Device for microscopic in-situ observation of mechanical response of sample

Abstract

The utility model provides a device for microscopic in-situ observation of mechanical response of a sample. The device comprises a sample processing device, a microscopic observation device and a water bath, the sample processing device is used for fixing a sample and applying force to the sample; the microscopic observation device is used for observing the reaction of the sample to the applied force in situ; 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] The utility model belongs to the technical field of bioengineering, and specifically relates to a device for microscopic in-situ observation of the mechanical response of samples. Background Art

[0002] When observing the mechanical response of biological or material samples by traditional methods, it is usually only possible to use non-in-situ observation methods. The problem of information distortion caused by non-in-situ sample processing is that the observation of the sample before and after being stressed is not carried out in the same environment, and only the information of the sample before and after being stressed can be observed, and the intermediate process cannot be reflected frame by frame.

[0003] Traditional mechanical testing equipment, such as universal material testing machines, is usually used to directly measure the mechanical properties of materials, but does not provide the in-situ observation of the material response mode and force change during the testing process. Moreover, the existing technologies usually aim to measure the mechanical properties of materials or biological samples, such as tensile strength, compressive strength, etc. By using specific fixtures to fix the sample and then applying force by using a motor or other driving mechanisms, although these technologies can also provide certain mechanical property data, they cannot provide in-situ observation, so the subtle changes of the sample during the stress process cannot be accurately captured.

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

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

[0006] To solve the above problems, the present utility model provides a device for microscopic in-situ observation of the mechanical response of a sample. A force is applied to the sample in a water bath to cause deformation, and the reaction of the sample to the applied force is observed in-situ through an optical microscope; the sample needs to be controlled to an appropriate size and first stained with fluorescence, and the staining reagent contains NHS active ester; the force-applying device includes a clamping component and a force-applying component. The clamping component fixes one end of the sample, and the other end undergoes displacement under the action of the force-applying component. The optical microscope records the changes of the sample under the force in-situ, can save images frame by frame, and can also use the supporting DIC or PIV to analyze the mechanical response of the sample, which is especially suitable for the in-situ observation of biological samples that need to simulate the physiological environment, so that the biological samples always maintain a wet and biologically active state, thereby obtaining more accurate data.

[0007] On the one hand, the present utility model provides a device for microscopic in-situ observation of the mechanical response of a sample. The device 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 a force to the sample; the microscopic observation device is used to observe in-situ the reaction of the sample to the applied force; the sample is located in the liquid in the water bath and within the observation range of the microscopic observation device.

[0008] The prior art usually involves non-in-situ sample processing, which means that the observation of the sample before and after being subjected to force is not carried out in the same environment, which may lead to distortion of the mechanical response information of the sample.

[0009] The device provided by the present utility model that can be used under a microscope includes a stable sample clamping system, an accurate force application mechanism, and an observation window compatible with the microscope, and the sample is located in a water bath. Through this device, in-situ observation can be carried out while applying a mechanical load. By realizing the in-situ observation of the mechanical response of the sample under the microscope, the present utility model can retain the in-situ information of the sample, allowing frame-by-frame analysis of the response without damaging the sample.

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

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

[0012] During the detection of the mechanical response of biological samples, due to leaving the original physiological environment, water loss is likely to occur.

[0013] The device for microscopic in-situ observation of the mechanical response of a sample provided by the present utility model enables the sample to be observed in a water bath. The water bath is filled with a liquid that can simulate the physiological environment for the sample or keep it active, and the sample is immersed in the liquid, so as to ensure that the water content in the sample does not change during the process of the sample reacting to the applied force.

[0014] The water bath described in the present utility model is not limited to immersing the sample in pure water only, but different liquids can be used for the water bath according to different samples.

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

[0016] In some ways, the water bath tank is made of transparent glass material and can transmit light. The thickness of the glass is in the range of 0.1 - 1 cm, so that the sample can still be clearly observed when the water bath tank is located within the microscope observation window. It can be understood that the glass thickness of the water bath tank only needs to ensure that it can be used to hold the water bath liquid.

[0017] Furthermore, 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 a force to the sample.

[0018] Furthermore, the microscopic observation device is an optical microscope.

[0019] Compared with an electron microscope, an optical microscope is more suitable for in - situ photographing of samples in a water bath environment. It not only allows simulating a physiological test environment but also does not cause damage to the samples.

[0020] 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 displace under the action of the force - applying component, so that the sample deforms.

