Method for capturing cells based on single-beam focused sound field three-dimensional space

Abstract

The invention relates to the field of ultrasonic control particles and microfluids, in particular to a method for capturing cells in a three-dimensional space based on a single-beam focused sound field, which comprises the following steps: selecting a biocompatible solution, regulating and controlling an acoustical contrast factor, designing a single-beam focused sound field with excellent focusing performance, and forecasting a three-dimensional sound radiation force. According to the present invention, the acoustic contrast factor of the cell is modulated by changing the concentration of the iodixanol solution with good biological compatibility, and particularly the acoustic contrast factor is changed into the negative number, such that the non-contact type three-dimensional single cell capture is achieved, and the activity of the cell can be maintained. The method effectively excavates the potential of three-dimensional cell capture of the single-beam focused sound field, and has important application value in the fields of precise measurement of cell mechanics, tissue engineering, particle assembly and the like.

Description

Technical Field

[0001] The invention belongs to the field of ultrasonic manipulation of particles and microfluids, and in particular relates to a method for capturing cells in three-dimensional space based on a single-beam focused acoustic field. Background Art

[0002] Precision manipulation of single cells is an essential part of technologies such as assisted reproduction, tissue engineering, and cell mechanics, and is crucial to the field of life sciences. Currently, single-cell manipulation is generally achieved clinically by relying on microtubule technology, which directly extracts cells through the pressure difference inside and outside the microtubule. However, this contact manipulation method inevitably causes physical damage to cells and may cause chemical cross-contamination, thus limiting its wide application and safety.

[0003] In view of the above limitations of contact manipulation technology, non-contact manipulation technologies based on various physical fields have emerged, including optical tweezers, magnetic tweezers, and acoustic tweezers. Among these non-contact manipulation technologies, ultrasonic-based acoustic tweezers technology has attracted more and more attention in the field of cell manipulation due to its gentleness, non-invasiveness, no need for cell labeling, and strong manipulation force.

[0004] There are different types of acoustic tweezers technology. Among them, single-beam acoustic tweezers based on focused acoustic fields have the natural advantage of selective manipulation. However, in practical applications, due to the positive acoustic contrast factor of cells in water, cells are often captured at the minimum acoustic pressure amplitude, which contradicts the characteristic of single-beam focused acoustic fields that the acoustic pressure amplitude is the largest at the focus, thus hindering the three-dimensional capture of typical human cells by single-beam focused acoustic fields. It is worth noting that focused acoustic waves have been widely used in the field of ultrasound medicine, but this specific limitation in cell manipulation needs to be addressed urgently. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the present invention proposes a method for capturing cells in three-dimensional space based on a single-beam focused acoustic field, using a special cell-friendly solution. This solution can reverse the acoustic contrast factor of the cell, so that the cell can be effectively captured at the focus of the single-beam focused acoustic field, that is, the position where the sound pressure amplitude is the largest. By selecting a solution with good biocompatibility to regulate the acoustic contrast factor, not only the three-dimensional capture of typical human cells using a single-beam focused acoustic field is achieved, but also the activity of the cell is ensured to be maintained during the manipulation process.

[0006] The technical solution of the present invention is as follows:

[0007] A method for capturing cells in three-dimensional space based on a single-beam focused acoustic field, which is characterized by:

[0008] S1. Select a solution with good biocompatibility and adjust the solution so that the acoustic contrast factor of the cells in the solution is reversed from a positive value to a negative value;

[0009] S2. Design single beam focused sound field;

[0010] S3. Calculate the three-dimensional acoustic radiation force under the action of the single-beam focused acoustic field to achieve three-dimensional capture of cells.

[0011] Preferably, the solution in step S1 is an iodixanol solution, which can achieve the reversal of the cell acoustic contrast factor, and the reversal effect becomes more obvious as the concentration of the solution increases.

[0012] Preferably, the step S1 further comprises performing a parameter study on the concentration of the solution to determine the quantitative relationship between the cell acoustic contrast factor and the solution concentration.

[0013] Preferably, the step S2 designs a single-beam focused sound field, designs a finite aperture focused sound field using the Fresnel principle, and adjusts the aperture size and the excitation frequency.

[0014] 8. Preferably, the frequency of the single-beam focused sound field is 40 MHz, the lateral side lobe is 0.26 times the main lobe, and the axial side lobe is 0.23 times the main lobe, which has the advantages of good focusing and low side lobes.

[0015] Preferably, the single-beam focused sound can form a high-quality focused sound field at the design frequency. When the driving frequency is higher than the design frequency, the focus and the cell capture position move away from the sound source. When the driving frequency is lower than the design frequency, the focus and the cell capture position move toward the sound source.

[0016] Preferably, the three-dimensional acoustic radiation force is calculated in step S3, based on the angular spectrum method, by decomposing the acoustic field into a superposition of a series of plane waves, and using the prior solution of the plane waves to calculate the cell under any acoustic field incidence.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) By selecting a solution with good biocompatibility and modulating its concentration to change the acoustic contrast factor of the cells, the three-dimensional capture of typical human cells can be achieved using a conventional single-beam focused acoustic field, without the need for special forms of acoustic fields such as focused acoustic vortices.

