WO2022160988A1 - Ultrasonic cell neuro-modulation system - Google Patents

Abstract

An ultrasonic cell neuro-modulation system, comprising: a cell containing apparatus (100) for containing marked cells, wherein the cell containing apparatus (100) comprises a base (110), and a light-transmitting opening is formed in the base (110); an ultrasonic stimulation apparatus (200) comprising an ultrasonic transducer (230), wherein the ultrasonic transducer (230) is used for emitting ultrasonic waves to the marked cells; and a cell imaging apparatus (300) comprising a laser light source (310), an objective lens (350) and an image collector (370), wherein the laser light source (310) emits laser light to the marked cells through the objective lens (350) and the light-transmitting opening, and the image collector (370) collects a fluorescence image of the marked cells through the objective lens (350). By means of the modulation system, neural activity of cells can be observed from a microscopic perspective, and instantaneous and continuous real-time dynamic imaging and monitoring can be performed on the cells. A new strategy is provided for ultrasonic neuro-modulation of in-vitro cell imaging, such that the system can be applied to future ultrasonic-mediated neuro-modulation more safely and effectively.

Description

A Cellular Ultrasound Neuromodulation System technical field

The present application relates to the field of instruments used in medicine or experiments, and in particular, to a cell ultrasonic neuromodulation system.

Background technique

Neuromodulation technology is a biomedical engineering technology that uses implantable or non-implantable technology to change the activity of the central nervous system, peripheral nerve or autonomic nervous system by means of electrical stimulation or drugs to improve the symptoms of the sick population and improve the quality of life. .

Ultrasound neuromodulation technology based on the mechanical effect of ultrasound is a new non-invasive brain stimulation and regulation technology that has emerged in recent years. It uses ultrasound of different intensities, frequencies, pulse repetition frequencies, pulse widths and durations to stimulate or stimulate the central nervous system at the stimulation site. Inhibitory effect, a reversible change in bidirectional regulation of neural function. Ultrasound-based non-invasive neuromodulation technology is considered to be one of the next-generation neuromodulation technologies with the most promising clinical translation. It has the advantages of non-invasiveness, large penetration depth, and high spatiotemporal distribution rate. Its regulatory effect has been verified on various scale targets such as neurons, nematodes, mice, and non-human primates. It modulates neural activity in deep brain regions such as the thalamus, making it a possible treatment for brain disorders such as Parkinson's, epilepsy, and depression.

Current ultrasound neuromodulation devices usually cannot provide timely and complete information on cell changes, and there is still a lack of instantaneous and continuous real-time dynamic imaging monitoring of cells.

technical problem

The present application provides a cell ultrasonic neuromodulation system, which can observe the neural activity of cells from a microscopic perspective, and can perform instantaneous and continuous real-time dynamic imaging monitoring of cells. The present application forms an integrated design by building the ultrasonic stimulation device on the cell imaging device, which can simplify the experimental steps and improve the experimental efficiency.

technical solutions

In order to solve the above problems, the technical solutions provided in the embodiments of the present application are: a cell ultrasonic neuromodulation system, comprising: a cell containing device for containing labeled cells, the cell containing device comprising a base, the A light-transmitting port is formed on the base; an ultrasonic stimulation device includes an ultrasonic transducer, and the ultrasonic transducer is used to emit ultrasonic waves to the marked cells; a cell imaging device includes a laser light source, an objective lens and an image collector, and the The laser light source emits laser light to the labeled cells through the objective lens and the light-transmitting port, and the image collector collects a fluorescent image of the labeled cells through the objective lens.

According to the cellular ultrasonic neuromodulation system provided by the embodiments of the present application, the labeled cells are stimulated by ultrasonic waves, and laser light is further emitted to the labeled cells, and then the fluorescent images of the labeled cells are collected by the image collector, thereby enabling The neural activity of cells can be observed from a microscopic perspective, and instantaneous and continuous real-time dynamic imaging monitoring of cells can be performed, providing reliable data support for neural regulation research.

The cell ultrasonic neuromodulation system provided in the embodiment of the present application uses the mechanical effect of ultrasonic waves to change the tension of the cell membrane, and the ultrasonic radiation force acts on the cell phospholipid bilayer, and induces the opening of mechanosensitive ion channels by ultrasonic stimulation, causing ion influx ( That is, causing a reversible increase in intracellular ion concentration), a direct observation cell fluorescence imaging system was built on the ultrasound-stimulated system to enhance the safety and effectiveness of ultrasound neuromodulation.

The present application provides a new research strategy for the ultrasonic neuromodulation technology of cell imaging, so that it can be applied more safely and effectively in the future ultrasound-mediated neuromodulation. The cellular ultrasound neuromodulation system provided in the embodiments of the present application can make observations in ultrasound-related research more intuitive and accurate, which is conducive to a large number of cell experiments, and can further promote the vigorous development of ultrasound neuromodulation technology.

The present application forms an integrated design by building the ultrasonic stimulation device on the cell imaging device, which can directly observe the neural activity of cells through one device, which can simplify the experimental steps, improve the experimental efficiency, and reduce the workload of scientific researchers.

In a possible design, the base includes a bottom wall and a peripheral wall, the peripheral wall is a hollow structure with two ends open, one end is fixed on the bottom wall, and a first light-transmitting hole is opened inside the bottom wall , the labeled cells are arranged on a transparent cell-climbing sheet, and the cell-climbing sheet is arranged in the hollow structure and blocks the first light-transmitting hole.