[0021] When observing the mechanical response of the sample in situ, it is necessary to control the magnitude of the force applied to the force - applying component. However, since the sample is in a water bath environment, when it is subjected to the force applied by the force - applying component, it is also easily affected by the resistance of water, and the slight shaking of the water bath will also bring certain forces in different directions. Therefore, it is difficult to accurately control from the magnitude of the applied force. By controlling the displacement distance and displacement direction of the force - applying component, so that the sample also undergoes the same - distance displacement and deformation in a specific direction, this method can more accurately control the deformation degree of the sample each time, and the detection result is also more accurate.

[0022] The force - applying component in the device provided by the present utility model can accurately control the displacement distance of the sample by applying a specific magnitude of micro - force to the sample.

[0023] Furthermore, the fixed end uses a splint and a screw to fix the first part of the sample; the movable end uses a magnetic splint to fix the second part of the sample.

[0024] Further, the magnetic splint has upper and lower clamping pieces. When the upper and lower clamping pieces approach each other, they are attracted by magnetism to clamp the sample, which can be conveniently opened and closed, thereby adjusting the clamping condition of the second part of the sample.

[0025] In some ways, the fixed end uses a plastic splint and screws to fix the sample, thereby having a better fixing effect.

[0026] In some ways, the mobile end uses a relatively flexible and convenient method to fix the sample, facilitating the observation and adjustment of the force and deformation of the sample at any time, and preventing sample damage.

[0027] In some ways, the mobile end uses a magnetic splint to fix the sample. The magnetic splint is more convenient to open and close. For example, inserting an operating rod between the magnetic splints and gently lifting it upwards can open it, and gently pressing it down can close it, which is convenient for operation and adjustment, and the magnetic force splint is not easy to damage the sample.

[0028] Further, a slide rail is provided below the mobile end. The mobile end can displace along the slide rail under the action of a force-applying component; the force-applying component includes a force-applying end and a control end.

[0029] In some ways, there are two slide rails below the mobile end. Two points determine a straight line. The two rails can make the displacement direction of the mobile end more stable and controllable, and the displacement distance more accurate.

[0030] Further, 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 displace 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 at the mobile end.

[0031] In some ways, since the sample is in the water bath, the mobile 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 into the water bath from outside the water bath through a certain connection method, thereby connecting to the force-applying end and applying force to it to accurately control its displacement distance.

[0032] Further, the thickness of the sample is less than 500 um, and the size is less than 2 x 2 cm; the liquid in the water bath is phosphate buffer solution.

[0033] In order to enable the sample in the water bath to be clearly observed at the microscope observation window, the size of the sample must be strictly controlled within an appropriate range.

[0034] In some ways, the length and width dimensions of the sample are 1 - 2 cm, and the thickness is 100 - 200 um.

[0035] In some ways, the sample is in a flake shape.

[0036] In some ways, the force applied by the force - applying component of the present utility model to the sample is a very tiny micro - force, which causes a tiny displacement and deformation of the sample, and then the whole process is photographed by a microscope and analyzed frame by frame.

[0037] When the liquid in the water - bath tank is phosphate buffer solution, it is more conducive to observing the sample than pure water, and is also more beneficial to keeping the sample in a biologically active state. Moreover, the sample is not easily damaged under the action of the force - applying device.

[0038] Further, the sample needs to be fluorescently stained, and the dye for the fluorescent staining contains NHS active ester (N - hydroxysuccinimide ester, N - hydroxysuccinimide ester, succinimidyl ester).

[0039] NHS active ester (N - hydroxysuccinimide ester, N - hydroxysuccinimide ester, succinimidyl ester) is the most commonly used active group in biolabeling reactions. It activates the carboxyl group in the sulfonated Cy3 molecule, enabling it to react with the amino group (primary amine or secondary amine) on the target biomolecule to form a stable amide bond, thereby labeling the dye molecule onto the biological macromolecule. Since free amino groups are common functional groups on the surface of proteins, antibodies, and polypeptides (from the lysine side chain), sulfonated Cy3 - NHS can react directly with them.

[0040] Research has shown that when the dye for fluorescent staining of the sample contains NHS active ester, compared with other dyes, it can significantly improve the clarity during microscope photography. The reason may be that after the reaction and labeling of NHS active ester with the amino group on the sample molecule, the sample can still maintain good clarity when photographed in the water - bath.

[0041] Further, the lens of the optical microscope is 10 - 100x.

[0042] The selection of the lens of the microscope has an obvious impact on the clarity of photographing the mechanical response process of the sample.

