System and method for microscopic in-situ observation of mechanical response of sample

Abstract

The invention provides a system and a method for microscopic in-situ observation of mechanical response of a sample, force is applied to the sample in a water bath to enable the sample to deform, and the reaction of the sample to the applied force is observed in situ through an optical microscope; a sample needs to be controlled to be in a proper size and needs to be subjected to fluorescent staining firstly, and a staining reagent contains NHS activated ester; the force applying device comprises a clamping part and a force applying part, the clamping part fixes one end of a sample, the other end of the sample moves under the action of the force applying part, the optical microscope can record changes of the sample in the stress state in situ in a video recording or photographing mode, images can be stored frame by frame, and the mechanical response of the sample can be analyzed through DIC or PIV. The in-situ observation device is particularly suitable for in-situ observation of a biological sample needing to simulate an in-vivo physiological environment, so that the biological sample is always kept in a moist and bioactive state, and more accurate data can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and more specifically, relates to a system and method for microscopic in-situ observation of the mechanical response of a sample. Background Technology

[0002] Traditional methods for observing the mechanical response of biological or material samples usually only allow for non-in-situ observation. Non-in-situ sample processing may lead to information distortion. The observation of the sample before and after the stress is not carried out in the same environment, and only the information before and after the sample is subjected to the stress can be observed. The intermediate process cannot be shown frame by frame.

[0003] Traditional mechanical testing equipment, such as universal testing machines, is typically used to directly measure the mechanical properties of materials, but it does not provide in-situ observation of the material's response and stress changes during the testing process. Moreover, existing technologies are usually designed to measure the mechanical properties of materials or biological samples, such as tensile strength and compressive strength. This is achieved by fixing the sample with specific clamps and then applying force using a motor or other driving mechanism. Although these technologies can provide some mechanical property data, they cannot provide in-situ observation and therefore cannot accurately capture the subtle changes in the sample during the stress process.

[0004] EP0696351B1 provides an in-situ tensile testing machine based on scanning electron microscopy for studying the plastic deformation mechanism of composite materials with an aluminum matrix and reinforced with silicon carbide (SiC) particles. However, this method requires the sample to be completely dehydrated and cannot be used to observe the mechanical response of the sample under water bath conditions in situ. Biological samples (such as ligaments, tendons, etc.) usually need to be tested in a simulated physiological environment to obtain more accurate and realistic results. The in-situ tensile testing machine provided by this patent is difficult to achieve in-situ mechanical response observation of biological samples.

[0005] Therefore, there is an urgent need to find a device that can realize in-situ observation of the mechanical response of samples under a microscope, so as to preserve the in-situ information of the samples and allow frame-by-frame analysis of the response without damaging the samples. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a system and method for in-situ microscopic observation of the mechanical response of samples. A force is applied to a sample in a water bath to induce deformation, and the sample's response to the applied force is observed in situ using an optical microscope. The sample must be controlled to a suitable size and pre-stained with fluorescent dye containing NHS-activated esters. The force-applying device includes a clamping component and a force-applying component. The clamping component fixes one end of the sample, while the other end is displaced under the action of the force-applying component. The optical microscope records the changes in the sample's stress state in situ, saving images frame by frame. The mechanical response of the sample can also be analyzed using a matching DIC or PIV. This method is particularly suitable for in-situ observation of biological samples requiring simulated physiological environments, ensuring the biological sample remains moist and biologically active, thereby obtaining more accurate data.

[0007] On one hand, the present invention provides a system for in-situ microscopic observation of the mechanical response of a sample, the system comprising a sample processing device, a microscopic observation device, and a water bath; the sample processing device is used to fix the sample and apply force to the sample; the microscopic observation device is used to observe the sample's response to the applied force in situ; the sample is located in the liquid within the water bath and is within the observation range of the microscopic observation device.

[0008] Existing techniques typically involve non-in-situ sample processing, meaning that the sample is not observed in the same environment before and after being subjected to force, which may lead to distortion of the sample's mechanical response information.