The area of the cell climbing sheet should be larger than the first light-transmitting hole, so that the first light-transmitting hole can be closed to prevent the cell fluid from dripping through the first light-transmitting hole. The fluorescent image of the cells can dynamically observe the cell state in real time.

In a possible design, the cell holding device further includes a fitting cover, the fitting cover is used to cover the other end of the peripheral wall, and a second light-transmitting hole is opened on the fitting cover .

By covering the fitting cover on the base, the cells can be arranged in the space enclosed by the fitting cover and the base, thereby preventing the cells from being polluted by the environment. A second light-transmitting hole is opened on the chimeric cover, which can be used for the passage of light. At this time, the user can use a microscope to observe the fluorescent image of the cell with the naked eye through the second light-transmitting hole.

In a possible design, the fitting cover includes a cover body and a fitting block, the fitting block is arranged on one side of the cover body, and the fitting block is adapted to the cross section of the hollow structure match. By arranging the chimeric block, the chimeric cover can be reliably covered on the base to prevent relative displacement between the two and avoid adverse effects on cell experiments.

In a possible design, a rubber gasket is arranged between the cell climbing sheet and the bottom wall. The rubber gasket has a certain viscosity and surface friction coefficient. By setting the rubber gasket, it can not only prevent the relative displacement between the cell crawling sheet and the bottom wall, but also improve the sealing effect between the cell crawling sheet and the bottom wall.

In one possible design, the base and the fitting cover are made of stainless steel. Stainless steel material has the advantages of high strength and hardness, wear resistance and corrosion resistance, strong plastic deformation ability, good sealing performance, easy processing, transportation and installation, short production cycle, not easy to break, beautiful appearance and maintenance-free. In addition, the base and the fitting cover are made of stainless steel, which can also increase the propagation speed and distance of ultrasonic waves, which is beneficial to improve the experimental efficiency.

In a possible design, the first light-transmitting hole is a circular hole with a diameter of 2-5 cm, preferably 2-3 cm.

In a possible design, the cell holding device is arranged on a stage of the cell imaging device, the stage includes two spaced apart support rods, and the objective lens passes through the two support rods. The gaps between the support rods are aligned with the light-transmitting openings.

In a possible design, the ultrasonic transducer is fixed on the cell holding device, and the ultrasonic transducer irradiates the labeled cells at an angle of 15-60 degrees, preferably 40-60 degrees. 50 degrees.

In a possible design, the ultrasonic transducer is fixedly arranged on the outer sidewall of the cell holding device (100).

In a possible design, the cell climbing sheet is made of transparent glass, with a thickness of 0.1-0.3 mm, preferably 0.1-0.18 mm.

In a possible design, the ultrasonic stimulation device includes a signal generator, a power amplifier and the ultrasonic transducer connected in sequence.

In a possible design, the cell imaging device further includes a first pinhole, a galvanometer, and a beam splitter sequentially arranged between the laser light source and the objective lens, and the beam splitter has a reflective optical path with A second pinhole, a filter and the image collector are sequentially arranged on the transmitted light path of the objective lens and the spectroscope.

In a possible design, the image collector is a PMT detector or a CCD camera.

In one possible design, the cells are neural cells, tumor cells, immune cells, stem cells or primary cells.

The application also provides that the regulatory system described in the above scheme is used in the research of movement disorders, pain, epilepsy, Parkinson's disease, depression, drug addiction, sleep dysfunction, functional recovery after nervous system damage and mental diseases Applications.

Description of drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are For some embodiments of the present application, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.

FIG. 1 is a schematic diagram of the overall structure of the cellular ultrasonic neuromodulation system provided in the embodiment of the present application.

FIG. 2 is a schematic diagram of the overall structure of the cell containing device provided in the embodiment of the present application.

FIG. 3 is an exploded view of the cell containing device provided in the embodiment of the present application.

FIG. 4 is a schematic structural diagram of a fitting cover and a base provided by an embodiment of the present application.

Figure 5 is a calcium image of ultrasound stimulated _N2A cells.

FIG. 6 is a graph showing the change of the mean relative fluorescence intensity of N ₂ A cells stimulated by ultrasound with time.

Figure 7 is a graph of the maximum mean relative fluorescence intensity of _N2A cells stimulated and unstimulated with ultrasound.

Figure 8 is a calcium fluorescence imaging image of N ₂ A cells treated with different ultrasonic sound pressures.

Figure 9 is a graph showing the time fluorescence intensity of N ₂ A cells under different sound pressures.

Figure 10 is an analysis diagram of the average fluorescence intensity of Fluo-4M under different sound pressures.

Figure 11 is a graph of the relative fluorescence intensity of individual _N2A cells after inhibitor treatment as a function of time.

Figure 12 is a graph comparing the mean maximum relative fluorescence intensities of inhibitor-treated N ₂ A cells and control N ₂ A cells.

