Apparatus and method for joint imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity, and application

Abstract

Disclosed are an apparatus and method for the joint imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity, and an application. The method for the joint imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity comprises: providing muscle cells; performing calcium ion staining on the muscle cells; and applying, to the muscle cells, which have been subjected to calcium ion staining, mechanical stimulation conjugated with the sarcomere contraction length of the muscle cells and oriented perpendicular to a sarcomere direction, and using a high-speed spinning disk confocal microscope to perform observation.

Description

A device and method for combined imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity and application TECHNICAL FIELD

[0001] The present application relates to the technical field of cell mechanics testing, in particular to a device and method for combined imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity and application. BACKGROUND

[0002] The three types of muscle cells in the human body are cardiac muscle, skeletal muscle and smooth muscle, which bear different mechanical sensations and have different mechanisms. Mechanical stimulation can be transmitted to different parts of the cell through the cell membrane and the cytoskeleton structure, causing a series of activities and inducing a series of complex transcriptomics and cellomics processes, further driving changes in cell morphology and function, such as maintaining cell morphology, growth, proliferation, apoptosis, migration, differentiation, gene expression, and protein synthesis. Calcium ions, as the second messenger in cells, play a core role in the process of mechanical transduction. Exploring the effects of mechanical stimulation on cells has the following significance: first, mechanical stimulation can promote muscle cell proliferation and migration, effectively regulate cell differentiation in stem cell research and regenerative medicine; second, it can regulate cell arrangement direction and spatial structure, enabling oriented 3D printing of cells in tissue engineering; and third, it can help to understand cell signaling, gene expression and cell microenvironment.

[0003] Force signals are directional vectors, and muscle cells also have a clear spatial structure. Sarcomeres are the basic units of rhythmic contraction of cells, so it is necessary to study the biological response of sarcomeres in muscle cells to mechanical stimulation of different directions, frequencies and amplitudes. The biological response mainly revolves around the activity of calcium ions on the sarcoplasmic reticulum. However, there is currently no device or method that can observe the activity of calcium ions on the sarcoplasmic reticulum under different mechanical stimulation. The existing technology cannot simulate the mechanical stimulation of the sarcomere contraction length and frequency conjugation of cells in the actual physiological environment, making it difficult to accurately monitor the activity of calcium ions on the sarcoplasmic reticulum.

[0004] Therefore, the prior art still needs to be improved. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a device and method for combined imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction, and application, which aims to solve the problem that the existing technology cannot simulate the actual mechanical stimulation of cells in the physiological environment, making it difficult to accurately monitor the activity of calcium ions on the sarcoplasm.

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

[0007] In a first aspect of the present application, a method for combined imaging of high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity conjugated with sarcomere contraction is provided, comprising the steps of: (1) providing a muscle cell; (2) performing calcium ion staining treatment on the muscle cell; (3) applying mechanical stimulation to the calcium ion staining treated muscle cell, which is conjugated with the sarcomere contraction length of the muscle cell and perpendicular to the direction of the sarcomere, and observing using a high-speed rotating disk confocal microscope.

[0008] Optionally, the calcium ion fluorescent probe used in the calcium ion staining treatment comprises one of Fluo-4 AM or Fluo-8 AM.

[0009] In a second aspect of the present application, a device for combined imaging of high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity conjugated with sarcomere contraction is provided, comprising: a cell regulation culture assembly comprising a bottom wall and a peripheral wall extending from the bottom wall, the bottom wall and the peripheral wall surrounding a groove, the bottom wall being provided with a through hole, and a glass slide being embedded in the through hole; a mechanical stimulation generation assembly arranged in the groove and close to the peripheral wall; and a microscopic observation assembly configured to observe the contents in the groove through the glass slide; when a calcium staining treated muscle cell is placed in the device for combined imaging of high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity conjugated with sarcomere contraction, the mechanical stimulation generation assembly applies mechanical stimulation to the calcium staining treated muscle cell, which is perpendicular to the direction of sarcomere contraction of the muscle cell and conjugated with the sarcomere contraction length of the muscle cell.