[0009] The present invention provides a microscope-compatible device comprising a stable sample holding system, a precise force application mechanism, and a microscope-compatible observation window, with the sample positioned in a water bath. This device allows for in-situ observation while applying mechanical loads. By enabling in-situ observation of the mechanical response of the sample under a microscope, the present invention preserves the in-situ information of the sample, allowing frame-by-frame analysis of the response without damaging the sample.

[0010] The sample described in this invention can be any sample or material that requires in-situ observation of the mechanical response.

[0011] In some approaches, the sample is a biological sample, such as ligaments, tendons, cartilage, muscles, etc.

[0012] Biological samples are prone to water loss during mechanical response testing because they are separated from their original physiological environment.

[0013] The system for microscopic in-situ observation of the mechanical response of a sample provided by this invention enables the sample to be observed in a water bath containing a liquid that can simulate the physiological environment of the sample or keep it active. The sample is immersed in the liquid, thereby ensuring that the water content of the sample does not change during the process of the sample responding to the applied force.

[0014] The water bath described in this invention is not limited to immersing the sample in pure water, but can be performed using different liquids depending on the sample.

[0015] In some methods, phosphate buffer or saline solution can be added to the water bath to ensure that biological samples do not lose water.

[0016] In some embodiments, the water bath is made of transparent glass, allowing light to pass through, with a glass thickness ranging from 0.1 to 1 cm. This allows the sample to be clearly seen even when the water bath is located within the microscope's observation window. It is understood that the glass thickness of the water bath only needs to be sufficient to hold the water bath liquid.

[0017] Furthermore, the sample processing device includes a clamping component and a force-applying component, wherein the clamping component is used to fix the sample and the force-applying component is used to apply force to the sample; the microscopic observation device is an optical microscope.

[0018] Compared to electron microscopes, optical microscopes are more suitable for in-situ imaging of samples in a water bath environment, which allows for the simulation of physiological testing environments without damaging the samples.

[0019] Furthermore, the clamping component includes a fixed end and a movable end; the fixed end is used to clamp and fix the first part of the sample; the movable end is used to clamp the second part of the sample and is displaced under the action of the force-applying component, thereby deforming the sample.

[0020] When observing the mechanical response of a sample in situ, it is necessary to control the magnitude of the force applied by the force-applying component. However, because the sample is in a water bath environment, it is susceptible to water resistance in addition to the force applied by the force-applying component, and the slight shaking of the water bath can also introduce forces in different directions. Therefore, it is difficult to accurately control the magnitude of the applied force. By controlling the displacement distance and direction of the force-applying component, the sample can also undergo displacement of the same distance in a specific direction, thus deforming. This method can more accurately control the degree of sample deformation each time, and the detection results are more accurate.

[0021] The force-applying component in the device provided by the present invention can precisely control the displacement distance of the sample by applying a micro-force of a specific magnitude to the sample.

[0022] Furthermore, the fixed end uses a clamp and screws to fix the first part of the sample; the movable end uses a magnetic clamp to fix the second part of the sample. When the upper and lower clamps of the magnetic clamp are close together, they attract each other due to magnetism and clamp the sample, which can be easily opened and closed, thereby adjusting the clamping situation of the second part of the sample.

[0023] In some methods, the fixing end uses plastic clamps and screws to fix the sample, thereby achieving a better fixing effect.

[0024] In some methods, the mobile device uses a more flexible and convenient way to fix the sample, making it easy to observe and adjust the stress and deformation of the sample at any time, and preventing sample damage.

[0025] In some methods, the mobile device uses magnetic clips to fix the sample. Magnetic clips are easier to open and close. For example, inserting an operating rod between the magnetic clips and gently lifting upwards will open them, while gently pressing downwards will close them. This makes operation and adjustment convenient, and the magnetic clips are less likely to damage the sample.

[0026] Furthermore, a slide rail is provided below the mobile end, and the mobile end can be displaced along the slide rail under the action of the force-applying component; the force-applying component includes a force-applying end and a control end.

[0027] In some embodiments, there are two slide rails below the mobile terminal. Two points determine a straight line. The two rails make the displacement direction of the mobile terminal more stable and controllable, and the displacement distance more precise.

