is a flow chart (I) of a laser ablation purification method according to one embodiment of the present application;

FIG. 7 is a schematic view of a culture imaging dish according to an embodiment of the present application;

FIG. 8 is a flow chart (II) of a laser ablation purification method according to an embodiment of the present application;

FIG. 9 is a flow chart of calculation of location information of ineffective sonoporated cells according to one embodiment of the present application;

FIG. 10 is a schematic diagram of a timing diagram of an isolated cell without perforation according to one embodiment of the present application;

FIG. 11 is a schematic diagram (I) of a timing diagram of an ex vivo cell of a repairable sonoporation according to one embodiment of the present disclosure;

FIG. 12 is a schematic diagram (II) of a timing diagram of an ex vivo cell of a repairable sonoporation according to one embodiment of the present disclosure;

FIG. 13 is a schematic diagram of a timing diagram of an isolated cell of an unrepairable sonoporation according to one embodiment of the present disclosure;

FIG. 14 is a schematic diagram of a timing diagram of a microbubble region of interest provided in one embodiment of the present application;

FIG. 15 is a schematic diagram of cell area data and a first fitted curve provided in one embodiment of the present application;

FIG. 16 is a schematic diagram of area data of a microbubble region of interest and a second fitted curve according to an embodiment of the present disclosure;

FIG. 17 is a schematic view of a use state of a culture imaging dish according to an embodiment of the present application.

Reference numerals: 10-an ultrasound transmission system; 11-arbitrary wave signal generator; a 12-power amplifier; 13-an ultrasonic transducer; 20-a three-dimensional motion system; 21-an electric stage; 30-an image acquisition system; 31-an image detector; 32-a second optical component; 33-a second objective; 40-a laser ablation system; 41-a laser generator; 42-a first optical component; 421-optical isolator; 422-beam expander; 423-spatial filters; 43-a first objective lens; 50-a processing module; 51-an ultrasonic control module; 52-a motion control module; 53-an image processing module; 54-a laser control module; 100-culturing an imaging dish; 101-top cover; 102-a bottom box; 103-a culture chamber; 104-filling holes; 105-transducer placement hole.

Detailed Description

The invention will be described in further detail below with reference to the drawings by means of specific embodiments. Wherein like elements in different embodiments are numbered alike in association. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted, or replaced by other elements, materials, or methods in different situations. In some instances, some operations associated with the present application have not been shown or described in the specification to avoid obscuring the core portions of the present application, and may not be necessary for a person skilled in the art to describe in detail the relevant operations based on the description herein and the general knowledge of one skilled in the art.

Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.

The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning. The terms "coupled" and "connected," as used herein, are intended to encompass both direct and indirect coupling (coupling), unless otherwise indicated.

Ultrasonic refers to sound waves with vibration frequencies exceeding 20 kilohertz, and microbubbles are a micron-sized structure of an internal gas core external coating. Microbubble-mediated sonoporation refers to micron-sized damage to nearby cell membranes caused by vibration and explosion of microbubbles under the drive of ultrasonic periodic positive and negative sound pressure, and the micro-damage to cells caused by the sonoporation can promote the transmission of gene drugs to cells. When ultrasound is applied to cells and microbubbles, three results are produced: no perforation, repairable sonoporation, and non-repairable sonoporation. Wherein repairable sonoporation can facilitate delivery of intracellular gene drugs without causing death of the cell, so called active gene drug delivery, such cells are defined as active sonoporated cells; while non-repairable sonoporation eventually leads to cell death due to the inability of the integrity of the cell membrane to recover, it is collectively referred to as ineffective gene drug delivery with no perforation, and these cells are defined as ineffective sonoporated cells.

After the in vitro cells are treated, how to purify the effective sonoporated cells becomes a technical problem to be solved.

Applicants have found that ineffective sonoporation cells can be ablated by means of cell ablation, leaving effective sonoporation cells, as described below with respect to cell ablation.

Cell ablation is the inactivation of cells using laser, chemical, etc., which is one method of studying cell lineage and cellular function during development. Cell ablation can be used to reveal important roles for specific cell types and has an important role in exploring the working mechanisms of cells after microdamage repair. There are significant differences in the performance of different cell ablation methods in studying specific biological problems. Existing ways of cell ablation may include: laser ablation, optogenetic ablation, chemical ablation, etc.

Laser ablation refers to the conversion of intense light generated by a laser into heat in a short period of time, causing damage to cells and causing cell death in a few seconds. Laser-mediated cell ablation has the ability to ablate cells at any stage, and the extent of damage to cells is affected by factors such as pulse power, duration, etc. Laser ablation has been used to study the postablation regenerative mechanism system of zebra fish interneuron mast cells (incc).

Optogenetic ablation is a method of combining laser ablation and genetics, which promotes rapid ablation of specific cells under the action of laser light by using genetically encoded photosensitizers. The laser intensity required for it is lower than for laser ablation, so that the death of nonspecific cells can be reduced, but the premature introduction and expression of genes are required.

Chemical ablation means that some cell populations are particularly susceptible to specific chemicals, so that these chemicals can be targeted as toxins to destroy specific cells, and chemical ablation has found relevant application in animal model fabrication of the mechanism of parkinsonism potential molecules.

Applicant's research has found that laser ablation can achieve relatively high spatial resolution of cell ablation, while operating relatively simply, in contrast to optogenetic ablation and chemical ablation. The applicant provides the position information of the ineffective sonoporation cells for laser ablation in combination with the image recognition mode, so that accurate laser ablation is realized, and finally, the population purification of the effective sonoporation cells is realized.

As shown in fig. 1, an embodiment of the present application provides an sonoporation cell laser ablation purification apparatus, which may include: an ultrasound emission system 10, a three-dimensional motion system 20, an image acquisition system 30, a laser ablation system 40, and a processing module 50.