[0010] Optionally, the through hole is circular, the glass slide is circular, and the thickness of the glass slide is 0.1-0.3 mm.

[0011] Optionally, the mechanical stimulation generation assembly is a mechanical wave generator.

[0012] Optionally, the mechanical wave generator comprises a power supply, a signal generator, a power amplifier, and a planar piezoelectric ceramic, the power supply, the signal generator, the power amplifier, and the planar piezoelectric ceramic being electrically connected to each other; wherein the power amplifier has a power amplification decibel number of 20-40 dB; the signal generator has a frequency of 1-10 MHz, a peak-to-peak value of 2500, an impedance of 50 Ω, and a waveform comprising at least one of Sine, Square, Ramp, Pulse, Noise, Dualtone, Harm, or Arb.

[0013] Optionally, the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device conjugated with muscle sarcomere contraction comprises a limiting component arranged in the groove and abutting against the peripheral wall, and the mechanical stimulation generating component is fixedly connected with the limiting component.

[0014] In a third aspect, the application provides a method for monitoring calcium ion activity on the sarcoplasmic reticulum by using the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device according to the second aspect, which comprises the following steps: coating the side of the slide facing the groove with a fibronectin solution, adding Tyrode's solution into the groove, and attaching the muscle cells subjected to calcium ion staining to the slide.

[0015] Optionally, the concentration of fibronectin in the fibronectin solution is 5 μg / mL, and the solvent of the fibronectin solution is phosphate buffer.

[0016] In a fourth aspect, the application provides an application of the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging method according to the first aspect and the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device according to the second aspect in cell mechanics research.

[0017] The application has the following beneficial effects:

[0018] The high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device and method according to the application can better simulate the actual mechanical stimulation encountered by cells in a physiological environment by applying mechanical waves with different frequencies, different intensities and different wavelengths to the muscle cells subjected to calcium staining, the mechanical waves being perpendicular or parallel to the direction of muscle sarcomere, and the wavelength of the applied mechanical waves being conjugated with the contraction length of the muscle sarcomere, and the concentration of calcium ions on the sarcoplasmic reticulum near the muscle sarcomere can be dynamically observed in the process. The observation results have high reliability and correlation, and are of great significance to cell mechanics research. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced.

[0020] FIG. 1 is a structural schematic view of the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device conjugated with muscle sarcomere contraction provided by the application;

[0021] FIG. 2 is a diagram of the conjugation relationship between muscle sarcomere and stimulation force provided by the application;

[0022] FIG. 3 is a diagram of the monitoring of calcium ion activity on the sarcoplasmic reticulum of mouse myocardial cells provided by the application;

[0023] FIG. 4 is a diagram of the calcium concentration on the sarcoplasmic reticulum generated by different mechanical stimulations provided by the application.

[0024] BRIEF DESCRIPTION OF DRAWINGS

[0025] 100, high frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device conjugated with sarcomere contraction; 1, cell regulation culture assembly; 10, bottom wall; 11, peripheral wall; 12, through hole; 13, glass slide; 14, groove; 15, limiting assembly; 2, mechanical stimulation generation assembly; 3, microscopic observation assembly. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present application. In the case of no conflict, the following embodiments and features in the embodiments can be combined with each other.

[0027] It should be noted that if the description of "first", "second" and the like is involved in the embodiments of the present application, the "first", "second" and the like are only for the purpose of description, and cannot be understood as indicating or implying the relative importance and implicitly indicating the number of the indicated technical features. Therefore, the features limited by "first", "second" can explicitly or implicitly include at least one of the features. In addition, the technical solutions of each embodiment can be combined with each other, but the combination of the technical solutions must enable the person skilled in the art to realize the basis, and when the combination of the technical solutions appears contradictory or unachievable, it should be considered that the combination of the technical solutions does not exist, and is not within the protection scope claimed by the present application.

