High-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device and method conjugated with sarcometral systole and application

Abstract

The invention discloses a high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device and method conjugated with sarcoplasmic systole and application. The high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging method conjugated with sarcoplasmic systole comprises the steps that muscle cells are provided; carrying out calcium ion dyeing treatment on the muscle cells; and applying mechanical stimulation which is conjugated with the systolic length of the sarcometre of the muscle cell and is vertical to the direction of the sarcometre to the muscle cell subjected to the calcium ion dyeing treatment, and observing by using a high-speed turntable confocal microscope. The monitoring method provided by the invention can better simulate the physiological conditions of the cells and monitor the calcium ions on the myoplasmon of the muscle cells, and is of great significance to the research of cell mechanics.

Description

Technical Field

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

[0002] The three major muscle cells in the human body are cardiac muscle, skeletal muscle, and smooth muscle. The three types of muscle cells in the human body have different mechanical perceptions and different mechanisms. Mechanical stimulation can be transmitted to various parts of the cell through the cell membrane and the cytoskeleton structure, causing a series of activities, inducing a series of complex transcriptomics and cytomics processes, and further driving changes in morphology and cell functions, such as maintaining cell morphology, growth, proliferation, apoptosis, migration, differentiation, gene expression, protein synthesis, etc. Calcium ions, as the second messenger in the cell, 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 the proliferation and migration of muscle cells, and can effectively regulate cell differentiation in stem cell research and regenerative medicine; second, it can regulate the direction and spatial structure of cell arrangement, and can achieve cell orientation 3D printing in tissue engineering; third, studying the regulatory mechanism of mechanics on cells will help to gain a deeper understanding of cell signals, gene expression, and the exploration of cell microenvironment.

[0003] The force signal is a directional vector, and muscle cells are also a cell system with a clear spatial structure. The sarcomere is the basic unit of rhythmic cell contraction. Therefore, it is very necessary to study the biological response of mechanical stimuli of different directions, frequencies, and amplitudes and the sarcomere in muscle cells. The biological response mainly revolves around the activity of calcium ions on the sarcoplasmic reticulum of the sarcomere. However, there is currently no device or method that can observe the activity of calcium ions on the sarcoplasmic reticulum under different mechanical stimuli. The existing technology cannot simulate the mechanical stimulation of cells in the actual physiological environment that is conjugate with the sarcomere contraction length and frequency, and it is difficult to accurately monitor the activity of calcium ions on the sarcoplasmic reticulum.

[0004] Therefore, the prior art needs to be improved. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a device, method and application for joint imaging of high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity conjugated with sarcomere contraction, aiming to solve the problem that the prior art cannot simulate the actual mechanical stimulation of cells in a physiological environment, and thus it is difficult to accurately monitor the activity of calcium ions on the sarcoplasmic reticulum.

[0006] The technical solution of this application is as follows: In a first aspect of the present application, a method for combined imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity is provided, comprising the steps of: (1) providing muscle cells; (2) subjecting the muscle cells to calcium ion staining; (3) applying mechanical stimulation conjugated with the sarcomere contraction length of the muscle cells and in a direction perpendicular to the sarcomere direction to the muscle cells treated with calcium ion staining, and observing using a high-speed spinning disk confocal microscope.

[0007] Optionally, the calcium ion fluorescent probe used in the calcium ion staining treatment includes one of Fluo-4 AM and Fluo-8AM.

[0008] According to a second aspect of the present application, there is provided a device for combined imaging of high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity conjugated with sarcomere contraction, comprising: a cell conditioning and culture component, the cell conditioning and culture component comprising a bottom wall and a peripheral wall extending from the bottom wall, the bottom wall and the peripheral wall forming a groove, the bottom wall being provided with a through hole, a glass slide being embedded in the through hole; a mechanical stimulation generating component, disposed in the groove and close to the peripheral wall; a microscope observation component, the microscope observation component being configured to observe the contents in the groove through the glass slide; when a muscle cell treated with calcium staining 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 generating component applies mechanical stimulation perpendicular to the sarcomere contraction direction of the muscle cell and conjugated to the sarcomere contraction length of the muscle cell to the muscle cell treated with calcium staining.

[0009] Optionally, the through hole is circular, the glass slide is circular, and the thickness of the glass slide is 0.1-0.3 mm.