The ultrasound transmission system 10 is configured to transmit ultrasound waves to a preset ultrasound region. Specific ultrasonic parameters need to be determined according to specific microbubble types and can be obtained through limited experiments. The preset ultrasound region is set according to the culture chamber of the culture imaging dish.

The three-dimensional motion system 20 is configured to load the culture imaging dish 100, and drive the culture imaging dish 100 to perform three-dimensional motion; the culture imaging dish 100 is added with an isolated cell and a physiological saline solution containing microbubbles. Ultrasonic waves are transmitted to the microbubble and cell areas, and the ultrasonic waves induce cavitation of the microbubbles and induce cells to generate different degrees of acoustic perforation and microdamage. The three-dimensional motion system 20 may include a motorized stage 21, and the motorized stage 21 may be used to place an observed specimen (i.e., the culture imaging dish 100) and to determine the location of the imaging region. The cell culture imaging dish 100 is used as a culture chamber for pre-experimental cells, an experimental platform for achieving contact between cells and microbubbles, and an experimental platform for sonoporation.

The image acquisition system 30 is configured to perform image acquisition of the isolated cells of the culture imaging dish 100, outputting a label-free microscopic image; the method is used for detecting the cell state after acoustic perforation and collecting a time sequence chart. This is because the application requires image acquisition of cells, and conventional image detectors are not satisfactory for direct use, and require image acquisition in combination with a microscope, the resulting image being referred to as a microscopic image. A label-free microscopic image refers to an image of a cell that has not been labeled with a targeted drug or gene. The area corresponding to the culture chamber is the microscopic field (or imaging field) that the application requires for microscopic observation. The image acquisition system 30 may be a microscope infinity optics. The three-dimensional motion system 20 drives the culture imaging dish 100 to move with high precision so that the area of the culture imaging dish 100 where the image acquisition needs to be performed is aligned with the imaging area of the image acquisition system 30, and also so that the cells of the culture imaging dish 100 where the laser ablation needs to be performed are aligned with the laser irradiation points of the laser ablation system 40.

The laser ablation system 40 is configured to emit laser light at a preset shot region.

The processing module 50 is configured to control the ultrasound transmission system 10 to ultrasonically process the isolated cells in the culture imaging dish 100 with ultrasound of preset parameters; controlling the image acquisition system 30 to acquire images of the isolated cells; identifying effective sonoporation cells and ineffective sonoporation cells in the isolated cells according to the microscopic image, and outputting position information of the ineffective sonoporation cells in the culture imaging dish 100; based on the positional information, the laser ablation system 40 is controlled to laser ablate each ineffective sonoporation cell. During image acquisition and laser ablation, the three-dimensional movement system 20 is controlled to drive the culture imaging dish 100 to move, and the culture imaging dish 100 is moved to a designated position for image acquisition and laser ablation.

The application is mainly studied for three parts, namely an ultrasonic combined microbubble sonoporation part which generates three types of cells of no perforation, repairable sonoporation and non-repairable sonoporation; the second part is an ineffective perforated cell image acquisition and screening part, and the part performs image recognition through machine learning to realize the recognition of non-perforated and irreparable sound-induced perforated cells; the third section is an ineffective perforated cell laser ablation section that achieves non-perforated and non-repairable sonoporation cell removal and population purification of repairable sonoporation cells.

The sonoporation and data acquisition part comprises inoculating cells to a special culture imaging dish 100 for culture until the cells are attached, replacing cell culture solution with sonoporation buffer solution during the sonoporation experiment so that the cells are fully contacted with microbubbles, cross focusing of a microscope imaging area and initialization of coordinates of an electric stage 21, starting the sonoporation experiment after completing ultrasonic parameter setting, and acquiring time sequence images of the sonoporation cells.

The data processing and cell selective laser ablation part comprises the steps of calculating the coordinates of the cells of the ineffective acoustic perforation, setting the laser power to be intolerable to the laser power of the cells, moving the cells of the ineffective acoustic perforation to the center of an imaging area, completing laser ablation, and repeating the operation until all the cells of the ineffective acoustic perforation are ablated.

As shown in fig. 2, in some embodiments, the ultrasound transmission system 10 may include an arbitrary wave signal generator 11, a power amplifier 12, and an ultrasound transducer 13.

The ultrasonic transducer 13 may be a single-element planar ultrasonic transducer 13, and the center frequency may be 1MHz for converting an electrical signal into an acoustic signal.

The arbitrary-wave signal generator 11 is configured to generate a pulse signal of a preset period. The arbitrary wave signal generator 11 outputs an arbitrary waveform electric signal, and the electric signal is transmitted to the power amplifier 12.

The power amplifier 12 is configured to perform voltage amplification of the pulse signal, and to drive the ultrasonic transducer 13 to generate ultrasonic waves by the amplified pulse signal. The ultrasonic transducer 13 is excited by the electric signals obtained by the arbitrary wave signal generator 11 and the power amplifier 12 to realize electroacoustic conversion and emit ultrasonic waves.

For example, the ultrasound transmission system 10 uses an arbitrary wave signal generator 11, an ultrasound power amplifier 12, and a single element planar ultrasound transducer 13. The connection and working modes between the devices are as follows: a periodic pulse sine wave is generated using an arbitrary wave signal generator 11, the output of which is connected to the input of a power amplifier 12, the signal line of the transducer is connected to the output of the power amplifier 12, and the electrical signal is converted into an ultrasonic pulse signal to be output. The ultrasonic pulse parameters and the output ultrasonic energy are different, and the ultrasonic pulse repetition period, the pulse duration and the sound pressure are adjusted to be used as one of methods for controlling the micro-damage degree of the cell sound-induced perforation. Where pulse repetition period refers to the time interval between one pulse and the next, and pulse duration refers to the number of periods of a sine wave within a single pulse. Taking human cervical cancer cells Hela as an example, the sound pressure amplitude can be set to be 0.58MPa, the pulse duration is 100 sine cycles, and the pulse repetition period is 10ms. Different kinds of cells can be induced by adjusting the ultrasonic sound pressure amplitude (such as 0.3-1.0 MPa) to perform different degrees of sound-induced perforation damage.