[0028] Please refer to FIG. 1, the present application provides a kind of high frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device 100 conjugated with sarcomere contraction, it generally includes cell regulation culture assembly 1, mechanical stimulation generation assembly 2 and microscopic observation assembly 3 (not shown in drawing), wherein cell regulation culture assembly 1 includes bottom wall 10 and the peripheral wall 11 of the edge of bottom wall 10, the peripheral wall 11 is perpendicular to bottom wall 10 and is formed with the recess 14 by surrounding bottom wall 10, bottom wall 10 is provided with through hole 12, glass slide 13 is embedded in through hole 12. Mechanical stimulation assembly 2 is arranged in recess 14, and is close to peripheral wall 11. Microscopic observation assembly is configured to observe the contents in recess 14 through glass slide. Microscopic observation assembly 3 can be high-speed turntable confocal microscope. When calcium ion channel is detected by calcium ion probe in cell, the fluorescence of probe instantaneously generates and disappears, and then can be captured by high-speed turntable confocal microscope. 2+

[0029] ​When the calcium-stained muscle cells are placed in the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device 100 conjugated with the contraction of the sarcomere, the mechanical stimulation generating assembly 2 can apply mechanical stimulation to the calcium-stained muscle cells, which is perpendicular to the contraction direction of the sarcomere of the muscle cells and conjugated with the contraction length of the sarcomere of the muscle cells. This mechanical stimulation is similar to the mechanical stimulation actually received in the cell physiological environment, which can better simulate the physiological environment of the cell, and the calcium ion activity on the sarcoplasmic reticulum of the muscle cell is monitored through the microscopic observation assembly. The monitoring result has high reliability and correlation.

[0030] In some embodiments, the through hole on the bottom wall 10 is circular, and the glass sheet 13 is also circular. The glass sheet 13 is embedded in the through hole to maintain the integrity of the entire bottom wall 10 to achieve the loading of the liquid and cells in the groove 14. Preferably, the thickness of the glass sheet is 0.1-0.3mm. For example, the thickness of the glass sheet is 0.1mm, 0.15mm, 0.2mm, 0.25mm or 0.3mm, etc.

[0031] In some embodiments, the mechanical stimulation generating assembly 2 is selected from a mechanical wave generator. Further, when the mechanical stimulation generating assembly 2 is a mechanical wave generator, the power supply, signal generator, power amplifier, and planar piezoelectric ceramic are electrically connected. The power amplifier has a power amplification of 20-40dB, the signal generator has a frequency of 1-10MHz, a peak-to-peak value (VVp) of 2500, an impedance of 50Ω, and a waveform including at least one of Sine, Square, Ramp, Pulse, Noise, Dualtone, Harm, or Arb.

[0032] The signal generator connected to the power supply generates electrical signals of different frequencies, wavelengths, amplitudes, and waveforms, and the power amplifier amplifies the electrical signals by 20-40dB, so that the amplified electrical signals are sent to the planar piezoelectric ceramic. The piezoelectric ceramic generates different pressure waves under the action of the electrical signals, which act as force signals on the muscle cells.

[0033] In some embodiments, the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device includes a limiting assembly 15 arranged in the groove 14 and abutting against the peripheral wall 11, and the mechanical stimulation generating assembly 2 is fixedly connected with the limiting assembly 15. By connecting the limiting assembly 15 with the mechanical stimulation generating assembly 2 and fixing the mechanical stimulation generating assembly 2 in the groove, the direction of the mechanical stimulation released by the mechanical stimulation generating assembly 2 can be kept horizontal, so as to better simulate the physiological environment of the cell.

[0034] The application further provides a method for combined imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity, comprising the following steps:

[0035] (1) providing muscle cells;

[0036] (2) performing calcium ion staining treatment on the muscle cells;

[0037] (3) applying mechanical stimulation conjugated with the sarcomere contraction length of the muscle cells and perpendicular to the direction of the sarcomere to the muscle cells subjected to the calcium ion staining treatment, and observing using a high-speed rotating disk confocal microscope.

[0038] The relationship between the frequency of the mechanical wave of the mechanical stimulation conjugated with the sarcomere contraction length of the muscle cells and the sarcomere contraction length is shown in FIG. 2.