[0019] (2) The selected biocompatible solution (such as iodixanol solution) is not only cell-friendly and can reverse the acoustic contrast factor of cells, but also can maintain cell activity. This feature is crucial for cell manipulation experiments because it ensures that cells are not damaged during the capture process, thereby improving the success rate of the experiment and the reliability of the data.

[0020] (3) The finite aperture focused acoustic field is designed using the Fresnel principle. By adjusting the aperture size, excitation frequency and other parameters, a balance between the practicality and economy of the focused acoustic field is achieved. It not only has excellent focusing performance, but also can manipulate cells more precisely, improving the accuracy and stability of cell manipulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the control of the acoustic ratio factor;

[0022] Figure 2 It is the sound intensity distribution of a single beam focused sound field in water calculated based on the angular spectrum method, where a is the sound intensity distribution in the propagation plane and b is the sound intensity distribution in the focal plane;

[0023] Figure 3 An example of a focused acoustic field device.

[0024] Figure 4 is the calculation result of the three-dimensional acoustic radiation force in the Rayleigh zone, where a is the curve of the axial acoustic radiation force varying with the axial position, b is the Gorkov potential distribution in the propagation plane, c is the Gorkov potential distribution in the focal plane, and d is the curve of the lateral acoustic radiation force in the focal plane varying with the lateral position;

[0025] Figure 5 is the result of the calculation of the three-dimensional acoustic radiation force outside the Rayleigh zone, where a is a fixed position (x s ,y s ,z s )=(0,0,30)μm, the axial acoustic radiation force on the cell changes with the cell / wavelength size ratio, b is the fixed position (x s ,y s ,z s )=(8,0,0)μm, the lateral acoustic radiation force on the cell changes with the cell / wavelength size ratio, c is the axial acoustic radiation force changes with the axial position when the cell size is a=0.41λ, d is the lateral acoustic radiation force changes with the lateral position when the cell size is a=0.41λ, and the axial capture position is selected as z according to c. s =4.2μm;

[0026] Figure 6 It is a schematic diagram of the dynamic changes of focal length and cell capture position inside and outside the Rayleigh zone when the excitation signal frequency is changed, wherein a, d, and g are the sound field distributions in the propagation plane in the iodixanol solution at 38 MHz, 40 MHz, and 42 MHz, respectively; b, e, and h are the curves of the axial acoustic radiation force of the cells in the Rayleigh zone varying with the axial distance at 38 MHz, 40 MHz, and 42 MHz, respectively; c, f, and i are the curves of the axial acoustic radiation force of the cells outside the Rayleigh zone varying with the axial distance at 38 MHz, 40 MHz, and 42 MHz, respectively. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be fully and clearly described below in conjunction with the embodiments and drawings, but this should not limit the protection scope of the present invention.

[0028] This embodiment provides a method for capturing cells in three-dimensional space based on a single-beam focused acoustic field. The method achieves stable capture of cells in three-dimensional space by regulating the acoustic contrast factor and using a specially designed focused acoustic field. The method specifically includes the following steps:

[0029] S1. Adjust the sound contrast factor:

[0030] like Figure 1 As shown in the figure, the color of the pseudo-color image represents the size of the acoustic contrast factor. By regulating the properties of the solution, such as concentration and temperature, the acoustic contrast factor can be reversed from a positive value to a negative value, so that the three-dimensional acoustic radiation force plays a role of restoring force, thereby achieving three-dimensional capture of cells in the focal area.

[0031] S2. Produce a focused sound field:

[0032] The present invention adopts a specially designed sound field focusing device, such as Figure 3 As shown in the figure, the device intercepts the spherical focusing wave through a finite plane to obtain two sets of concentric positive and negative electrodes with opposite phases. These electrodes excite the piezoelectric material to vibrate, thereby forming a focused acoustic field. Figure 2 As shown, the spatial distribution of the sound intensity of the sound field generated by the focusing device has a good focusing structure, ensuring the precise application of the acoustic radiation force.

[0033] S3. Calculate the acoustic radiation force:

[0034] This embodiment is based on the angular spectrum method to calculate the acoustic radiation force on particles inside and outside the Rayleigh region. The general expression of the three-dimensional acoustic radiation force on particles of any size in any sound field is:

[0035]

[0036] Among them, the coefficients are defined as:

[0037]

[0038] When the particle is in the Rayleigh region, that is, a<<λ, the acoustic radiation force formula can be degenerated into the Gor'kov theory. The Gor'kov potential of small particles and their corresponding acoustic radiation force are calculated using the Gor'kov theory. For large particles outside the Rayleigh region, first calculate the s ,y s ,z s )=(0,0,30)μm and (x s ,y s ,zs )=(8,0,0)μm) changes with cell size / wavelength to verify whether the acoustic radiation force will be negative at a specific size, which is a necessary condition for achieving three-dimensional cell capture. Next, extract the cell size corresponding to the negative acoustic radiation force, and calculate the changes in the axial and lateral acoustic radiation forces of cells of this size with distance. Through this step, the capture effect can be verified and the capture parameters can be optimized.