As shown in fig. 3, in some embodiments, the laser ablation system 40 may include a laser generator 41, a first optical assembly 42, and a first objective lens 43.

The laser generator 41 is configured to generate laser light of a preset wavelength. The laser generator 41 may be a fiber pulse laser with a center wavelength of 800 nm.

The first optical assembly 42 may include at least one of an optical isolator 421, a spatial filter 423, a beam expander 422, and a short-pass dichroic mirror. Wherein, the optical isolator 421 is used for limiting unidirectional propagation of laser, and the optical isolator 421 can prevent damage to the light source when backward output light is generated in the optical path.

The spatial filter 423 is used to remove high-order modes and noise in the laser, and the spatial filter 423 can remove the high-order modes and noise in the laser, thereby improving the quality of the laser.

The beam expander 422 is used for expanding the beam diameter of the laser, the beam expander 422 can expand the diameter of the input parallel beam, the laser after beam expansion passes through the objective lens and is focused into smaller light spots of 1-2 μm), and the divergence angle of the laser beam is reduced.

The short-wave dichroic mirror is used for reflecting laser, and can almost completely reflect light in a long wave band (laser emitted by the pulse laser) and almost completely transmit light in other short wavelengths. When the position of the laser generator 41 needs to be adjusted, the laser can be reflected through the short-wave dichroic mirror, the laser transmitter does not need to be opposite to the culture imaging dish 100, meanwhile, a yielding space is provided for the image acquisition system 30, the effect that the laser ablation system 40 and the image acquisition system 30 share one objective lens can be achieved, and when cells in the acquired images are located on the alignment mark, the laser ablation system 40 also achieves alignment.

The first objective lens 43 is configured to focus the laser light to a preset irradiation area, and the first objective lens 43 can achieve micron-sized focusing of the laser light.

As shown in fig. 4, in some embodiments, the image acquisition system 30 may include an image detector 31 and a second objective lens 33; the reflected light of the isolated cells is collected by the image detector 31 via the second objective 33, and if necessary a corresponding second optical component 32 may be provided.

Alternatively, as shown in fig. 3, first optical assembly 42 may include a short-wave-pass dichroic mirror; the image acquisition system 30 may include an image detector 31; the reflected light from the isolated cells passes through the first objective lens 43 and is collected by the image detector 31 after passing through the short-wavelength dichroic mirror. In these embodiments, the image acquisition system 30 shares the same objective lens as the laser ablation system 40, and the laser is focused at the center of the imaging region of the image acquisition system 30.

In some embodiments, a short-wave pass dichroic mirror is coupled between an objective lens and a sleeve lens (tube lens) of a microscope infinity optical system (i.e., image acquisition system 30). The light outputted by the laser generator 41 firstly passes through the optical isolator 421, then enters the beam expander 422 to expand the diameter of the laser beam, the laser beam is outputted to the spatial filter 423 after being expanded, and the filtered parallel light enters the short-wave dichroic mirror coupled to the microscope system to realize almost total reflection to the infinity microscope objective. The sonicated cell culture imaging dish 100 is placed on the motorized stage 21 and the laser is focused in the culture imaging dish 100 and the image detector 31 transmits the image to the processing module 50.

According to the method, cell selective laser ablation is achieved under a microscope imaging area, real-time monitoring is conducted under the same imaging area, a laser collimation beam is focused on a sample in the center of the microscope imaging area, and cells die when the laser intensity is larger than a cell survival threshold value.

In some embodiments, as shown in fig. 5, the processing module 50 may include an ultrasound control module 51, a motion control module 52, an image processing module 53, and a laser control module 54. The four modules may be a plurality of functional modules implemented by the same processor, or may be one functional module implemented by one or more processors.

The ultrasonic control module 51 is used for controlling the ultrasonic transmission system 10 to carry out ultrasonic treatment on the isolated cells in the culture imaging dish 100 by adopting ultrasonic waves with preset parameters.

The motion control module 52 is used to control the operation of the three-dimensional motion system 20 to drive the culture imaging dish 100 to move relative to the image acquisition system 30 so that the region of the culture imaging dish 100 to be acquired is traversed.

The image processing module 53 is configured to control the image acquisition system 30 to acquire microscopic images of the isolated cells, identify valid sonoporated cells and non-valid sonoporated cells in the isolated cells according to the microscopic images, and output positional information of the non-valid sonoporated cells in the culture imaging dish 100.

The laser control module 54 is configured to control the three-dimensional movement system 20 to move each ineffective sonoporation cell into a preset irradiation region of the laser ablation system 40 according to the position information, and control the laser ablation system 40 to perform laser ablation on each ineffective sonoporation cell.

The following describes a specific procedure of the sonoporation cell laser ablation purification system for performing the sonoporation cell laser ablation purification method, as shown in fig. 6 and 8, and the method may include the following steps:

step 0, culturing the isolated cells in a culture imaging dish 100.

Cell culture includes fabrication of culture imaging dish 100, cell planting, and preparation of sonoporation buffer. Materials such as a cell culture imaging dish 100, a plastic transparent sheet, an ultrasonic cleaner 40kHz, an electrothermal blowing dry box, a carbon dioxide incubator, human cervical cancer cells (Hela), a buffer (e.g., ethane sulfonic acid, abbreviated as HEPES) 1m, ph7.2 to 7.4, DMEM medium (Dulbecco's Modified Eagle Medium, a medium containing various amino acids and glucose) may be used.