[0028] Furthermore, the force-applying component includes a force-applying end and a control end; the force-applying end is connected to the moving end, thereby causing the moving end to move together; the control end is used to control the displacement distance of the force-applying end, thereby controlling the displacement distance of the second part of the sample on the moving end.

[0029] In some methods, since the sample is in a water bath, the moving end of the clamping mechanism and the force-applying end of the force-applying component also need to be in the water bath, while the control end of the force-applying component needs to be outside the water bath. In order to accurately control the displacement of the force-applying end, the control end needs to extend from outside the water bath into the water bath through a certain connection method, so as to connect with the force-applying end and apply force to it, and accurately control its displacement distance.

[0030] Furthermore, the sample has a thickness of less than 500 μm and a size of less than 2 x 2 cm; the liquid in the water bath is a phosphate buffer solution.

[0031] In order for the sample in the water bath to be clearly observed through the microscope window, the size of the sample must be strictly controlled within an appropriate range.

[0032] In some embodiments, the sample has a length and width of 1 to 2 cm and a thickness of 100 to 200 μm.

[0033] In some embodiments, the sample is in the form of a sheet.

[0034] In some embodiments, the force applied to the sample by the force-applying component of the present invention is a very small microforce, causing the sample to undergo minute displacement and deformation, which is then captured by a microscope and analyzed frame by frame.

[0035] When the liquid in the water bath is phosphate buffer, it is more helpful for observing the sample than pure water, and it is also more conducive to maintaining the biological activity of the sample. In addition, the sample is less likely to be damaged under the action of force-applying equipment.

[0036] Furthermore, the sample needs to be fluorescently stained, and the fluorescent staining agent contains NHS activated ester (N-hydroxysuccinimide ester, succinimidyl ester).

[0037] NHS-activated ester (N-hydroxysuccinimide ester, succinimidylester) is the most commonly used activating group in biolabeling reactions. It activates the carboxyl group in sulfonated Cy3 molecules, allowing it to react with the amine group (primary or secondary amine) on the target biomolecule to form a stable amide bond, thereby labeling the dye molecule onto the biomolecule. Since free amine groups are common functional groups on the surface of proteins, antibodies, and peptides (derived from the lysine side chain), sulfonated Cy3-NHS can react directly with them.

[0038] Extensive research has demonstrated that when the staining agent used for fluorescent staining of samples contains NHS activated ester, it can significantly improve the clarity of microscopic imaging compared to other staining agents. This may be because the NHS activated ester reacts with the amino groups on the sample molecules to form a label, which allows the sample to maintain good clarity when photographed in a water bath.

[0039] Furthermore, the lens of the optical microscope has a resolution of 10 to 100x.

[0040] The choice of microscope lens has a significant impact on the clarity of images captured of the mechanical response process of a sample.

[0041] In some embodiments, the lens of the optical microscope is 20x, 50x, or 100x.

[0042] In some embodiments, the optical microscope has a 20x lens, which, compared to 50x and 100x, further improves the clarity of images captured of the mechanical response process of the sample.

[0043] On the other hand, the present invention provides a method for in-situ microscopic observation of the mechanical response of a sample. The method employs the system described above to observe the mechanical response of the sample in situ, and includes the following steps:

[0044] (1) Fix the sample in the clamping component;

[0045] (2) Set the sample movement distance at the control end of the force-applying component;

[0046] (3) Turn on the camera mode on the microscope;

[0047] (4) Turn on the motor of the force-applying component. The force-applying component causes the sample to deform. Record the changes in the sample under force in situ.

[0048] (6) The mechanical response of the sample is analyzed by software, and the in-situ mechanical response video of the sample can be analyzed frame by frame.

[0049] Furthermore, the software includes DIC (Digital Image Correlation Analysis) or PIV (Particle Image Velocimetry) analysis.

[0050] The DIC method is a method that creates feature points (speckle map) on the surface of the object under test, captures the movement of speckle features at the pixel level, and uses optimized 3D digital image correlation algorithms to provide full-view morphology, displacement and strain data measurement for the experiment in two-dimensional and three-dimensional space. It is usually divided into four steps: speckle creation, system calibration, strain measurement and image analysis.