Reference numerals: 100, cell holding device; 110, base; 111, first light-transmitting hole; 112, bottom wall; 113, peripheral wall; 120, fitting cover; 121, second light-transmitting hole; 122, cover body ; 123, chimeric block; 130, cell climbing sheet; 140, rubber gasket;

200, ultrasonic stimulation device; 210, signal generator; 220, power amplifier; 230, ultrasonic transducer;

300, cell imaging device; 310, laser light source; 320, first pinhole; 330, galvanometer; 340, beam splitter; 350, objective lens; 360, stage; 370, image collector; 380, filter; 390 , the second pinhole.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments It is a part of the embodiments of the present application, but not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

In the description of the embodiments of the present application, the terms "first" and "second" are only used for description purposes, and cannot be understood as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with "first", "second" may expressly or implicitly include at least one of that feature. In the description of the present application, "plurality" means at least two, such as two, three, etc., unless expressly and specifically defined otherwise. In this application, unless otherwise expressly specified and limited, the terms "installed", "connected", "connected", "fixed" and other terms should be understood in a broad sense, for example, it may be a fixed connection or a detachable connection , or integrated; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal communication between two elements or the interaction relationship between the two elements, unless otherwise clearly defined. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood according to specific situations.

In this application, unless otherwise expressly stated and defined, a first feature "on" or "under" a second feature may be in direct contact with the first and second features, or the first and second features indirectly through an intermediary touch. Also, the first feature being "above", "over" and "above" the second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is level higher than the second feature. The first feature being "below", "below" and "below" the second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature has a lower level than the second feature.

In the description of the present application, it should be understood that the orientation or positional relationship (if any) indicated by the terms "inside", "outside", "upper", "bottom", "front", "rear", etc. is The orientation or positional relationship shown in FIG. 1 is only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot It is construed as a limitation of this application.

Neuromodulation technology is a biomedical engineering technology that uses implantable or non-implantable technology to change the activity of the central nervous system, peripheral nerve or autonomic nervous system by electrical stimulation or drug means to improve the symptoms of the sick population and improve the quality of life.

Neuromodulation is one of the most rapidly developing disciplines in the field of medical science in the past two decades, bringing subversive changes to the treatment of many diseases. The principle of neuromodulation is a biomedical engineering technology that uses light, magnetism, electricity, ultrasound and other physical or chemical means to change the signal transmission of the nervous system, regulate the activity of neurons and the neural network where they are located, and ultimately cause changes in specific brain functions. At present, nerve regulation mainly includes electrical stimulation and drug methods, and it is also the most widely used technology in clinical practice. However, these methods are invasive and require specific devices to be implanted into the body, which can cause damage to the human body.

Specifically, for electrical stimulation, the most commonly used neuromodulation techniques include deep brain stimulation (Deep Brain Stimulation). Brain Stimulation (DBS), Spinal Cord Stimulation (SCS), Vagus Nerve Stimulation (VNS) and Sacral Neuromodulation (SNM), etc. The stimulation device is applied to the human brain, spinal cord and peripheral nerves, and uses electrical signals to stimulate the nerves to achieve the purpose of treatment.

On the one hand, the above-mentioned electrical stimulation neuromodulation technology is invasive, and electrodes need to be implanted in the human body, causing certain damage to the human body. On the other hand, the above-mentioned electrical stimulation neuromodulation technology cannot directly observe the behavior of cellular neural activity.

For drug means, the most commonly used neuromodulation technology is to inject drugs into the nervous system by implanting a drug micro-pump device, using slow-release technology in the nervous system, and injecting drugs into the nervous system to achieve the purpose of treatment, which can treat cancer pain, Parkinson's disease, Alzheimer's disease Alzheimer's disease (AD), refractory spasticity and other diseases.

However, the above-mentioned drug implantation is also invasive and will cause certain damage to the human body, and this method only achieves the purpose of treating and relieving neurological diseases by injecting drugs slowly into the nerve tissue for a long time. Neurological diseases are treated from the perspective of neuromodulation mechanism.

Combined with the above analysis, it becomes more and more urgent to develop a new neuromodulation method to achieve the goal of non-invasive and good stimulation effect.

Ultrasound neuromodulation technology based on the mechanical effect of ultrasound is a new non-invasive brain stimulation and regulation technology that has emerged in recent years. It uses ultrasound of different intensities, frequencies, pulse repetition frequencies, pulse widths and durations to stimulate or stimulate the central nervous system at the stimulation site. Inhibitory effect, a reversible change in bidirectional regulation of neural function. Ultrasound-based non-invasive neuromodulation technology is considered to be one of the next-generation neuromodulation technologies with the most promising clinical translation. It has the advantages of non-invasiveness, large penetration depth, and high spatiotemporal distribution rate. Its regulatory effect has been verified on various scale targets such as neurons, nematodes, mice, and non-human primates. It modulates neural activity in deep brain regions such as the thalamus, making it a possible treatment for brain disorders such as Parkinson's, epilepsy, and depression.

Chinese patent application CN112156381A discloses an ultrasonic nerve regulation device. By adjusting the frequency of the plane sound wave emitted by the plane ultrasonic transducer, the ultrasonic focusing depth can be adjusted, which provides a simple method for ultrasonic nerve regulation and stimulation of brain nuclei of different depths. method.

Chinese patent application CN109011228A discloses a high spatial resolution ultrasonic nerve regulation method and system. By using a lower ultrasonic sound intensity, the ultrasonic nerve regulation effect can be produced only in the area where the micro-nano materials gather, and the threshold of ultrasonic nerve regulation is lowered. Improves the spatial resolution of traditional ultrasound neuromodulation.

However, the ultrasonic neuromodulation devices disclosed in the above two patents cannot directly observe the neural activity of cells, and cannot precisely control the safety and effectiveness of ultrasonic regulation.

Chinese patent application CN109771855A invents a device and method for ultrasonic regulation of nerve tissue based on multimodal imaging. By combining CT/MRI multimodal imaging guidance, ultrasonic transmitting circuit, ultrasonic probe and human-computer interaction interface, non-invasive Under the premise of higher resolution, the neural tissue of different depths can be regulated.