As shown in fig. 7, embodiments of the present application further provide a culture imaging dish 100, which may include: a top cover 101 and a bottom box 102 mated with the top cover 101.

The top cover 101 is provided with a transducer placement hole 105, and the transducer placement hole 105 is used for mounting and fixing the ultrasonic transducer 13.

A bottom case 102 mated with the top case 101, the bottom case 102 being provided with a culture chamber 103, the culture chamber 103 having a liquid filling hole. The culture imaging dish may further comprise a stopper matching the pour hole 104 for sealing the culture chamber 103 so that liquid in the culture chamber 103 does not leak out when the bottom box 102 is inverted.

Wherein the culture imaging dish 100 has a first use state, a second use state and a third use state.

In the first use state, the top cover 101 is not assembled to the bottom case 102, the bottom case 102 is set upright, the culture chamber 103 is filled with a physiological saline solution containing ex vivo cells, and the ex vivo cells are cultured for a predetermined period of time and then attached to the bottom of the culture chamber 103.

In the second use state, the top cover 101 is not assembled to the bottom case 102, and after the first use state, the culture chamber 103 is filled with a physiological saline solution containing microbubbles, and the bottom case 102 is inverted so that the microbubbles float up to the bottom of the culture chamber 103 and are in sufficient contact with the isolated cells.

In the third use state, after the first use state, the bottom case 102 is set up, the top cover 101 is assembled to the bottom case 102, the ultrasonic transducer 13 is mounted on the transducer placement hole 105, and the action area of the ultrasonic transducer 13 covers the culture chamber 103. It can be seen that using the culture imaging dish 100 provided by the present application can make microbubbles more fully contact with cells, ensuring the effect of sonoporation. However, it should be noted that the purification method and apparatus provided in the present application are not necessarily limited to the culture imaging dish 100 provided in the present application, and other possible culture dishes may be used in the present application, which is also claimed in the present application.

As shown in fig. 7 (a), the culture imaging dish 100 may include a top cover 101 and a bottom case 102, and the top cover 101 is provided with a transducer placement hole 105 for fixing the ultrasonic transducer 13; the middle part of the bottom box 102 is provided with a culture chamber 103, for example, the bottom of the culture chamber 103 is a glass sheet with the thickness of <0.2mm, and the top is a plastic sheet with the thickness of <0.1 mm. The bottom glass sheet facilitates microscopic imaging and laser ablation. The liquid filling hole 104 of the culture chamber 103 can be used for adding liquid, and the whole culture imaging dish 100 is transparent and can be made of plastic or glass. Before use, the culture imaging dish 100 is put into an ultrasonic cleaner for cleaning for 3-4 times, and then put into an electrothermal blowing drying oven for drying.

As shown in (a) of fig. 17, the cell planting procedure may include: micro-inoculating cells in the cell subculture bottle into a culture chamber 103 of a bottom box 102 which is placed in a positive position; the bottom cassette 102 was placed in a carbon dioxide incubator for 24h until the cells adhered to the bottom glass sheet of the culture chamber 103.

As shown in fig. 17 (B), the preparation process of the sonoporation buffer may include: uniformly mixing HBSS solution containing 10mM HEPES to obtain physiological salt solution; and mixing the physiological saline solution and the microbubbles to obtain the required sonoporation buffer. After the cells adhere to the bottom glass sheet of the culture chamber 103, the sonoporation buffer is injected into the well-placed bottom box 102 through the injection hole of the culture chamber 103.

After the injection of the sonoporation buffer, as shown in fig. 17 (C), the bottom box 102 is inverted for about 5 to 8 minutes to allow the microbubbles to fully contact the cells, during which the microbubbles float up due to the gas core structure and fully contact the cells on the bottom glass sheet of the culture chamber 103.

As shown in fig. 17 (D), the bottom case 102 after the cells are sufficiently contacted with the microbubbles is being placed on the microscope stage (i.e., the motorized stage 21).

As shown in fig. 17 (E), the top cover 101 of the culture imaging dish 100 is fitted to the bottom case 102, and the ultrasonic transducer is mounted on the transducer placement hole 105, so that the position of the transducer can be fixed and the ultrasonic output is ensured to cover the culture chamber 103. Physiological saline solution is added to the culture imaging dish 100 (specifically outside the culture chamber 103, inside the bottom box 102) to completely submerge the ultrasound transducer 13 plane to complete the ultrasound coupling.

Step 1, performing ultrasonic treatment on isolated cells in the culture imaging dish 100 by adopting ultrasonic waves with preset parameters, wherein isolated cells and physiological salt solution containing microbubbles are added in the culture imaging dish 100, so that the microbubbles are cavitated and the cells generate different degrees of acoustic perforation micro-damage.

For example, the acoustic energy output by the single-element planar ultrasound transducer 13 is uniform across the cell region, and the cell culture imaging dish 100 is placed in a uniform field of ultrasonic energy by adjusting the x, y, z axes of the three-dimensional movement during the experiment.

Step 2, acquiring microscopic images of the isolated cells, identifying effective sonoporated cells and ineffective sonoporated cells in the isolated cells according to the microscopic images, and outputting the position information of the ineffective sonoporated cells in the culture imaging dish 100.

In some embodiments, the culture imaging dish 100 (corresponding to the microscopic view of the culture chamber 103) has at least one region to be processed, and each region to be processed has a set of microscopic images, and a set of microscopic images may include a plurality of time-series images, which are formed by photographing the region to be processed according to a preset photographing time sequence.