[0010] Optionally, the mechanical stimulation generating component is a mechanical wave generator.

[0011] Optionally, the mechanical wave generator includes a power supply, a signal generator, a power amplifier, and a planar piezoelectric ceramic, and the power supply, the signal generator, the power amplifier and the planar piezoelectric ceramic are electrically connected to each other; wherein the power amplification decibel number of the power amplifier is 20-40 dB; the frequency of the signal generator is 1-10 MHz, the peak-to-peak value is 2500, the impedance is 50 Ω, and the waveform includes at least one of Sine, Square, Ramp, Pulse, Noise, Dualtone, Harm or Arb.

[0012] Optionally, the high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity combined imaging device includes a limiting component, the limiting component is arranged in the groove and abuts against the outer peripheral wall, and the mechanical stimulation generating component is fixedly connected to the limiting component.

[0013] In the third aspect of the present application, there is provided a method for monitoring calcium ion activity on the sarcoplasmic reticulum by combining high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity with an imaging device according to the second aspect of the present application, comprising the steps of: coating an adhesion protein solution on the side of the glass slide facing the groove, adding Tyrode's solution into the groove, and attaching muscle cells treated with calcium ion staining to the glass slide.

[0014] Optionally, the concentration of the adhesion protein in the adhesion protein solution is 5 μg / mL, and the solvent of the adhesion protein solution is phosphate buffer.

[0015] The fourth aspect of the present application provides a method for combined imaging of high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity conjugated with sarcomere contraction according to the first aspect of the present application and a device for combined imaging of high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity conjugated with sarcomere contraction according to the second aspect of the present application for use in cell mechanics research.

[0016] The beneficial effects of this application are: The high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity combined imaging device method of the present application applies mechanical waves of different frequencies, different intensities, and different wavelengths to the muscle cells treated with calcium staining, which can be perpendicular or parallel to the direction of the sarcomere, and the applied force wavelength is conjugated with the sarcomere contraction length, so as to better simulate the actual mechanical stimulation encountered by cells in the 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, which is of great significance to the study of cell mechanics. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments.

[0018] Figure 1 A schematic diagram of the structure of a device for high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity combined imaging provided in an embodiment of the present application; Figure 2 A diagram showing the conjugate relationship between the sarcomere and the stimulus force provided in the embodiment of the present application; Figure 3 A graph for monitoring calcium ion activity on the sarcoplasmic reticulum of mouse myocardial cells provided in the examples of the present application; Figure 4 This is a diagram of sarcoplasmic reticulum calcium concentration produced by different mechanical stimuli provided in the examples of the present application. Description of Reference Numerals

[0019] 100. A device for combined imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity; 1. Cell conditioning culture component; 10. Bottom wall; 11. Peripheral wall; 12. Through hole; 13. Glass slide; 14. Groove; 15. Limiting component; 2. Mechanical stimulation generating component; 3. Microscopic observation component. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0021] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0022] See also Figure 1 , an embodiment of the present application provides a high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device 100 conjugated with sarcomere contraction, which generally includes a cell regulation culture component 1, a mechanical stimulation generating component 2 and a microscopic observation component 3 (not shown in the figure), wherein the cell regulation culture component 1 includes a bottom wall 10 and a peripheral wall 11 extending from the edge of the bottom wall 10, the peripheral wall 11 is perpendicular to the bottom wall 10 and is surrounded by the bottom wall 10 to form a groove 14, and a through hole 12 is provided on the bottom wall 10, and a glass slide 13 is embedded in the through hole 12. The mechanical stimulation component 2 is arranged in the groove 14 and is close to the peripheral wall 11. The microscopic observation component is configured to observe the contents in the groove 14 through the glass slide. The microscopic observation component 3 can be a high-speed rotating disk confocal microscope. The calcium ion channel detected by the calcium ion probe in the cell rapidly releases Ca 2+ The fluorescence of the probe is generated and disappears instantaneously, and can be captured by a high-speed spinning disk confocal microscope.

[0023] When the muscle cells treated with calcium staining are placed in the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device 100 that is conjugated with the sarcomere contraction, the mechanical stimulation generating component 2 can apply mechanical stimulation that is perpendicular to the contraction direction of the sarcomere of the muscle cells and conjugated with the sarcomere contraction length of the muscle cells to the muscle cells treated with calcium staining. This mechanical stimulation is similar to the actual mechanical stimulation in the physiological environment of the cell, can better simulate the physiological environment of the cell, and monitor the calcium ion activity on the sarcoplasmic reticulum of the muscle cell through the microscopic observation component, and the monitoring results have high reliability and correlation.