[0039] In some embodiments, the muscle cells can be obtained from experimental animals including mice, rats, rabbits, etc. For example, the mice can be anesthetized, the heart tissue can be obtained through thoracotomy surgery, and the precipitated muscle cells can be separated through gravity sedimentation.

[0040] In some embodiments, the step of performing calcium ion staining treatment on the muscle cells comprises using a calcium ion fluorescent probe to perform calcium ion staining on the muscle cells, and the calcium ion fluorescent probe can be selected from one of Fluo-4AM or Fluo-8 AM.

[0041] In some embodiments, the specific step of (3) comprises coating a fibronectin solution on the side of the slide of the device for combined imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity as described in the application above facing the groove, adding Tyrode's solution into the groove, and attaching the muscle cells subjected to the calcium ion staining treatment to the slide.

[0042] The concentration of fibronectin in the fibronectin solution is 5 μg / mL, and the solvent of the fibronectin solution is phosphate buffer (PBS buffer).

[0043] The application further provides the use of the device for combined imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity as described above and / or the method for combined imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity as described above in cell mechanics research. For example, the mechanical conduction process of muscle cells can be researched by monitoring the calcium ion activity on the sarcoplasmic reticulum.

[0044] The application will be further described in detail through specific embodiments.

[0045] Embodiment 1

[0046] (1) Mice are anesthetized with isoflurane mask at 8-12 weeks of age, then thoracotomy is performed, and the descending aorta and inferior vena cava are cut. Then EDTA buffer is injected into the right ventricle to flush the heart to remove blood. Then the ascending aorta is clamped with a hemostatic forceps (Reynolds forceps), and the heart is transferred to a tray containing EDTA buffer. In the tray, inject EDTA buffer into the left ventricle, and observe the patency of the coronary artery circulation. Next, the heart is transferred to a perfusion buffer, and the left ventricle is flushed with perfusion buffer. Then the heart is transferred to a tray containing collagenase buffer, and collagenase buffer is injected for digestion. After digestion is complete, the heart is transferred to a new tray, and the left ventricular tissue is torn and dissociated. The cell suspension is filtered through a cell strainer, and then washed with stop buffer. The cell suspension is passed through a 100 μL cell strainer, and the tissue is filtered out, and then the cell strainer is washed with 5 mL of stop buffer. At this time, the cell suspension is about 15 mL, and the cell suspension is aliquoted into two 15 mL centrifuge tubes (the bottom is steeper and easier to settle), and then the cells are allowed to settle by gravity for 20 min, and most of the muscle cells are precipitated into granules, and the non-muscle cells are suspended in the upper layer. The precipitated muscle cells are transferred to a 15 mL centrifuge tube (the yellowish-white precipitate at the bottom of the test tube can be seen after successful extraction by gravity settling, which is the precipitate of cardiac muscle cells). The precipitated muscle cells are isolated. Additional gravity settling is performed using perfusion buffer to purify the muscle cells. Finally, the cardiac muscle cells are resuspended and transferred to a muscle cell conditioning culture system for culture.

[0047] (2) Take 1 mL of the cardiac muscle cell suspension prepared in (1), add 10 μL of Fluo-8 AM stock solution (calcium ion fluorescent probe, the concentration of Fluo-8 AM is 10 μM), and keep it in the dark at room temperature for 10 min. Then centrifuge at 300 rpm for 2 min, remove the supernatant, and gently suspend the cells in Tyrode's solution without indicator. Wait for 20 min to allow the Fluo-8 AM to completely delipidize, and complete the calcium staining of the cardiac muscle cells. Then the calcium ion-stained cardiac muscle cells are passed through a double 90 cell strainer to obtain the muscle cells to be monitored.