For example, as shown in fig. 7 (B) and (C), the microscopic field of view on the bottom glass of the culture chamber 103 may be divided into a plurality of areas to be processed, each of which has a size corresponding to the size of the imaging area of the image acquisition system 30. The clear image of the culture imaging dish 100 is made visible by adjusting the angle of the objective lens of the image acquisition system 30 before image acquisition. According to the central position of each area to be processed, the position of the culture imaging dish 100 relative to the image acquisition system 30 is determined every time the image acquisition is carried out, the three-dimensional movement system 20 drives the electric object stage 21 to move, and the electric object stage 21 moves according to the set parameters. For example, the culture imaging dish 100 is composed of 25 small square imaging areas, i.e., 25 small square areas with a size of 2mm x 2mm, constituting a panoramic image of the cell culture imaging dish 100. The image acquisition frequency is 0.1 second/area, the acquisition process lasts for 15 minutes, and the acquisition process can be divided into three stages: 30 seconds before the ultrasonic action, 30 seconds during the ultrasonic action and 14 minutes after the ultrasonic action. At this time, 25 sets of microscopic images may be acquired in total, each set including a plurality of time-series images corresponding to the three phases described above.

In some embodiments, in step 2, identifying valid sonoporated cells and non-valid sonoporated cells in the ex vivo cells from the microscopic image may comprise:

step 210, identifying the effective sonoporation cells and the ineffective sonoporation cells in the isolated cells by identifying the microscopic image by adopting a semantic segmentation model and a classification model.

In some embodiments, the identifying the microscopic image by machine learning using the semantic segmentation model and the classification model may include:

step 211, performing semantic segmentation processing on a microscopic image corresponding to the isolated cells by adopting a semantic segmentation model, reserving a cell area corresponding to the isolated cells in the microscopic image, and removing non-cell areas except the isolated cells.

And 212, carrying out feature extraction on the acoustic perforation on the microscopic image by adopting a preset screening rule to obtain a cell area feature set and a microbubble region of interest feature set. The features of the cell area feature set are related to the area data of the cell and the features of the microbubble region of interest feature set are related to the area data of the microbubble region of interest. The preset screening rules are set for the correlation of cell area and microbubble area.

Step 213, classifying each isolated cell according to the cell area feature set and the microbubble area feature set by using a classification model, and identifying whether the current isolated cell is a valid sonoporated cell or a non-valid sonoporated cell.

In the method, microscopic images are identified in a machine learning mode, and in terms of a segmentation algorithm, a traditional threshold segmentation algorithm, a segmentation algorithm based on region growth or a segmentation algorithm based on deep learning, such as U-net or Mask R-CNN, can be adopted. In terms of classification algorithms, conventional machine learning algorithms such as Support Vector Machines (SVMs), random Forest (Random Forest), artificial neural networks, or the like may be used. Alternatively, deep learning based classification algorithms such as Convolutional Neural Networks (CNNs) or Recurrent Neural Networks (RNNs) may be used.

In some embodiments, in step 212, feature extraction on sonoporation of the microscopic image using a preset screening rule may include:

1) And obtaining a first fitting curve of the area data of the cells and the time, and obtaining a second fitting curve of the area data of the microbubble region of interest and the time.

2) Obtaining a maximum cell area value and a maximum slope of the first fitting curve according to the first fitting curve, taking the maximum cell area value as a first characteristic, and taking time corresponding to the maximum slope of the first fitting curve as a second characteristic; the set of cell area features may include a first feature and a second feature.

3) Extracting a subsequence of the area data of the cell, obtaining a third fitting curve of the subsequence and time, obtaining the maximum value of the subsequence and the sum of slopes corresponding to a plurality of preset time points of the third fitting curve according to the third fitting curve, taking the maximum value of the subsequence as a third characteristic, and taking the sum of slopes corresponding to a plurality of preset time points of the third fitting curve as a fourth characteristic.

4) And obtaining the sum of slopes corresponding to the second fitting curve at a plurality of preset time points according to the second fitting curve, and taking the sum of slopes corresponding to the second fitting curve at the plurality of preset time points as a fifth characteristic. The set of microbubble region of interest features may include a fifth feature.

5) And interpolating the area data of the cells by adopting an interpolation method, performing smooth curve processing to obtain a fourth fitting curve of the area data of the cells and time by adopting the interpolation method, and taking the peak value of the fourth fitting curve as a sixth characteristic.

6) And obtaining a corresponding seventh characteristic according to the sonoporation state of the isolated cells and the second fitting curve.

7) And calculating the inter-frame difference value of the images before and after the sound induced perforation of the microbubble region of interest by adopting a pixel inter-frame difference method as an eighth feature.

In summary, the set of cell area features may include a first feature, a second feature, a third feature, a fourth feature, and a sixth feature; the set of microbubble region of interest features may include a fifth feature, a seventh feature, and an eighth feature.

As will be described in more detail below with reference to one embodiment, as shown in FIG. 9, images acquired during sonoporation are acquired, each of which represents a small square area of the cell culture imaging dish 100 at a given time. The functions that can be realized in step 2 of the present application include data preprocessing, semantic segmentation, and cell classification, and the specific functions are as follows.

Data preprocessing: the acquired images are sorted according to time sequence images of the area to be processed (corresponding to each small square area) and classified according to groups. Brightness and contrast adjustment is performed on the image, for example, the output time-series image data is subjected to brightness and contrast adjustment by using g (x) =f (x) ×alpha+beta, alpha is called gain, beta is called bias, f (x) is an input image, and g (x) is a final output result.

Semantic segmentation: inputting the preprocessed time sequence image into a trained semantic segmentation model to obtain a corresponding semantic segmentation binary image, for example, adopting a U-Net++ semantic segmentation model.