[0024] In some embodiments, the through hole on the bottom wall 10 is circular, and the glass slide 13 is also circular. The glass slide 13 is embedded in the through hole to maintain the integrity of the entire bottom wall 10, so as to accommodate liquid and cells in the groove 14. Preferably, the thickness of the glass slide is 0.1-0.3 mm. For example, the thickness of the glass slide is 0.1 mm, 0.15 mm, 0.2 mm, 0.25 mm or 0.3 mm, etc.

[0025] In some embodiments, the mechanical stimulation generating component 2 is selected from a mechanical wave generator. Further, when the mechanical stimulation generating component 2 is a mechanical wave generator, it includes a power supply, a signal generator, a power amplifier, and a planar piezoelectric ceramic, and the power supply, the signal generator, the power amplifier, and the planar piezoelectric ceramic are electrically connected. Among them, the power amplifier has a power amplification decibel of 20-40 dB, the frequency of the signal generator is 1-10 MHz, the peak-to-peak value (VVp) is 2500, the impedance is 50 Ω, and the waveform includes at least one of Sine, Square, Ramp, Pulse, Noise, Dualtone, Harm, or Arb.

[0026] A signal generator connected to a power supply generates electrical signals of different frequencies, wavelengths, amplitudes, and waveforms, which are amplified by 20-40 dB through a power amplifier. The amplified electrical signals are then sent to the planar piezoelectric ceramics. Under the action of the electrical signals, the piezoelectric ceramics generate different pressure waves, which act as force signals on the muscle cells.

[0027] In some embodiments, the high-frequency mechanical stimulation conjugated with the sarcomere contraction and the sarcoplasmic reticulum calcium activity combined imaging device includes a limiting component 15, the limiting component 15 is arranged in the groove 14 and abuts against the peripheral wall 11, and the mechanical stimulation generating component 2 is fixedly connected to the limiting component 15. By connecting the limiting component 15 to the mechanical stimulation generating component 2 and fixing the mechanical stimulation generating component 2 in the groove, it can be ensured that the direction of the mechanical stimulation released by the mechanical stimulation generating component 2 remains horizontal, so as to better simulate the physiological environment of the cell.

[0028] The present application also provides a method for combined imaging of high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity conjugated with sarcomere contraction, comprising the steps of: (1) Provide muscle cells; (2) Calcium ion staining of muscle cells; (3) A mechanical stimulus conjugated to the sarcomere contraction length of the myocytes and perpendicular to the sarcomere direction was applied to the myocytes treated with calcium ion staining, and the myocytes were observed using a high-speed spinning disk confocal microscope.

[0029] Among them, the relationship between the frequency of the mechanical wave of the mechanical stimulus conjugate with the sarcomere contraction length of the muscle cell and the sarcomere contraction length is as follows: Figure 2 shown.

[0030] In some embodiments, the muscle cells can be obtained from experimental animals, including mice, rats, rabbits, etc. For example, the mouse can be anesthetized, the heart tissue can be obtained through thoracotomy, and the precipitated muscle cells can be separated by gravity sedimentation.

[0031] In some embodiments, the step of performing calcium ion staining on the muscle cells includes: 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-4 AM and Fluo-8 AM.

[0032] In some embodiments, the specific step of (3) includes: coating the glass slide of the high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity combined imaging device as described above in the embodiment of the present application with an adhesion protein solution on the side facing the groove, adding Tyrode's solution into the groove, and attaching the muscle cells treated with calcium ion staining to the glass slide.

[0033] The concentration of the adhesion protein in the adhesion protein solution is 5 μg / mL, and the solvent of the adhesion protein solution is phosphate buffered saline (PBS buffer).

[0034] The present application also provides the application of the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device and / or the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging method conjugated with sarcomere contraction in cell mechanics research. For example, by monitoring the calcium ion activity on the sarcoplasmic reticulum, the mechanical conduction process of muscle cells can be studied.

[0035] The following is further described through specific implementation methods.