However, multimodal imaging guidance by CT/MRI can only guide tissue positioning at the macroscopic level, and cannot observe the neural activity behavior of cells at the microscopic level. In addition, CT/MRI imaging has disadvantages such as high cost, long appointment period, inability to long-term and real-time imaging monitoring.

To sum up, the current ultrasound neuromodulation devices usually use ultrasound or sonodynamic therapy to detect cells at different time points to study the mechanism of ultrasound on cells, but they cannot provide timely and complete information on cell changes. Transient and continuous real-time dynamic imaging monitoring of cells is still lacking.

In order to be able to observe the changes of cells caused by ultrasonic stimulation in real time, the embodiments of the present application provide a cell ultrasonic neuromodulation system. The cell ultrasonic neuromodulation system is a real-time imaging device for ultrasonically stimulated cells based on a fluorescence microscope. The neural activity of the cells is observed, and the cells can be monitored instantaneously and continuously with real-time dynamic imaging.

FIG. 1 is a schematic diagram of the overall structure of the cellular ultrasonic neuromodulation system provided in the embodiment of the present application. FIG. 2 is a schematic diagram of the overall structure of the cell storage device 100 provided by the embodiment of the present application. FIG. 3 is an exploded view of the cell storage device 100 provided by the embodiment of the present application. FIG. 4 is a schematic structural diagram of the fitting cover 120 and the base 110 provided by the embodiment of the present application.

As shown in Figure 1-4, the cellular ultrasound neuromodulation system includes:

The cell containing device 100 is used for containing the marked cells. The cell containing device 100 includes a base 110 , and a light-transmitting opening is formed on the base 110 .

The ultrasonic stimulation device 200 includes an ultrasonic transducer 230, and the ultrasonic transducer 230 is used for transmitting ultrasonic waves to the marked cells.

The cell imaging device 300 includes a laser light source 310, an objective lens 350 and an image collector 370. The laser light source 310 emits laser light to the marked cells through the objective lens 350 and the light-transmitting port, and the image collector 370 collects the marked cells through the objective lens 350. Fluorescence image.

Specifically, the cell containing device 100 is used for containing labeled cells, and here, the labeled cells may also be referred to as dyed cells, for example, by calcium ion fluorescent probes, potassium ion fluorescent probes, Stained with sodium ion fluorescent probes or other ion fluorescent probes.

Under the action of ultrasonic waves emitted by the ultrasonic transducer 230 of the ultrasonic stimulation device 200, the laser light source 310 of the cell imaging device 300 emits a specific wavelength of laser light to illuminate the marked cells, and the image collector 370 can capture the fluorescence of the cells Images, and then can observe the neural activity of cells from a microscopic point of view, and can perform instantaneous and continuous real-time dynamic imaging monitoring of cells.

The cell holding device 100 includes a base 110, on which the marked cells are arranged, a light-transmitting opening is formed on the base 110, and the objective lens 350 of the cell imaging device 300 is facing the light-transmitting opening, through which the laser light can pass. The light port illuminates the cells, and the collector 370 collects fluorescence images through the light-transmitting port. The light-transmitting port allows light to pass through to reach the cells inside. The present application does not limit the formation method of the light-transmitting port.

For example, the entire base 110 may be made of a transparent material such as glass, plastic or resin, in which case the entire base 110 constitutes the light-transmitting opening.

For another example, only the light-transmitting opening is made of a transparent material, and the other parts of the base are made of an opaque material.

For another example, as shown in FIGS. 2-4 , the entire base 110 is made of an opaque material, and a first light-transmitting hole 111 can be opened on the base 110 . constitute the light-transmitting port.

The ultrasonic stimulation device 200 includes an ultrasonic transducer 230 for emitting ultrasonic waves to the labeled cells. The ultrasound transducer 230 may be an ultrasound probe. For example, it can be a focused ultrasound probe.

Optionally, the ultrasonic transducer 230 may be any of a single-element ultrasonic transducer, a linear array ultrasonic transducer, an area array ultrasonic transducer, a phased array ultrasonic transducer, an interdigital transducer, and the like. A sort of.

Optionally, the frequency of the ultrasonic transducer 230 is 1 to 3 MHz, preferably 2 to 3 MHz, the ultrasonic sound pressure of the emission is 0.01 to 0.50 MPa, preferably 0.03 to 0.17 MPa, and the power is 40 to 60 dB, preferably 50 to 60 dB .

As shown in FIG. 1 , the ultrasonic stimulation device 200 includes a signal generator 210 , a power amplifier 220 and the ultrasonic transducer 230 which are connected in sequence.

The signal generator 210 is used for generating sine wave pulses, the power amplifier 220 is used for amplifying the sine wave pulses, and the ultrasonic transducer 230 is used for converting the amplified sine wave pulses into ultrasonic waves, and irradiating the cells.

Optionally, the ultrasonic stimulation device 200 may further include an electronic control device, which can be used to set the operating parameters of the signal generator 210 and the power amplifier 220 , and to turn on and off the ultrasonic transducer 230 .

The cell imaging device 300 is used to generate fluorescence of the labeled cells and acquire fluorescence images. For example, the cell imaging device 300 may be a fluorescence microscope or a confocal laser.