[0051] Particle image velocimetry (PIV) is a method that uses multiple images to record the positions of particles in a flow field and analyzes the captured images to determine the flow velocity. Its basic principle is to scatter tracer particles in the flow field and use a pulsed laser sheet as a light source to illuminate the region of the flow field to be measured. Through two or more consecutive exposures, the images of the particles are recorded on film or a CCD camera.

[0052] However, conventional DIC experimental methods have the following drawbacks, which are optimized in the apparatus provided by this invention:

[0053] 1. Low precision and low resolution. Typical DIC experimental methods do not rely on microscope imaging, but on ordinary cameras, whose image and video data resolution is lower than that of the microscope lens used in this invention.

[0054] 2. The sample being tested cannot contain water. In general DIC methods, the creation and capture of feature points on the sample surface cannot tolerate moisture in the sample, which is not suitable for testing biological samples.

[0055] 3. It cannot provide a physiologically similar environment for biological samples. In the DIC method, biological samples are extremely prone to dehydration and inactivation during long-term testing, but the device of this invention can provide an aquatic environment, keeping biological samples moist and biologically active at all times.

[0056] The present invention has the following beneficial effects:

[0057] (1) The provided device allows direct observation of the sample during the stress process, enabling in-situ observation and more accurately capturing the true behavior of the sample during the stress process. Compared with non-in-situ observation, it reduces the distortion that may occur during sample processing.

[0058] (2) It supports frame-by-frame analysis of the mechanical response of samples, providing more detailed and in-depth data analysis capabilities than traditional techniques, and making it possible to gain a deeper understanding of the mechanical behavior of materials or biological samples.

[0059] (3) The sample clamping device allows the entire device to be water bathed. Phosphate buffer or physiological saline can be added to the water bath to ensure that the sample does not lose water and to provide the sample with conditions similar to a physiological environment.

[0060] (4) It can achieve non-destructive testing, allowing real-time in-situ observation of mechanical response without damaging the sample. During the application of mechanical load, it can ensure that the sample is not damaged, which is crucial for maintaining the natural state of the sample and obtaining accurate data. It is especially suitable for samples that cannot withstand traditional mechanical testing methods and is of great significance for precious or non-renewable samples.

[0061] (5) By adjusting various parameters of the device, the stability of the sample is ensured when mechanical load is applied;

[0062] (6) Good compatibility, not only applicable to biomechanics or materials science research, but also can be combined with research and analysis methods in other fields. For example, the device can be used in conjunction with existing microscope systems and analysis methods such as digital image correlation (DIC) and particle tracking velocimetry (PIV), thus expanding its application scope. Attached Figure Description

[0063] Figure 1 This is a schematic diagram of the microscopic in-situ observation device for the mechanical response of the sample in Example 1;

[0064] Figure 2 This is an overall structural diagram of the microscopic in-situ observation device for the mechanical response of the sample in Example 1;

[0065] Figure 3 This is a structural diagram of the sample processing device in Example 1;

[0066] Figure 4 This is a structural diagram of the device inside the water bath in Example 1;

[0067] Figure 5 This is a schematic diagram illustrating the process of displacement of the second part of the sample in Example 1;

[0068] Figure 6 The image shows the results of local displacement and strain data of the sample obtained from PIV analysis in Example 2. Detailed Implementation

[0069] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate understanding of the present invention and are not intended to limit it in any way. The raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0070] Example 1: Microscopic in-situ observation sample mechanical response device provided by the present invention

[0071] A schematic diagram of the microscopic in-situ observation sample mechanical response device provided in this embodiment is shown below. Figure 1 See the overall structure diagram. Figure 2 The structural diagram of the sample processing device is shown below. Figure 3 The structure of the device inside the water bath is shown in [reference]. Figure 4 A schematic diagram of the displacement process of the second part of the sample is shown below. Figure 5 .