[0039] Example

[0040] The following is an example of using single-beam focused acoustic tweezers to achieve three-dimensional cell capture.

[0041] The design frequency of the single-beam focusing acoustic tweezers device is 40MHz, the focal length is 1mm, and the number of electrode circles n is 26. The captured cell type is MCF-7 breast cancer cells, and the solution with good biocompatibility is selected as a 60% iodixanol solution, which can achieve the inversion of the acoustic contrast factor while maintaining the activity of the cells.

[0042] The density of iodixanol solution and the speed of sound satisfy the following relationship: Figure 1 As shown in the black curve, as the concentration of iodixanol solution increases (from left to right), the acoustic contrast factor gradually reverses from a positive value to a negative value. When the cell size is a = 0.0375 μm, the Gor'kov potential of the small cell and its corresponding acoustic radiation force can be calculated using the Gor'kov formula, as shown in Figure 4 As shown in , the acoustic radiation force can play a restoring role and achieve three-dimensional capture of MCF-7 cells in the Rayleigh region. Outside the Rayleigh region, since the long wavelength limit is not met, the general expression of the acoustic radiation force is needed to perform capture calculations. Figure 5 (a) and 5(b), first calculate the s ,y s ,z s )=(0,0,30)μm and (x s ,y s ,z s )=(8,0,0)μm, the acoustic radiation force on the cells at two fixed positions varies with the cell size / wavelength. It is found that when the cell radius is a=15.375μm, the axial acoustic radiation force and the lateral acoustic radiation force are both negative, which meets the necessary conditions for three-dimensional cell capture. Next, calculate the axial acoustic radiation force ( Figure 5 (c)) and lateral acoustic radiation force ( Figure 5(d) As the distance changes, it is found that both the axial acoustic radiation force and the lateral acoustic radiation force can act as a restoring force, allowing the MCF-7 cells to be captured three-dimensionally in the focal area. When the electrodes are stimulated near the design frequency of the focusing acoustic tweezers device, 40 MHz (38 MHz-42 MHz), it can be found that the focal length and the cell capture position inside and outside the Rayleigh region will change dynamically with the excitation frequency. When the excitation frequency is greater than the design frequency, the focal position and the capture position move away from the device, and when the excitation frequency is less than the design frequency, the focal position and the capture position move toward the device.

[0043] It is explained that in this embodiment, single-beam focused acoustic tweezers can be used to achieve three-dimensional capture of MCF-7 cells.

Claims

1. A method for capturing cells in three-dimensional space based on a single-beam focused acoustic field, characterized in that: include: S1. Select a solution with good biocompatibility and adjust the solution so that the acoustic contrast factor of the cells in the solution is reversed from a positive value to a negative value; S2. Design single beam focused sound field; S3. Calculate the three-dimensional acoustic radiation force under the action of the single-beam focused acoustic field to achieve three-dimensional capture of cells.

2. The method for capturing cells in three-dimensional space based on single-beam focused acoustic field according to claim 1, characterized in that: The solution in step S1 is an iodixanol solution, which can achieve the reversal of the cell acoustic contrast factor, and the reversal effect becomes more obvious as the concentration of the solution increases.

3. The method for capturing cells in three-dimensional space based on single-beam focused acoustic field according to claim 1, characterized in that: The step S1 also includes performing a parameter study on the concentration of the solution to determine the quantitative relationship between the cell acoustic contrast factor and the solution concentration.

4. The method for capturing cells in three-dimensional space based on single-beam focused acoustic field according to claim 1, characterized in that: The step S2 designs a single-beam focused sound field, uses the Fresnel principle to design a finite aperture focused sound field, and adjusts the aperture size and the excitation frequency.

5. The method for capturing cells in three-dimensional space based on single-beam focused acoustic field according to claim 4, characterized in that: The single-beam focused sound field frequency is 40 MHz, the lateral side lobe is 0.26 times the main lobe, and the axial side lobe is 0.23 times the main lobe, which has the advantages of good focusing and low side lobes.

6. The method for capturing cells in three-dimensional space based on single-beam focused acoustic field according to claim 4, characterized in that: The single-beam focused sound can form a high-quality focused sound field at the design frequency. When the driving frequency is higher than the design frequency, the focus and the cell capture position move away from the sound source. When the driving frequency is lower than the design frequency, the focus and the cell capture position move toward the sound source.

7. The method for capturing cells in three-dimensional space based on single-beam focused acoustic field according to claim 1, characterized in that: The three-dimensional acoustic radiation force is calculated in step S3 based on the angular spectrum method by decomposing the acoustic field into a superposition of a series of plane waves and using the prior solution of the plane waves to calculate the acoustic radiation force on particles such as cells under any incident acoustic field.

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