As shown in FIG. 1 , the cell imaging device 300 further includes a first pinhole 320 , a galvanometer 330 and a beam splitter 340 arranged between the laser light source 310 and the objective lens 350 in sequence, and the objective lens is provided on the reflected light path of the beam splitter 340 350 , a second pinhole 390 , a filter 380 and the image collector 370 are sequentially arranged on the transmitted light path of the beam splitter 340 .

The image collector 370 is used to collect fluorescence images, for example, a charge-coupled device (Charge-coupled device) Device, CCD) camera. In this embodiment of the present application, the image collector 370 is a photomultiplier tube (Photomultiplier Tube, PMT) detector.

Optionally, the image collector 370 can also be electrically connected to a computer, and can send the collected fluorescence image to the computer for secondary processing and display.

The laser light source 310 is used to emit the light source. The galvanometer 330 is composed of an X-Y optical scanning head, an electronic drive amplifier and an optical reflection lens. The objective lens 350 is used to magnify the specimen for the first time, and it is the most important component that determines the performance of the microscope - the level of resolution. The beam splitter 340 is capable of reflecting laser wavelengths. The filter 380 has a frequency-selective circuit or an arithmetic processing system, and has the functions of filtering out noise and separating different signals. PMT detector is a commonly used photodetector, which is a device that uses the photoelectric effect to convert optical signals into electrical signals, and is an important part of optoelectronic systems.

The first pinhole 320 and the second pinhole 390 may also be referred to as excitation pinholes and probe pinholes, respectively. The excitation pinhole and the detection pinhole are conjugated (confocal), the confocal point is the detected point, and the plane where the detected point is located is the confocal plane. The excitation light becomes a point light source after passing through the excitation pinhole, and then is reflected on the cells by the beam splitter 340 and focused by the objective lens 350, and the fluorescence generated by the excited cells is focused on the detection pinhole through the same objective lens. Behind the detection pinhole is a PMT detector, and light that is not in the focal plane cannot be detected by the PMT detector through the pinhole.

According to the cellular ultrasonic neuromodulation system provided in the embodiment of the present application, the labeled cells are stimulated by ultrasonic waves, and laser light is further emitted to the labeled cells. The neural activity of cells can be observed from a microscopic perspective, and instantaneous and continuous real-time dynamic imaging monitoring of cells can be performed, providing reliable data support for neural regulation research.

The cell ultrasonic neuromodulation system provided in the embodiment of the present application uses the mechanical effect of ultrasonic waves to change the tension of the cell membrane, and the ultrasonic radiation force acts on the cell phospholipid bilayer, and induces the opening of mechanosensitive ion channels by ultrasonic stimulation, causing ion influx ( That is, causing a reversible increase in intracellular ion concentration), a direct observation cell fluorescence imaging system was built on the ultrasound-stimulated system to enhance the safety and effectiveness of ultrasound neuromodulation.

The present application provides a new research strategy for ultrasound neuromodulation technology for in vitro cell imaging, making it safer and more effective for future ultrasound-mediated neuromodulation. The cellular ultrasound neuromodulation system provided in the embodiments of the present application can make observations in ultrasound-related research more intuitive and accurate, which is conducive to a large number of cell experiments, and can further promote the vigorous development of ultrasound neuromodulation technology.

The present application forms an integrated design by building the ultrasonic stimulation device on the cell imaging device, which can directly observe the neural activity of cells through one device, which can simplify the experimental steps, improve the experimental efficiency, and reduce the workload of scientific researchers.

The cellular ultrasound neuromodulation system provided in the embodiments of the present application can be applied to movement disorders, pain, epilepsy, Parkinson's disease, depression, drug addiction, sleep dysfunction, functional recovery after nervous system damage, mental diseases or in the study of other systemic diseases.

Optionally, the cells here (sometimes also referred to simply as cells) can be nerve cells in the deep brain region or nerve cells in the superficial brain region, but are not limited to nerve cells, and can also be tumor cells, immune cells and other cells.

Optionally, the mechanosensitive ion channel here can be a piezo1 channel, or other exogenous/endogenous mechanosensitive ion channels, such as MscL ion channel and so on.

As shown in Figures 2-4, the base 110 includes a bottom wall 112 and a peripheral wall 113. The peripheral wall 113 has a hollow structure with two open ends, one end is fixed on the bottom wall 112, and a first light-transmitting hole 111 is opened inside the bottom wall 112. The labeled cells are arranged on the transparent cell climbing sheet 130 , and the cell climbing sheet 130 is arranged in the hollow structure and blocks the first light-transmitting hole 111 .

Here, the area of the cell crawling sheet 130 should be larger than the first light-transmitting hole 111, so that the first light-transmitting hole 111 can be closed to prevent the cell fluid from dripping through the first light-transmitting hole 111, and the image collector 370 can pass through the first light-transmitting hole 111. The light-transmitting hole 111 collects the fluorescence images of the cells on the cell climbing sheet 130 in real time, so that the state of the cells can be dynamically observed in real time.

Specifically, at this time, the cells can be seeded on the cell slide 130 and stained with an ionic fluorescent reagent. The ultrasonic transducer 230 arranged on the side wall of the cell holding device 100 starts ultrasonic stimulation, and the ultrasonic energy is irradiated on the cell slide 130, and the mechanosensitive ion channel of the cell (such as the Piezo1 channel, introduced by Coste et al. in mice in 2010). A mechanosensitive ion channel found in neuroma cells N ₂ A) opens, causing an increase in the influx of ions (eg, calcium ions) that can be visualized using a cell fluorescence imaging system (ie, cell imaging device 300 ).