[0048] (3) Place the muscle cells to be monitored obtained in (2) in the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device conjugated with muscle sarcomere contraction in the embodiments of the present application, the bottom wall of the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device conjugated with muscle sarcomere contraction is spin-coated with a laminin solution, and the recess is filled with Tyrode's solution. The Tyrode's solution includes NaCl 8.0 g, 10% KCl 2.0 ml (0.2 g), 10% MgSO4·7H2O 2.6 ml (0.26 g), 5% NaH2PO4·2H2O 1.3 ml (0.065 g), NaHCO3 1.0 g, 1M CaCl2 1.8 ml (0.2 g), and glucose 1.0 g. The Tyrode's solution is a low Reynolds number solution, and under the action of the Tyrode's solution, the direction of the muscle cells to be monitored can be changed to ensure that the mechanical stimulation is perpendicular to the direction of the muscle sarcomeres of the muscle cells.

[0049] (4) Select the rod-shaped muscle cells with clear stripes and no active spontaneous contraction (less than once per minute) as healthy Ca 2+ resistant cells under a microscope, adjust the focal plane to enable the muscle sarcomeres to be observed, and image to enable the muscle sarcomeres to be observed, so as to record the instantaneous changes of calcium ions by a fast high-resolution rotating disk laser confocal microscope, and realize the monitoring of Ca 2+ .

[0050] (5) Set the laser intensity of the high-speed rotating disk confocal microscope to 5%, the exposure time to 50 ms, and take pictures in a fastest time sequence mode for 60 s. Open the time sequence data obtained by taking pictures in the imaris software, export the video as a TIFF format original drawing, save the original drawing in a folder, write the data path, input the cell diameter, use omnipose to segment the first frame of the video, draw the minimum circumscribed ellipse of the contour of each cell, set the length and short axis ratio of the ellipse, filter the cells whose length and short axis ratio do not belong to the range, calculate the area of each contour, set the area range, and filter out the cells whose ratio does not conform to the range.

[0051] Use the SAM2 model to track the contours of the cells that meet the conditions after segmenting the first frame, and the tracking process outputs a gif. Record the fluorescence intensity of each cell in each frame, the average fluorescence intensity of the cell, the maximum fluorescence intensity of the cell, the minimum fluorescence intensity of the cell, the angle of the cell, and the length of the long and short axes of the cell, and output these data as different tables.

[0052] (6) Adjust the exposure time to increase the imaging frequency as much as possible under the premise of keeping each experiment consistent. The background fluorescence value is greater than 0, the fluorescence intensity of the resting cell should be in the range of 5-10 times the background fluorescence, and the background fluorescence is set to 10. Ensure that the calcium spark imaging is selected under the same focal plane, the spark state presents a crescent shape, and the fluorescence intensity at the front end of the spark is the largest, as shown in FIG. 3.

[0053] (7) Put the myocardial cells prepared in (2) into the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device in the embodiments of the present application which is conjugated with the sarcomere contraction, apply different mechanical stimulation, and record the changes of calcium ion concentration on the sarcoplasmic reticulum, and the final results are shown in FIG. 4, wherein over-conjugate indicates that the wavelength of mechanical stimulation exceeds the length of sarcomere, and under-conjugate indicates that the wavelength of mechanical stimulation is less than the length of sarcomere. As can be seen from FIG. 4, under different frequencies of mechanical stimulation, the changes of calcium ion concentration on the sarcoplasmic reticulum can be observed obviously.

[0054] In summary, the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging method conjugated with sarcomere contraction of the present application can apply mechanical waves of different frequencies, different intensities and different wavelengths to the muscle cells treated by calcium staining, which can be perpendicular or parallel to the direction of sarcomere, and the wavelength of applied force is conjugated with the length of sarcomere contraction, better simulating the actual mechanical stimulation encountered by cells in physiological environment. In this process, the concentration of calcium ions on the sarcoplasmic reticulum near the sarcomere can be dynamically observed. The observation results have high reliability and correlation, and are of great significance to the study of cell mechanics.

[0055] It should be understood that the application of the present application is not limited to the above examples, and those of ordinary skill in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the claims attached to the present application.

Claims

1. A method for combined imaging of high-frequency mechanical stimulation conjugate to sarcomere contraction and sarcoplasmic reticulum calcium activity, characterized in that, Including the following steps: (1) Provide muscle cells; (2) Calcium ion staining treatment of muscle cells; (3) Apply a mechanical stimulus to the calcium ion-stained muscle cells that is conjugate to the contraction length of the sarcomere and perpendicular to the direction of the sarcomere, and observe it using a high-speed rotating confocal microscope.