[0036] Example 1 (1) Mice were anesthetized continuously with an isoflurane mask at 8-12 weeks of age, and then a thoracotomy was performed to cut the descending aorta and the inferior vena cava. EDTA buffer was then injected into the right ventricle and the heart was flushed to remove blood. The ascending aorta was then clamped with a hemostatic forceps (Reynolds forceps) and the heart was transferred to a tray containing EDTA buffer. EDTA buffer was injected into the left ventricle in the tray to observe the patency of the coronary artery circulation. Next, the heart was transferred to a perfusion buffer and the left ventricle was flushed with perfusion buffer. The heart was then transferred to a tray containing collagenase buffer and collagenase buffer was injected for digestion. After digestion was completed, the heart was transferred to a new tray, the left ventricular tissue was torn and cells were dissociated. The cell suspension was filtered through a cell sieve and then washed with stop buffer. The cell suspension was passed through a 100 μL cell sieve to filter out the tissue, and the cell sieve was washed with 5 mL stop buffer. At this point, the cell suspension is about 15 mL. Divide the cell suspension into two 15 mL centrifuge tubes (the bottom is steeper and easier to settle), and then let the cells settle by gravity for 20 min. Most of the muscle cells will settle into particles, and non-muscle cells will be suspended in the upper layer. Transfer the precipitated muscle cells to a 15 mL centrifuge tube (if the extraction is successful, you can see the yellow-white precipitate at the bottom of the test tube by gravity sedimentation, which is the cardiomyocyte precipitation). Separate the precipitated muscle cells. Use perfusion buffer for additional gravity sedimentation to purify the muscle cells. Finally, resuspend the cardiomyocytes and transfer them to the muscle cell conditioning culture system for culture.

[0037] (2) Take 1 mL of the cardiomyocyte suspension prepared in (1), add 10 μL of Fluo-8 AM stock solution (calcium ion fluorescent probe, Fluo-8 AM concentration is 10 μM), and keep 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 solution without indicator. Wait for 20 min to allow Fluo-8 AM to completely degrease and complete the calcium staining of the cardiomyocytes. Then pass the calcium ion stained cardiomyocytes through a double 90 cell sieve to obtain the muscle cells to be monitored.

[0038] (3) The muscle cell to be monitored obtained in (2) is placed in the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device conjugated with sarcomere contraction in the embodiment of the present application. The bottom wall of the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device conjugated with sarcomere contraction is spin-coated with adhesion protein solution, and the groove is filled with Tyrode's solution. 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˙2H2O1.3 ml (0.065 g), NaHCO3 1.0 g, 1M CaCl2 1.8 ml (0.2 g), and glucose 1.0 g. As a low Reynolds coefficient solution, Taishiye can change the direction of the muscle cell to be monitored under its action to ensure that the mechanical stimulation is perpendicular to the direction of the sarcomere of the muscle cell.

[0039] (4) Select rod-shaped myocytes with clear striations and no active spontaneous contraction (less than once per minute) under microscopic observation as healthy Ca 2+ Tolerocytes, imaging requires the ability to adjust the focal plane to see the sarcomere, so that the transient changes of calcium ions can be recorded by fast high-resolution spinning disk laser confocal microscopy to achieve Ca 2+ monitoring.

[0040] (5) Set the laser intensity of the high-speed spinning disk confocal microscope to 5%, the exposure time to 50ms, and shoot in the fastest time series mode for 60s. Open the captured time series data in Imaris software, export the video as a TIFF format original image and save it in a folder. Write the data path, enter the cell diameter, use Omnipose to segment the first frame of the video, draw the minimum circumscribed ellipse of the outline of each cell, set the major and minor axis ratio of the ellipse, and use parameters to filter out cells whose major and minor axis ratios do not fall within this range. Calculate the area of ​​each outline, set the area range, and filter out cells whose ratios do not meet this range.

[0041] After the first frame is segmented, the SAM2 model is used to track the contours of cells that meet the conditions, and the tracking process is output as a gif. 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, the angle of the cell, the long axis and the short axis length of the cell are recorded, and these data are output as different tables.

[0042] (6) Under the premise of keeping each experiment consistent, adjust the exposure time and try to increase the imaging frequency. The background fluorescence value should be greater than 0, and the fluorescence intensity of resting cells should be in the range of 5-10 times the background fluorescence, and the background fluorescence should be set to 10. Ensure that calcium spark imaging is selected under the same focal plane, the spark state is crescent-shaped, and the fluorescence intensity at the front end of the spark is the largest, such as Figure 3 shown.