The cross-sectional shape of the first light-transmitting hole 111 may be any shape such as a circle, a rectangle, a trapezoid, a parallelogram, a racetrack, etc., which is not limited in this application.

The shape of the cell climbing sheet 130 and the cross-sectional shape of the first light-transmitting hole 111 may be the same or different. For example, the shape of the cell crawling sheet 130 can also be any shape such as a circle, a rectangle, a trapezoid, a parallelogram, a racetrack shape, and the like.

In the embodiment of the present application, the first light-transmitting hole 111 is a circular hole with a diameter of 2-5 cm, preferably 2-3 cm. The cell climbing sheet 130 is also circular, and its diameter is larger than that of the first light-transmitting hole 111 .

The shape, size, thickness and material of the cell climbing sheet 130 in this application can be flexibly designed. The round thin glass used in this embodiment has a thickness of 0.1-0.3 mm, preferably 0.1-0.18 mm, which is convenient for cells For the imaging observation, other transparent materials for easy imaging can also be used, for example, plastic or resin materials can be used.

As shown in FIGS. 2-4 , in the embodiment of the present application, the shape of the bottom wall 112 (that is, the shape of the opening of the peripheral wall 113 ) is a circle. In other embodiments, the shape of the bottom wall 112 can also be a rectangle or a trapezoid. , parallelogram, racetrack shape and other arbitrary shapes.

As shown in FIGS. 2-4 , in the embodiment of the present application, the cell containing device 100 further includes a fitting cover 120 , the fitting cover 120 is used to cover the other end of the peripheral wall 113 , and the fitting cover 120 is provided with a first Two light-transmitting holes 121 .

By covering the fitting cover 120 on the base 110 , the cells can be placed in the space enclosed by the fitting cover 120 and the base 110 , thereby preventing the cells from being polluted by the environment. A second light-transmitting hole 121 is opened on the fitting cover 120 for the passage of light. At this time, a user can observe the fluorescent image of the cell with the naked eye through the second light-transmitting hole 121 using a microscope.

The second light-transmitting hole 121 is disposed opposite to the first light-transmitting hole 111 , and the shapes and sizes of the two may be the same or different. In the embodiment of the present application, the second light-transmitting hole 121 is also a circular hole, and has the same size as the first light-transmitting hole 111 .

In the embodiment of the present application, the base 110 and the fitting cover 120 are made of stainless steel.

Stainless steel material has the advantages of high strength and hardness, wear resistance and corrosion resistance, strong plastic deformation ability, good sealing performance, easy processing, transportation and installation, short production cycle, not easy to break, beautiful appearance and maintenance-free. In addition, the base and the fitting cover are made of stainless steel, which can also increase the propagation speed and distance of ultrasonic waves, which is beneficial to improve the experimental efficiency.

Optionally, the base 110 and the fitting cover 120 may also be made of other metal materials (eg, aluminum alloy or copper alloy).

Optionally, the base 110 and the fitting cover 120 may also be made of other transparent or opaque materials such as plastic, rubber, resin, glass, and the like.

As shown in FIGS. 2-4 , in the embodiment of the present application, the fitting cover 120 includes a cover body 122 and a fitting block 123 , the fitting block 123 is arranged on one side of the cover body 122 , and the fitting block 123 is connected to the hollow structure. Sections fit. At this time, the second light-transmitting hole 121 penetrates through the cover body 122 and the fitting block 123 .

By arranging the fitting block 123 , the fitting cover 120 can be reliably covered on the base 110 to prevent relative displacement between the two and avoid adverse effects on the cell experiment.

As shown in FIGS. 2-4 , in the embodiment of the present application, a rubber gasket 140 is disposed between the cell crawling sheet 130 and the bottom wall 112 .

The rubber gasket 140 has a certain viscosity and surface friction coefficient. By arranging the rubber gasket 140, the relative displacement between the cell crawling sheet 130 and the bottom wall 112 can not only be prevented, but also the sealing between the cell crawling sheet 130 and the bottom wall 112 can be improved. Effect.

Optionally, the rubber gasket 140 includes two, one is located between the cell climbing sheet 130 and the bottom wall 112 , and the other is located inside the cell climbing sheet 130 . At this time, the cell-climbing sheet 130 is placed between the two rubber washers 140 , which can better fix the cell-climbing sheet 130 .

As shown in FIG. 1 , the cell holding device 100 is disposed on the stage 360 of the cell imaging device 300 , the stage 360 includes two supporting rods arranged at intervals, and the objective lens 350 is aligned through the gap between the two supporting rods The light-transmitting port (ie, the first light-transmitting hole 111 ).

As shown in FIG. 2 , the ultrasonic transducer 230 is fixed on the cell holding device 100 , and the ultrasonic transducer 230 irradiates the cells at an angle of 15-60 degrees.

Specifically, the ultrasonic transducer 230 is fixedly disposed on the outer side wall of the cell holding device 100 (eg, on the outer side wall of the fitting cover 120 ), and the distance between the emission direction of the ultrasonic transducer 230 and the vertical direction is The included angle is 15-60 degrees, so that the cells can be irradiated better, preferably 40-50 degrees. For example, the ultrasound transducer 230 illuminates the labeled cells at a 45 degree angle.

Optionally, in other embodiments, the ultrasonic transducer 230 may also be fixedly disposed at other positions of the cell storage device 100 , for example, disposed on the outer sidewall of the cell storage device 100 .

Optionally, the ultrasonic transducer 230 may be fixedly disposed on the cell holding device 100 in any manner, such as binding with a bandage, adhesive tape, glue, and clipping.