[0072] like Figure 2 The microscopic in-situ observation device 1 for the mechanical response of a sample includes a sample processing device 2, a microscopic observation device 3, and a water bath 4. The sample processing device 2 is used to fix the sample 5 and apply force to it. The microscopic observation device 3 is used to observe the response of the sample 5 to the applied force in situ. The sample 5 is located in the liquid 6 within the water bath 4 and is within the observation range of the microscopic observation device 3. The sample 5 can be a biological sample. During the mechanical response detection process, it is prone to dehydration due to being removed from its original physiological environment. This device 1 allows the sample 5 to be observed in the water bath 4, which contains a liquid 6 that simulates the physiological environment of the sample or keeps it active. The sample 5 is immersed in the liquid 6, thus ensuring that the water content of the sample 5 does not change during the response to the applied force. The liquid in the water bath can be phosphate buffer or physiological saline to ensure that the biological sample does not lose water. The water bath 4 is made of transparent glass, allowing light to pass through, and is approximately 0.3 cm thick, allowing the sample 5 to be clearly observed even when the water bath 4 is within the observation window of the observation device 3.

[0073] like Figures 2-4 The sample processing device 2 includes a clamping component 7 and a force-applying component 8. The clamping component 7 is used to fix the sample 5, and the force-applying component 8 is used to apply force to the sample 5. The microscopic observation device 3 is an optical microscope 9 (Nikon upright fluorescence microscope Ni-E) with a camera 22. Compared with an electron microscope, the optical microscope 9 is more suitable for in-situ imaging of samples in a water bath environment, which will not damage the microscope, and the image clarity is also higher.

[0074] like Figure 3The clamping component 7 includes a fixed end 10 and a movable end 11. The fixed end 10 is used to clamp and fix the first part 12 of the sample 5; the movable end 11 is used to clamp the second part 13 of the sample 5 and displace it under the action of the force-applying component 8, thereby deforming the sample 5. When observing the mechanical response of the sample 5 in situ, it is necessary to control the magnitude of the force applied by the force-applying component 8. However, since the sample 5 is in a water bath environment, it is easily affected by the resistance of the water while being subjected to the force applied by the force-applying component 8, and the slight shaking process of the water bath will also bring certain forces in different directions, making it difficult to accurately control the magnitude of the applied force. By controlling the displacement distance and displacement direction of the force-applying component, the sample will also undergo displacement of the same distance in a specific direction, thereby deforming. This method can more accurately control the force application method each time, and the detection results are more accurate. The force-applying component 8 in the device 1 of this embodiment can accurately control the displacement distance of the sample 5 by applying a specific amount of micro-force to the sample 5.

[0075] Preferably, the fixed end 10 uses a plastic clamp 14 and screws 15 to fix the first part 12 of the sample 5; the movable end 11 uses a magnetic clamp 16 to fix the second part 13 of the sample 5. When the upper and lower clamps 17 of the magnetic clamp 16 are close together, they attract each other magnetically and clamp the sample 5, which can be easily opened and closed, thereby adjusting the clamping situation of the second part 13 of the sample 5. The fixed end 10 uses a plastic clamp 14 and screws 15 to fix the sample, thus achieving a better fixing effect. The movable end 11 uses a more flexible and convenient magnetic clamp 16 to fix the sample 5. The magnetic clamp 16 is easier to open and close. For example, inserting an operating rod between the upper and lower clamps 17 of the magnetic clamp 16 and gently lifting it upwards will open it, and gently pressing it downwards will close it, which is convenient for operation and adjustment. Moreover, the magnetic clamp 16 is less likely to damage the sample 5, and it is also convenient to observe and adjust the force and deformation of the sample 5 at any time to prevent damage to the sample 5.

[0076] like Figure 2 and 4The mobile end 11 is provided with a slide rail 18 below it. Under the action of the force-applying component 8, the mobile end 11 can move along the slide rail 18. The force-applying component 8 includes a force-applying end 19 and a control end 20. The force-applying end 19 is connected to the mobile end 11, thereby driving the mobile end 11 to move together. The control end 20 is used to control the displacement distance of the force-applying end 19, thereby controlling the displacement distance of the sample 5 second part 13 of the mobile end 11. There are two slide rails 18 below the mobile end 11. Two points determine a straight line. The two rails 18 can make the displacement direction of the mobile end 11 more stable and controllable, and the displacement distance more accurate. Since sample 5 is in water bath 4, the moving end 11 of clamping component 7 and the force-applying end 19 of force-applying component 8 also need to be in water bath 4. However, the control end 20 of force-applying component 8 needs to be outside water bath 4. To accurately control the displacement of force-applying end 19, control end 20 needs to extend from outside water bath 4 into water bath 4 via a specific connection method, thereby connecting with force-applying end 19 and applying force to it to precisely control its displacement distance. Control end 20 precisely controls the displacement of force-applying end 19 by the advancing distance of rocker arm 21.