[0043] (7) The myocardial cells to be monitored prepared in (2) are placed in the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device conjugated with sarcomere contraction in the embodiment of the present application, different mechanical stimuli are applied, and the changes in the calcium ion concentration on the sarcoplasmic reticulum are recorded. The final results are as follows: Figure 4 As shown in Figure 2, hyperconjugation means that the wavelength of the mechanical stimulus exceeds the length of the sarcomere, and underconjugation means that the wavelength of the mechanical stimulus is less than the length of the sarcomere. Figure 4 It can be seen that under mechanical stimulation of different frequencies, changes in calcium ion concentration on the sarcoplasmic reticulum can be clearly observed.

[0044] In summary, the high-frequency mechanical stimulation conjugated with sarcomere contraction and the combined imaging method of sarcoplasmic reticulum calcium activity of the present application, by applying mechanical waves of different frequencies, different intensities, and different wavelengths to the muscle cells treated with calcium staining, which can be perpendicular or parallel to the direction of the sarcomere, and the applied force wavelength is conjugated with the sarcomere contraction length, better simulates the actual mechanical stimulation encountered by cells in the physiological environment, and can dynamically observe the concentration of calcium ions on the sarcoplasmic reticulum near the sarcomere in this process. The observation results have high reliability and correlation, which is of great significance to the study of cell mechanics.

[0045] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. A method for combined imaging of high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity, characterized in that: Includes steps: (1) Provide muscle cells; (2) Calcium ion staining of muscle cells; (3) A mechanical stimulus conjugated to the sarcomere contraction length of the myocytes and perpendicular to the sarcomere direction was applied to the myocytes treated with calcium ion staining, and the myocytes were observed using a high-speed spinning disk 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 one of Fluo-4 AM and Fluo-8 AM.

3. A device for high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity combined imaging, characterized in that: include: A cell conditioning and culture component, the cell conditioning and culture component comprising a bottom wall and a peripheral wall extending from the bottom wall, the bottom wall and the peripheral wall are arranged to form a groove, the bottom wall is provided with a through hole, and a glass slide is embedded in the through hole; A mechanical stimulation generating component is disposed in the groove and close to the outer peripheral wall; a microscope observation component, wherein the microscope observation component is configured to observe the contents in the groove through the glass slide; When the muscle cells treated with calcium staining are placed in the high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity combined imaging device conjugated with sarcomere contraction, the mechanical stimulation generating component applies mechanical stimulation perpendicular to the sarcomere contraction direction of the muscle cells and conjugated with the sarcomere contraction length of the muscle cells to the muscle cells treated with calcium staining, and the limited observation component can obtain fluorescent imaging of calcium ions on the sarcoplasmic reticulum of the muscle cells.

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

6. The device for high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity combined imaging according to claim 5, characterized in that: The mechanical wave generator comprises 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; Wherein, the power amplification decibel number of the power amplifier is 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 including at least one of Sine, Square, Ramp, Pulse, Noise, Dualtone, Harm, or Arb.

7. The device for high-frequency mechanical stimulation conjugated with sarcomere contraction and sarcoplasmic reticulum calcium activity combined imaging according to claim 3, characterized in that: The high-frequency mechanical stimulation conjugate with sarcomere contraction and sarcoplasmic reticulum calcium activity combined imaging device comprises a limiting component, wherein the limiting component is arranged in the groove and abuts against the peripheral wall, and the mechanical stimulation generating component is fixedly connected to the limiting component.

8. A method for monitoring calcium ion activity on the sarcoplasmic reticulum using the high-frequency mechanical stimulation conjugated with sarcomere contraction and the sarcoplasmic reticulum calcium activity combined imaging device as described in any one of claims 3 to 7, characterized in that: The method comprises the following steps: 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 treated with calcium ion staining onto the glass slide.

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

10. Use of a method for combined imaging of high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity conjugated with sarcomere contraction as described in claim 1 or 2, and a device for combined imaging of high-frequency mechanical stimulation and sarcoplasmic reticulum calcium activity conjugated with sarcomere contraction as described in claims 3 to 7 in cell mechanics research.

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