The research team of the inventor has used the cellular ultrasound neuromodulation system provided in the examples of this application for experimental verification, and the mouse neuroma cell (N ₂ A cell) experiment was carried out through the cellular ultrasound neuromodulation system, which can be observed from a microscopic perspective. Ultrasound to N ₂ A cells regulates neural activity behavior, and can perform instantaneous and continuous real-time dynamic imaging monitoring of N ₂ A cells, providing reliable data support for subsequent research.

The technical solutions in the present application will be clearly and completely described below with reference to the embodiments in the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

Example 1: Ultrasound stimulated cellular calcium fluorescence imaging

①Culture and inoculate mouse neuroma cells (N ₂ A cells), trypsinize N ₂ A cells in log phase, count the cells, and inoculate regulated cells at a density of (4~6)×10 ⁵ cells/ml in 6 In the well plate (cell slides have been placed in the 6-well plate), culture in a 37-degree constant temperature incubator for 24-48 hours.

②Ca ²⁺ fluorescent probe Fluo 4-AM to label cells: add an appropriate amount of calcium ion fluorescent indicator Fluo-4 AM to 20% Pluronic F-127 to prepare a 0.5 mmol/L stock solution, which should be diluted to 2.5~3uM probe working solution. When N ₂ A cells grow to 75~85%, discard the medium and wash twice with Phosphate Buffer Saline (PBS). Then add probe working solution to each cell slide, and stain at room temperature for 15-30 min in the dark. After staining, the dye solution was aspirated, washed with PBS, and added to Hank's Balanced Salt Solution (HBSS) for detection. Then place the cell slide on the base.

③Ultrasonic stimulation: Start the ultrasonic stimulation system to stimulate cells to climb the nerve cells on the slice.

④ Cell calcium fluorescence imaging: Cell calcium fluorescence imaging based on fluorescence microscope was used to monitor the changes of cytoplasmic calcium ion content induced by ultrasound stimulation of N ₂ A, and the imaging of Ca ²⁺ fluorescent probes was directly observed under the microscope.

⑤Experimental results: Fig. 5 is a calcium imaging image of N ₂ A cells stimulated by ultrasound. Among them, the first column (BF column) in Figure 5 is the brightfield image of ultrasound-stimulated N2A cells, the _second column (Fluo-4 column) is the Fluo-4 staining map, and the third column (PI column) is the PI Staining map, the fourth column (Merge column) is a fusion map of Fluo-4 staining and PI staining. In Figure 5, US(-) indicates no ultrasound stimulation, US(+) indicates ultrasound stimulation, and US(+5min) indicates five minutes after ultrasound stimulation. FIG. 6 is a graph showing the change of the mean relative fluorescence intensity of N ₂ A cells stimulated by ultrasound with time. Among them, US means receiving ultrasound stimulation, and Control means not receiving ultrasound stimulation. Figure 7 is a graph of the maximum mean relative fluorescence intensity of _N2A cells stimulated and unstimulated with ultrasound.

As shown in Figures 5-7, the results showed that the calcium fluorescence of N ₂ A cells was significantly enhanced immediately after ultrasonic stimulation, and the fluorescence intensity decreased after 5 min. Among the cells with elevated calcium, there were few PI-positive cells, indicating that ultrasound irradiation did not cause cell death and that calcium influx was not caused by increased cell membrane permeability. In contrast, _N2A cells that were not irradiated with ultrasound showed no obvious fluorescence changes.

Example 2: Sound pressure-dependent increase of cytoplasmic free calcium levels in N ₂ A cells stimulated by ultrasound

①The mouse neuroma cells (N ₂ A cells) were inoculated on the cell slides, and then the cells were labeled with the Ca ²⁺ fluorescent probe Fluo 4-AM. The specific method was the same as that in Example 1.

②Ultrasonic stimulation: Start the ultrasonic stimulation system and set different ultrasonic sound pressures. In this example, 0.03, 0.06, 0.11 and 0.17 Mpa were used, and then the changes of calcium ion concentration were observed by cell calcium fluorescence imaging.

③Experimental results: Fig. 8 is the calcium fluorescence imaging diagram of N ₂ A cells treated with different ultrasonic sound pressures; Fig. 9 is the time fluorescence intensity curve diagram of N ₂ A cells under different sound pressures. Figure 10 is an analysis diagram of the average fluorescence intensity of Fluo-4M under different sound pressures.

As shown in Figure 8-10, the results show that when the sound pressure is 0.03 MPa, the maximum average relative fluorescence intensity value of N ₂ A cells is 0.03±0.03, and when the sound pressure is 0.06 MPa, the maximum average relative fluorescence intensity value of N ₂ A cells is 0.09±0.05, when the sound pressure was 0.11 MPa, the maximum mean relative fluorescence intensity value of N ₂ A cells was 0.68±0.15 (P=0.001<0.05), when the sound pressure was 0.17 MPa, the maximum mean relative fluorescence intensity value of N ₂ A cells was was 1.48±0.19 (P=1.1x10-5<0.05). The maximum mean relative fluorescence intensity of cells increased significantly when the sound pressure was 0.11 MPa and 0.17 MPa. The maximum mean relative fluorescence intensity of cells increased with increasing sound pressure.

Example ₃ : The role of piezo1 channels on the cell membrane in ultrasound-stimulated elevation of calcium levels in N2A cells

①The mouse neuroma cells (N ₂ A cells) were inoculated on the cell slides, and then the cells were labeled with the Ca ²⁺ fluorescent probe Fluo 4-AM. The specific method was the same as that in Example 1.