Cell classification:

1) According to the bright field image and the semantic segmentation result, each isolated cell is extracted and positioned, for example, the method of searching a connected domain is used for realizing region connection, a time sequence image corresponding to the isolated cell is obtained, and the cell center point position (x, y) is recorded. As shown in fig. 10 to 13, according to the size of the isolated cells, microscopic images corresponding to each isolated cell can be captured for each microscopic image of the area to be treated, and a set of microscopic images can be obtained for each isolated cell.

2) And extracting features according to the bright field time sequence image of the isolated cells and the semantic segmentation result. The data of the characteristics mainly comprise two aspects, namely, the area data R (i) of the cells is obtained through calculation according to the semantic segmentation result. Setting the sliding window step length as 10 and extracting the subsequence R2 (i) of R (i) with the window size of 3. On the other hand, the coordinates of the center point of the microbubbles adhered to the cells are calculated, the region of interest of the microbubbles (or called as the microbubble ROI region) is cut out according to the coordinates, the size of the cutting frame is defined as one third or one fifth of the size of the input image, and the cutting ensures that the microbubbles are basically positioned at the center position of the cutting frame. Taking the cell shown in fig. 12 as an example, a time-series image corresponding to the microbubble ROI area data R3 (i) is calculated and obtained from the semantic segmentation result corresponding to the time-series image as shown in fig. 14.

3) Curve fitting is performed on R (i), R2 (i), and R3 (i), for example, using a smoothing spline function in matlab, and the cell shown in fig. 12 is taken as an example, the R (i) fitting curve is shown in fig. 15, and the peak value of the fitting curve is extracted as a first feature μ1 and the time corresponding to the maximum slope is extracted as a second feature μ2. The peak value and the slope of the R2 (i) fitting curve are extracted and used as a third characteristic mu 3 and a fourth characteristic mu 4. The R3 (i) fitted curve is shown in FIG. 16, and the slope of the R3 (i) fitted curve is extracted as a fifth feature μ 5. The sum of the slopes of the fitted curve is the sum of the slopes of the fitted curve corresponding to a plurality of preset time points, for example, ten time points of-05 s, 0s, 10s … s and the like shown in fig. 12, or other time points.

4) The R (i) is smoothed by interpolation, and the curve peak is extracted as a sixth feature μ 6, for example, by interpolation using a make_interpolation_spline function in the scipy library.

5) Obtaining a seventh feature mu 7 according to the sonoporation state of the isolated cells and the second fitting curve, for example, calculating the average slope_mean (i) of the slope sum of the R3 (i) fitting curve of each type of cells in the classification data set, wherein i=1, 2,3,4 respectively represent a perforation death type, a membrane foaming type, a membrane shrinkage type and an unsuccessful perforation type, and extracting the seventh feature mu 7 according to the formula 1-2;

6) The inter-frame difference of the images before and after the sonoperforation of the microbubble ROI area is calculated using the pixel inter-frame difference method as the eighth feature μ8.

7) And inputting the extracted feature vectors mu 1-mu 8 into a trained classification network to obtain a classification result.

8) Based on the classification result, the abscissa (position information) corresponding to the ineffective sonoporated cells is output to the processing module 50.

In order to improve the accuracy of image recognition, experiments are carried out on the established various extractable characteristics, different characteristic combinations are adopted as the basis of machine learning, 8 characteristics associated with isolated cells and microbubbles are finally reserved, and according to the 8 characteristics, high-accuracy cell type classification can be realized.

The applicant proposes features relating to sonoporation by analysis according to the process of sonoporation. In addition to the 8-dimensional features used in the present application, cell roundness time series data and cell eccentricity time series data are also extracted, standard deviations corresponding to the two sets of time series data are calculated and obtained as features mu 9 and mu 10, and the number of microbubbles adhered to cells and the diameter of the largest microbubbles are extracted as features mu 11 and mu 12. It is also attempted to extract the average value of the image optical flow speed, and the maximum value as parameters and then make feature selection. The applicant provides actual measurement experiments and analysis to obtain an optimal feature subset, namely 8-dimensional feature combinations used in the application, and finally selects the 8-dimensional feature with highest recognition accuracy by increasing or decreasing various features and combining experiments.

In order to realize rapid screening of a large number of cells, the application uses a machine learning method to select and mark the position to be subjected to laser ablation. The cell state after sonoporation is divided into effective sonoporation and ineffective sonoporation, and a visualization system is provided for screening and marking cells of ineffective sonoporation under the effect of sonoporation, and the flow chart of calculating the coordinates of cells of ineffective sonoporation is shown in fig. 9.

And step 3, carrying out laser ablation on each ineffective sound-induced perforation cell according to the position information.

In some embodiments, prior to laser ablating each ineffective sonoporated cell, further comprising:

step 300, performing laser irradiation on a reference cell by adopting the minimum output power, and judging the laser ablation effect of the current output power according to the motion state of the reference cell; the reference cell is a null sonoporated cell or a new ex vivo cell.

If the laser ablation effect of the current output power does not meet the preset ablation effect, gradually increasing the output power of the laser according to the preset change amplitude, carrying out laser irradiation on the reference cells after adjusting the output power each time, and judging the laser ablation effect of the adjusted output power again according to the motion state of the reference cells until the laser ablation effect meets the preset ablation effect.

In order to achieve the best effect of cell selective laser ablation, the application uses cells after sonoporation to explore laser parameters (laser power), and the specific steps are as follows:

1) Adjusting the laser focus position: the empty cell culture imaging dishes 100 of the same size and material are placed on the motorized stage 21, the laser is tuned to a very low energy (about 3-5 mv), a small bright spot indicating the focal position of the laser is observed in the computer display, and the small bright spot is located in the center of the imaging area, i.e., aligned with the alignment mark shown in fig. 7 (C).