[0077] Preferably, the thickness of sample 5 is less than 500 μm and the size is less than 2 x 2 cm; the liquid in the water bath 4 is phosphate buffer. To ensure that sample 5 in the water bath 4 can be clearly observed through the microscope window, the size of sample 5 must be strictly controlled within a suitable range. Preferably, the length and width of sample 5 are 1–2 cm, and the thickness is 100–200 μm. Sample 5 is in sheet form.

[0078] The method for performing in-situ microscopic observation of the mechanical response of a sample using the apparatus provided in this embodiment includes the following steps:

[0079] (1) Fix sample 5 in clamping component 7;

[0080] (2) Set the sample movement distance at the control end 20 of the force application component 8;

[0081] (3) Turn on the camera mode of the optical microscope 9;

[0082] (4) Turn on the motor of the force application component 8. The force application component 8 causes the sample 5 to deform. Record the changes of the sample 5 under the force state in situ.

[0083] (5) The mechanical response of sample 5 was analyzed using software. The in-situ mechanical response video of sample 5 could be analyzed frame by frame. The analysis software included DIC (Digital Image Correlation Analysis) or PIV (Particle Image Velocimetry) analysis.

[0084] Example 2: Observation of ligament tissue deformation under micro-force.

[0085] This embodiment uses the microscopic in-situ observation system for the mechanical response of samples provided in Example 1 to observe the deformation of ligament tissue under micro-force. The main process is as follows:

[0086] 1. Select a fresh porcine ligament-bone sample slice, 1-2 cm in length and width, and 100-200 μm in thickness. Sample pretreatment: Stain with sulfonated Cy3-NHS (polyfluorescent bio-based) fluorescent staining agent, which contains NHS activated ester.

[0087] 2. Place the ligament sample on the stage and sample clamping system of the optical microscope of the present invention, ensuring that the sample and the stepper motor applying the force are on the same horizontal line. The liquid in the water bath is phosphate buffer (composed of 137mM sodium chloride, 2.7mM potassium chloride, 8mM disodium hydrogen phosphate and 2mM potassium dihydrogen phosphate).

[0088] 3. Apply five known displacements (to the lengths corresponding to 4%, 8%, 12%, 16%, and 18% strain of the sample, which is equivalent to lengthening the sample by 4%, 8%, 12%, 16%, and 18%) to the ligament end of the ligament-bone sample using a stepper motor.

[0089] 4. In-situ observation: The deformation of the ligament sample under stress is observed in real time using the high-resolution microscope equipped with the device. The microscope lens is 20x.

[0090] 5. Data recording: Record the deformation process of the sample, including the size, rate and recovery of local deformation, and save it in the form of video recording.

[0091] 6. Analysis: The mechanical response of the sample was analyzed using PIV, and the local deformation characteristics of the ligament tissue were calculated.

[0092] The results of the local displacement and strain data obtained from the PIV analysis are shown in [the table below]. Figure 6 It can be seen that when the strain is less than or equal to 12%, the ligament region far from the bone end deforms first. After the strain reaches 16%, the ligament-bone interface region begins to deform, proving that there is a protective mechanism of delayed deformation in this interface region.

[0093] Example 3: The effect of sample pretreatment on the analytical results of samples observed in a water bath under microscopic in situ.

[0094] This embodiment uses the apparatus provided in Embodiment 1 and follows the method provided in Embodiment 2 to perform in situ microscopic observation of ligament samples (derived from pigs) in a water bath. The following staining agents are used for sample pretreatment:

[0095] 1. As in Example 2, the fluorescent staining dye sulfonated Cy3-NHS activated ester (Multi-Fluorescent Bio) contains NHS activated ester.