② piezo1 inhibitor-treated cells: N ₂ A cells were treated with 3uM piezo1 channel-specific blocker GsMTx-4 for 30 min.

③Ultrasonic stimulation: Start the ultrasonic stimulation system, set the ultrasonic frequency to 2 MHz and the sound pressure of 0.17 MPa to stimulate the N ₂ A cells for 10 s, and then observe the change of calcium ion concentration by cell calcium fluorescence imaging.

④Experimental results: Fig. 11 is a graph showing the change of relative fluorescence intensity of single N ₂ A cells with time after inhibitor treatment. Figure 12 is a graph comparing the mean maximum relative fluorescence intensities of inhibitor-treated N ₂ A cells and control N ₂ A cells.

As shown in Figures 11 and 12, the results showed that the average maximum relative fluorescence intensity of N ₂ A cells treated with GsMTx-4 was 1.08 ± 0.07, and the average maximum relative fluorescence intensity of control group N ₂ A cells was 1.36 ± 0.06. The results showed that the piezo1 channel-specific blocker GsMTX-4 reduced the increase of calcium levels in ultrasound-stimulated N ₂ A cells (p=0.004<0.05), indicating that piezo1 channels were also involved in the increase of calcium levels in ultrasound-stimulated N ₂ A cells. high.

The above are only specific embodiments of the present application, but the protection scope of the present application is not limited to this. should be covered within the scope of protection of this application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims (16)

A cellular ultrasonic neuromodulation system, characterized in that, comprising: A cell containing device (100) for containing marked cells, the cell containing device (100) includes a base (110), and a light-transmitting opening is formed on the base (110); an ultrasonic stimulation device (200), comprising an ultrasonic transducer (230), the ultrasonic transducer (230) is used to emit ultrasonic waves to the labeled cells; A cell imaging device (300), comprising a laser light source (310), an objective lens (350) and an image acquisition device (370), the laser light source (310) is directed to the marker through the objective lens (350) and the light-transmitting port After the cells emit laser light, the image collector (370) collects a fluorescent image of the labeled cells through the objective lens (350). The regulation system according to claim 1, wherein the base (110) comprises a bottom wall (112) and a peripheral wall (113), the peripheral wall (113) is a hollow structure with two ends open, and one end is fixed to the On the bottom wall (112), a first light-transmitting hole (111) is opened inside the bottom wall (112), and the marked cells are arranged on the transparent cell crawling sheet (130). A climbing sheet (130) is arranged in the hollow structure and blocks the first light-transmitting hole (111). The regulation system according to claim 2, characterized in that, the cell containing device (100) further comprises a fitting cover (120), and the fitting cover (120) is used to cover the peripheral wall (113) ), a second light-transmitting hole (121) is opened on the fitting cover (120). The regulation system according to claim 3, characterized in that, the fitting cover (120) comprises a cover body (122) and a fitting block (123), and the fitting block (123) is disposed on the cover body On one side of (122), the fitting block (123) is adapted to the cross-section of the hollow structure. The regulation system according to any one of claims 2-4, characterized in that, a rubber gasket (140) is provided between the cell crawling sheet (130) and the bottom wall (112). The regulation system according to claim 3 or 4, characterized in that, the base (110) and the fitting cover (120) are made of stainless steel. The control system according to any one of claims 2-4, wherein the first light-transmitting hole (111) is a circular hole with a diameter of 2-5 cm. The regulation system according to any one of claims 1-4, characterized in that, the cell containing device (100) is arranged on the stage (360) of the cell imaging device (300), so The object stage (360) includes two supporting rods arranged at intervals, and the objective lens (350) is aligned with the light-transmitting port through a gap between the two supporting rods. The regulation system according to any one of claims 1-4, characterized in that, the ultrasonic transducer (230) is fixedly arranged on the cell containing device (100), and the ultrasonic transducer (230) irradiating the labeled cells at an angle of 15-60 degrees. The regulation system according to any one of claims 1-4, characterized in that, the ultrasonic transducer (230) is fixedly arranged on the outer side wall of the cell holding device (100). The regulation system according to any one of claims 2-4, wherein the cell climbing sheet (130) is made of transparent glass, and has a thickness of 0.1-0.3 mm. The regulation system according to any one of claims 2-4, wherein the ultrasonic stimulation device (200) comprises a signal generator (210), a power amplifier (220) and the ultrasonic transducer connected in sequence device (230). The regulation system according to any one of claims 1-4, characterized in that, the cell imaging device (300) further comprises a laser light source (310) and the objective lens (350) arranged in sequence between the laser light source (310) and the objective lens (350). a first pinhole (320), a galvanometer (330) and a beam splitter (340), the objective lens (350) is provided on the reflected light path of the beam splitter (340), and the transmitted light of the beam splitter (340) A second pinhole (390), a filter (380) and the image collector (370) are arranged on the road in sequence. The control system according to any one of claims 1-4, characterized in that, the image acquisition device (370) is a PMT detector or a CCD camera. The regulatory system according to any one of claims 1-4, wherein the cells are nerve cells, tumor cells, immune cells, stem cells or primary cells. The regulatory system according to any one of claims 1-15 in movement disorders, pain, epilepsy, Parkinson's disease, depression, drug addiction, sleep dysfunction, functional recovery after nervous system damage, and psychiatric disorders applications in research.