2. The method according to claim 1, characterized in that, The calcium ion fluorescent probe used in the calcium ion staining treatment includes either Fluo-4 AM or Fluo-8 AM.

3. A device for combined imaging of high-frequency mechanical stimulation conjugate to sarcomere contraction and sarcoplasmic reticulum calcium activity, characterized in that, include: A cell regulation culture component, the cell regulation culture component including a bottom wall and an outer peripheral wall extending from the bottom wall, the bottom wall and the outer peripheral wall forming a groove, the bottom wall having a through hole, and a glass slide being embedded in the through hole; A mechanical stimulation generating component is disposed in the groove and close to the outer peripheral wall; A microscopic observation assembly, wherein the microscope observation assembly is configured to observe the contents within the groove through the glass slide; When calcium-stained muscle cells are placed in the combined imaging device of high-frequency mechanical stimulation conjugate to sarcomere contraction and sarcoplasmic reticulum calcium activity, the mechanical stimulation generating component applies a mechanical stimulus to the calcium-stained muscle cells that is perpendicular to the direction of sarcomere contraction and conjugate to the length of sarcomere contraction. The limiting observation component can acquire fluorescence imaging of calcium ions on the sarcoplasmic reticulum of the muscle cells.

4. The imaging device for high-frequency mechanical stimulation conjugate to sarcomere contraction and combined imaging of sarcoplasmic reticulum calcium activity according to claim 3, characterized in that, The through hole is circular, the glass slide is circular, and the thickness of the glass slide is 0.1-0.3 mm.

5. The imaging device for high-frequency mechanical stimulation conjugate to sarcomere contraction and combined imaging of sarcoplasmic reticulum calcium activity according to claim 3, characterized in that, The mechanical stimulation generating component is a mechanical wave generator.

6. The imaging device for high-frequency mechanical stimulation conjugate to sarcomere contraction and combined imaging of sarcoplasmic reticulum calcium activity according to claim 5, characterized in that, The mechanical wave generator includes a power supply, a signal generator, a power amplifier, and a planar piezoelectric ceramic, wherein the power supply, the signal generator, the power amplifier, and the planar piezoelectric ceramic are electrically connected to each other. The power amplifier has a power amplification factor of 20-40 dB. The signal generator has a frequency of 1-10MHz, a peak-to-peak value of 2500, an impedance of 50Ω, and a waveform including at least one of Sine, Square, Ramp, Pulse, Noise, Dualtone, Harm, or Arb.

7. The imaging device for high-frequency mechanical stimulation conjugate to sarcomere contraction and combined imaging of sarcoplasmic reticulum calcium activity according to claim 3, characterized in that, The high-frequency mechanical stimulation conjugate to sarcomere contraction and the combined imaging device for sarcoplasmic reticulum calcium activity includes a limiting component, which is disposed in the groove and abuts against the outer peripheral wall, and the mechanical stimulation generating component is fixedly connected to the limiting component.

8. A method for monitoring calcium dissociation activity on the sarcoplasmic reticulum using a combined imaging device for high-frequency mechanical stimulation conjugate to sarcomere contraction and sarcoplasmic reticulum calcium activity as described in any one of claims 3 to 7, characterized in that, The steps include: coating the side of the glass slide facing the groove with an adhesion protein solution, adding Tyrode's solution into the groove, and attaching the muscle cells that have undergone calcium ion staining to the glass slide.

9. The method according to claim 8, characterized in that, The concentration of the cohesin in the cohesin solution is 5 μg / mL, and the solvent of the cohesin solution is phosphate buffer.

10. The application of the high-frequency mechanical stimulation conjugate to sarcomere contraction and the combined imaging method of sarcoplasmic reticulum calcium activity as described in claim 1 or 2, and the high-frequency mechanical stimulation conjugate to sarcomere contraction and the combined imaging device of sarcoplasmic reticulum calcium activity as described in claims 3 to 7, in cell mechanics research.

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