2) The culture imaging dish 100, which is used only for positioning, is removed and the cell culture imaging dish 100 containing sonicated cells is placed on the motorized stage 21.

3) Based on the result of sorting the effective sonoporated cells, the motorized stage 21 is controlled based on the positional information to move the ineffective sonoporated cells to the center of the imaging region.

4) Adjusting the current value of the laser generator 41 changes the output power of the laser, adjusts the laser average power in 10mW to 100mW in 10mW increments, analyzes the effect of laser ablation by observing the movement state of the cells after each set of experiments is completed, and repeats three experiments at each laser power.

5) And analyzing the result to determine the laser power which can realize laser in-situ ablation and has minimum damage to surrounding cells, and completing the subsequent laser ablation experiment.

After laser parameter adjustment and exploration of a test part, intolerable laser power of cells is successfully obtained, invalid sonoporation cell position information is traversed, an electric object stage 21 is controlled, cells are aligned by using an alignment mark of an imaging area, laser is focused at the center of the invalid sonoporation cells, and finally selective laser ablation of unmarked cells is realized.

And 4, re-collecting microscopic images of the isolated cells, determining that all the ineffective sonoporation cells are ablated, and finishing the population purification of the effective sonoporation cells.

In summary, the present application acquires a plurality of time-series images by performing image acquisition on isolated cells, then uses a trained semantic segmentation and classification network model to screen ineffective sonoporation cells on the acquired bright field image, completes classification of sonoporation cells on the image layer, and marks the positions of the ineffective sonoporation cells as reference information for cell laser ablation.

The laser ablation part uses a laser and an optical element to generate focused high-energy laser in the center of a microscopic imaging area, and further moves ineffective sonoporated cells to the center of the microscopic imaging area through the electric objective table 21, so that the ineffective sonoporated cells are eliminated by laser ablation, and the effective sonoporated cells are kept to be continuously cultured to realize the purification of transfected cell populations.

The application provides a selective laser ablation mode of label-free cells, and external labels (such as genes and targets) are not needed. The intelligent method is used for realizing the sorting of the effective sonoporation cells and the marking of the ineffective sonoporation cells, solves the defect that the fluorescent dye is used for positioning the ineffective sonoporation cells, and has the following advantages: the method realizes rapid screening of a large number of sound-induced perforation cells, reduces the time for observing and selecting by naked eyes, and improves the classification accuracy of effective sound-induced perforation cells and ineffective sound-induced perforation cells by using a machine learning mode.

Reference is made to various exemplary embodiments herein. However, those skilled in the art will recognize that changes and modifications may be made to the exemplary embodiments without departing from the scope herein. For example, the various operational steps and components used to perform the operational steps may be implemented in different ways (e.g., one or more steps may be deleted, modified, or combined into other steps) depending on the particular application or taking into account any number of cost functions associated with the operation of the system.

While the principles herein have been shown in various embodiments, many modifications of structure, arrangement, proportions, elements, materials, and components, which are particularly adapted to specific environments and operative requirements, may be used without departing from the principles and scope of the present disclosure. The above modifications and other changes or modifications are intended to be included within the scope of this document.

The foregoing detailed description has been described with reference to various embodiments. However, those skilled in the art will recognize that various modifications and changes may be made without departing from the scope of the present disclosure. Accordingly, the present disclosure is to be considered as illustrative and not restrictive in character, and all such modifications are intended to be included within the scope thereof. Also, advantages, other advantages, and solutions to problems have been described above with regard to various embodiments. The benefits, advantages, solutions to problems, and any element(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature. The terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, system, article, or apparatus. Furthermore, the term "couple" and any other variants thereof are used herein to refer to physical connections, electrical connections, magnetic connections, optical connections, communication connections, functional connections, and/or any other connection.

Those skilled in the art will recognize that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention. Accordingly, the scope of the invention should be determined only by the following claims.

Claims

1. A method for purifying sonoporation cells by laser ablation, comprising:

collecting microscopic images of isolated cells in a culture imaging dish, identifying effective sonoporation cells and ineffective sonoporation cells in the isolated cells according to the microscopic images, and outputting position information of the ineffective sonoporation cells in the culture imaging dish; the microscopic field of the culture imaging dish is added with isolated cells and physiological salt solution containing microbubbles, and is subjected to ultrasonic treatment, so that the microbubbles are cavitated and the cells generate different degrees of acoustic perforation and micro damage;

performing laser ablation on each ineffective sound-induced perforation cell according to the position information;

2. The method of claim 1, wherein the microscopic field of view of the culture imaging dish has at least one area to be processed, each of the areas to be processed has a set of the microscopic images, and a set of the microscopic images includes a plurality of time-series images, the plurality of time-series images are formed by photographing the area to be processed according to a preset photographing time sequence;

Identifying valid sonoporated cells and non-valid sonoporated cells in the isolated cells from the microscopic image, comprising:

and identifying the microscopic image by adopting a semantic segmentation model and a classification model, and identifying effective sonoporation cells and ineffective sonoporation cells in the isolated cells.

3. The method of claim 2, wherein identifying the microimage using a semantic segmentation model and a classification model comprises:

performing semantic segmentation processing on the microscopic image corresponding to one isolated cell by adopting a semantic segmentation model, reserving a cell area corresponding to the isolated cell in the microscopic image, and removing non-cell areas except the isolated cell;

carrying out feature extraction on the microscopic image about sonoporation by adopting a preset screening rule to obtain a cell area feature set and a microbubble region of interest feature set; the features of the cell area feature set are related to the area data of the cell, and the features of the microbubble region of interest feature set are related to the area data of the microbubble region of interest;

and classifying each isolated cell by adopting a classification model according to the cell area characteristic group and the microbubble area characteristic group, and identifying whether the current isolated cell is an effective sonoporated cell or an ineffective sonoporated cell.