[0096] 2. Type I collagen antibody (1:200 dilution, catalog number ab88147, Abcam) and fluorescent secondary antibody Goat Anti-Mouse IgG H&L (1:400 dilution, Alexa) 488, catalog number ab150113 (Abcam), does not contain NHS activated esters, and is specifically labeled with type I collagen, the most abundant component in tissues, by immunofluorescence.

[0097] 3. Cy3 carboxylic acid (multi-fluorescent bio-), which does not contain NHS activated ester, contains a carboxyl group (-COOH) in its molecular structure. The principle of labeling is that CY3 carboxylic acid forms a covalent bond with amino acid residues (especially amino groups) in proteins, thus attaching the fluorescent dye to the protein molecule.

[0098] 4. Cy3-NHS activated ester (Multi-fluorescent Bio), which contains NHS activated ester. Cy3-NHS activated ester has low water solubility, so an organic co-solvent is required in the aqueous labeling reaction system.

[0099] Samples stained with the above three staining agents were observed in situ under a water bath. The sharpness, fluorescence intensity, signal-to-noise ratio, and color saturation of the images captured by the optical microscope were examined. Sharpness was measured using OpenCV to calculate the Laplacian sharpness of the image. Fluorescence intensity, signal-to-noise ratio, and color saturation were quantified using ImageJ software. The results were then compared with clear static images of the sample (without the water bath) and normalized. The results are shown in Table 1.

[0100] Table 1. Effects of different staining agents on analytical results

[0101]

[0102] As shown in Table 1, the image quality of the dye containing NHS activated ester is significantly better than that of the dye without NHS activated ester. This may be because NHS activated ester can react with the amino groups in the sample. Samples prepared by this reaction principle are more suitable for imaging in a water bath, resulting in better color development and improved image clarity. Although both dyes 1 and 4 contain NHS activated ester, the image quality of dye 4 is lower than that of dye 1. This may be because the water solubility of Cy3-NHS activated ester is not as good as that of sulfonated Cy3-NHS activated ester, thus affecting its fluorescent labeling efficiency. Therefore, dye 1 is the preferred dye.

[0103] Example 4: The effect of phosphate buffer on the analytical results of samples in a water bath for in-situ microscopic observation.

[0104] This embodiment uses the apparatus provided in Example 1 and follows the method provided in Example 2 to perform in-situ microscopic observation of ligament samples (derived from pigs) in a water bath. The liquids in the water bath are phosphate buffer, pure water, and physiological saline (the buffer concentrations are the same as in Example 1). The clarity, fluorescence intensity, signal-to-noise ratio, and color saturation of the images captured during in-situ microscopic observation of the mechanical response of the samples in the water bath were examined. The detection results are shown in Table 2.

[0105] Table 2. Effects of different staining agents on analytical results

[0106]

[0107] As can be seen from Table 2, immersion of samples in different liquids has different effects on the quality of images captured when observing the mechanical response in a water bath under microscopic in situ. The best choice is phosphate buffer, which may be because biological samples can better maintain their biological activity in phosphate buffer, thus making it more conducive to microscopic observation.

[0108] Example 5: The Influence of Sample Size on the Analytical Results of Samples Observed in a Water Bath under Microscopic In-situ

[0109] This embodiment uses the apparatus provided in Embodiment 1 and follows the method provided in Embodiment 2 to perform microscopic in-situ water bath observations on the following types of samples, wherein the sample sizes are as follows:

[0110] 1. Ligament sample, length and width 1-2cm, thickness 100-200um;

[0111] 2. Ligament sample, length and width 2-3cm, thickness 200-300um;

[0112] 3. Ligament sample, length and width 3-4cm, thickness 300-400um;

[0113] 4. Ligament sample, length and width 0-1cm, thickness 50-90um.

[0114] The above four different sized samples were observed in a water bath under a microscope to examine the clarity, fluorescence intensity, signal-to-noise ratio, and color saturation of the images captured by the optical microscope. The test results are shown in Table 3.