4. A method according to claim 3, wherein the feature extraction of the microimages with respect to sonoporation using preset screening rules comprises:

obtaining a first fitting curve of area data and time of cells, and obtaining a second fitting curve of area data and time of a microbubble region of interest;

obtaining a maximum cell area and a maximum slope of the first fitting curve according to the first fitting curve, taking the maximum cell area as a first characteristic, and taking time corresponding to the maximum slope of the first fitting curve as a second characteristic; the set of cell area features includes the first feature and the second feature;

obtaining the sum of slopes corresponding to the second fitting curve at a plurality of preset time points according to the second fitting curve, and taking the sum of slopes corresponding to the second fitting curve at the plurality of preset time points as a fifth characteristic; the set of microbubble region of interest features includes the fifth feature.

5. The method of claim 2, further comprising, prior to laser ablating each of the ineffective sonoporated cells:

performing laser irradiation on a reference cell by adopting the minimum output power, and judging the laser ablation effect of the current output power according to the motion state of the reference cell; the reference cell is the ineffective sonoporation cell or a new ex vivo cell;

6. An sonoporation cell laser ablation purification apparatus, comprising:

an ultrasound transmission system (10) configured to transmit ultrasound waves to a preset ultrasound region;

a three-dimensional motion system (20) configured to load a culture imaging dish (100), driving the culture imaging dish (100) into three-dimensional motion; an isolated cell and a physiological salt solution containing microbubbles are added into the culture imaging dish (100);

an image acquisition system (30) configured to perform image acquisition of isolated cells of the culture imaging dish (100) and output a microscopic image;

a laser ablation system (40) configured to emit laser light at a preset shot region;

a processing module (50) configured to control an ultrasound transmission system (10) to ultrasonically process the isolated cells in the culture imaging dish (100) with ultrasound of preset parameters; controlling an image acquisition system (30) to acquire images of the isolated cells; identifying valid sonoporated cells and non-valid sonoporated cells in the isolated cells from the microscopic image and outputting positional information of the non-valid sonoporated cells in the culture imaging dish (100); controlling a laser ablation system (40) to perform laser ablation on each of the ineffective sonoporation cells according to the position information.

7. The apparatus of claim 6, wherein the laser ablation system (40) comprises a laser generator (41), a first optical assembly (42), and a first objective lens (43);

the laser generator (41) is configured to generate laser light of a preset wavelength;

the first optical component (42) comprises at least one of an optical isolator (421), a spatial filter (423), a beam expander (422), and a short-wave-pass dichroic mirror;

the first objective lens (43) is configured to focus laser light to the preset irradiation area;

wherein the optical isolator (421) is configured to limit unidirectional propagation of the laser light;

the spatial filter (423) is used for removing high-order modes and noise in laser;

the beam expander (422) is used for expanding the beam diameter of the laser;

the short-wave-pass dichroic mirror is used for reflecting laser.

8. The apparatus according to claim 7, wherein the image acquisition system (30) comprises an image detector (31) and a second objective (33); the reflected light of the ex-vivo cell is collected by the image detector (31) through the second objective (33);

alternatively, the first optical assembly (42) comprises the short-wave pass dichroic mirror;

the image acquisition system (30) comprises an image detector (31); the reflected light of the isolated cells is collected by the image detector (31) after passing through the first objective lens (43) and the short-wave-communication dichroic mirror.

9. The apparatus according to any one of claims 6-8, wherein the processing module (50) comprises an ultrasound control module (51), a motion control module (52), an image processing module (53) and a laser control module (54);

the ultrasonic control module (51) is configured to control an ultrasonic emission system (10) to carry out ultrasonic treatment on the isolated cells in the culture imaging dish (100) by adopting ultrasonic waves with preset parameters;

the motion control module (52) is configured to control the three-dimensional motion system (20) to work, and drive the culture imaging dish (100) to move relative to the image acquisition system (30) so that a region to be acquired of the culture imaging dish (100) is traversed;

the image processing module (53) is configured to control the image acquisition system (30) to acquire microscopic images of the isolated cells, identify valid sonoporated cells and non-valid sonoporated cells in the isolated cells from the microscopic images, and output positional information of the non-valid sonoporated cells in the culture imaging dish (100);

the laser control module (54) is configured to control the three-dimensional movement system (20) to move each of the ineffective sonoporation cells into a preset irradiation region of the laser ablation system (40) according to the position information, and control the laser ablation system (40) to perform laser ablation on each of the ineffective sonoporation cells.

10. A culture imaging dish, comprising:

a top cover (101), wherein the top cover (101) is provided with a transducer placement hole (105), and the transducer placement hole (105) is used for installing and fixing an ultrasonic transducer (13);

a bottom box (102) matched with the top cover (101), wherein the bottom box (102) is provided with a culture chamber (103), and the culture chamber (103) is provided with a liquid injection hole (104);

the culture imaging dish has a first use state, a second use state and a third use state;

in the first use state, the top cover (101) is not assembled on the bottom box (102), the bottom box (102) is arranged in the right direction, the culture chamber (103) is injected with physiological saline solution containing isolated cells, and the isolated cells are adhered to the bottom of the culture chamber (103) after being cultured for a preset time period;

in the second use state, the top cover (101) is not assembled to the bottom box (102), after the first use state, the culture chamber (103) is injected with physiological saline solution containing microbubbles, and the bottom box (102) is inverted so that the microbubbles float to the bottom of the culture chamber (103) and are fully contacted with the isolated cells;

in the third use state, after the first use state, the bottom box (102) is arranged in the right direction, the top cover (101) is assembled on the bottom box (102), the ultrasonic transducer (13) is installed on the transducer placement hole (105), and the action area of the ultrasonic transducer (13) covers the culture chamber (103).