[0115] Table 3. Effects of different sample sizes on the analysis results

[0116]

[0117] As can be seen from Table 3, the size of the sample significantly affects the quality of the images captured when observing the mechanical response in a water bath under a microscope. When the sample size is too large, it will be difficult for the microscope to focus accurately. When the sample size is too small, the requirements for image clarity will be higher, resulting in a decrease in image quality. Therefore, the optimal sample size is 1-2 cm in length and width and 100-200 μm in thickness.

[0118] Example 6: Selection of Optical Microscope Lens

[0119] This embodiment uses the apparatus provided in Embodiment 1 and follows the method provided in Embodiment 2 to perform in-situ microscopic observation of the following samples in a water bath. The static optical microscopes used are 10x, 20x, 50x, and 100x. The samples were observed in the water bath using different lenses to examine the clarity, fluorescence intensity, signal-to-noise ratio, and color saturation of the images captured by the optical microscope. The results are shown in Table 4.

[0120] Table 4. The Influence of Different Lenses on the Analysis Results

[0121]

[0122] As shown in Table 4, the choice of lens significantly affects the image quality when observing mechanical responses in a water bath using microscopy. This is likely because different lenses have different focusing effects, resulting in noticeable differences in image quality when photographed in a water bath. A 10X lens offers a wide field of view but has limited resolution and sharpness; while 50X and 100X lenses offer high resolution, their narrow focusing range makes them prone to out-of-focusing, failing to meet observation requirements. Therefore, the optimal lens is 20X.

[0123] While the present invention has been disclosed above, it is not limited thereto. Its applications in medicine can be expanded accordingly. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A system for in-situ microscopic observation of the mechanical response of a sample, characterized in that, It includes a sample processing device, a microscopic observation device, and a water bath; the sample processing device is used to fix the sample and apply force to the sample; the microscopic observation device is used to observe the sample's response to the applied force in situ; the sample is located in the liquid in the water bath and is within the observation range of the microscopic observation device.

2. The system as described in claim 1, characterized in that, The sample processing device includes a clamping component and a force-applying component. The clamping component is used to fix the sample, and the force-applying component is used to apply force to the sample. The microscopic observation device is an optical microscope.

3. The system as described in claim 2, characterized in that, The clamping component includes a fixed end and a movable end; the fixed end is used to clamp and fix a first part of the sample; the movable end is used to clamp a second part of the sample and is displaced under the action of the force-applying component, thereby deforming the sample.

4. The system as described in claim 3, characterized in that, The fixed end uses a clamp and screws to fix the first part of the sample; the movable end uses a magnetic clamp to fix the second part of the sample. When the upper and lower clamps of the magnetic clamp are close together, they attract each other due to magnetism and clamp the sample, which can be easily opened and closed, thereby adjusting the clamping situation of the second part of the sample.

5. The system as described in claim 4, characterized in that, The mobile end is provided with a slide rail below, and the mobile end can be displaced along the slide rail under the action of the force-applying component; the force-applying component includes a force-applying end and a control end; the force-applying end is connected to the mobile end, thereby driving the mobile end to move together; the control end is used to control the displacement distance of the force-applying end, thereby controlling the displacement distance of the second part of the sample on the mobile end.

6. The system as described in claim 1, characterized in that, The sample has a thickness of less than 500 μm and a size of less than 2 x 2 cm; The liquid in the water bath is a phosphate buffer solution.

7. The system as described in claim 1, characterized in that, The sample needs to be fluorescently stained, and the fluorescent staining agent contains NHS activated ester.

8. The system as described in claim 1, characterized in that, The lens of the optical microscope is 10 to 100x.

9. A method for in-situ microscopic observation of the mechanical response of a sample, characterized in that, The method of observing the mechanical response of a sample in situ under a microscope using the system described in any one of claims 1 to 8 includes the following steps: (1) Fix the sample in the clamping component; (2) Set the sample movement distance at the control end of the force-applying component; (3) Turn on the camera mode on the microscope; (4) Turn on the motor of the force-applying component, and the force-applying component will cause the sample to deform; (5) The mechanical response of the sample was analyzed using software.

10. The method as described in claim 9, characterized in that, The software mentioned in step (5) includes DIC or PIV analysis.

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