[0043] In some ways, the lens of the optical microscope is 20x, 50x or 100x.

[0044] In some ways, the lens of the optical microscope is 20x, which can further improve the clarity of photographing the mechanical response process of the sample compared with 50x and 100x.

[0045] The method for micro - in - situ observation of the mechanical response of a sample using the above - mentioned device includes the following steps:

[0046] (1) Fix the sample in the clamping component;

[0047] (2) Set the moving distance of the sample at the control end of the force - applying component;

[0048] (3) Turn on the camera mode of the microscope;

[0049] (4) Turn on the motor of the force - applying component. The force - applying component drives the sample to deform, and record the changes of the sample under the force in situ;

[0050] (1) Use software to analyze the mechanical response of the sample. The in - situ mechanical response video of the sample can be analyzed by taking screenshots frame by frame.

[0051] Further, the software includes DIC (Digital Image Correlation Analysis) or PIV (Particle Image Velocimetry Analysis).

[0052] The DIC method is a method of making characteristic points (speckle pattern) on the surface of the object to be measured, capturing the movement of speckle features at the pixel level, and using an optimized 3D digital image correlation algorithm to provide measurement of two - dimensional and three - dimensional full - field topography, displacement, and strain data for the experiment. It is usually divided into four steps: speckle making, system calibration, strain measurement, and image analysis.

[0053] PIV (Particle Image Velocimetry Analysis) is a method of using multiple cameras to record the positions of particles in the flow field and analyzing the captured images to measure the flow velocity. Its basic principle is to distribute tracer particles in the flow field and use a pulsed laser sheet light source to irradiate the measured flow field area. Through two or more consecutive exposures, the images of the particles are recorded on the film or CCD camera.

[0054] However, the general DIC experimental method has the following disadvantages, which are optimized in the device provided by the present utility model:

[0055] 1. Low accuracy and low resolution. The general DIC experimental method does not rely on microscope imaging but on ordinary cameras, and the resolution of its image and video data is lower than that of the microscope lens used in the present utility model.

[0056] 2. It does not allow the sample to be measured to contain water. In the general DIC method, making and capturing the characteristic points on the surface of the sample do not allow the sample to have moisture, which is not friendly to the testing of biological samples.

[0057] 3. It cannot provide a physiological - like environment for biological samples. In the DIC method, biological samples are extremely prone to water loss and inactivation during long - term testing, but the device of the present utility model can provide an aqueous environment to keep biological samples always moist and biologically active.

[0058] The present utility model has the following beneficial effects:

[0059] (1) The provided device allows for the direct observation of a sample during the force application process, enabling in-situ observation, more accurately capturing the true behavior of the sample during the force application process, and reducing the distortion that may occur during the sample processing compared to non-in-situ observation;

[0060] (2) It supports frame-by-frame analysis of the mechanical response of the sample, providing more detailed and in-depth data analysis capabilities than traditional techniques, and making it possible to deeply understand the mechanical behavior of materials or biological samples;

[0061] (3) The sample clamping device allows for the water bath of the entire device. Phosphate buffer solution or physiological saline can be added to the water bath tank to ensure that the sample does not lose water, providing conditions similar to the physiological environment for the sample;

[0062] (4) It can achieve non-destructive testing, allowing for in-situ observation of the immediate 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, and is especially suitable for samples that cannot withstand traditional mechanical testing methods, and is of great significance for precious or non-renewable samples;

[0063] (5) By debugging various parameters of the device, the stability of the sample during the application of mechanical load is ensured;

[0064] (6) It has 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, this device can be used in combination with existing microscope systems and analysis methods such as digital image correlation method (DIC) and particle image velocimetry (PIV), expanding its application scope. Description of the Drawings

[0065] Figure 1 It is a schematic diagram of the device for microscopic in-situ observation of the mechanical response of the sample in Example 1;

[0066] Figure 2 It is the overall structure diagram of the device for microscopic in-situ observation of the mechanical response of the sample in Example 1;

[0067] Figure 3 It is the structure diagram of the sample processing device in Example 1;

[0068] Figure 4 It is the structure diagram of the device inside the water bath tank in Example 1;

[0069] Figure 5 It is a schematic diagram of the process of displacement of the second part of the sample in Example 1;

[0070] Figure 6 It is the result diagram of the local displacement and strain data of the sample obtained by PIV analysis in Example 2. Detailed implementation manners

[0071] The preferred embodiments of the present utility model will be further described in detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate the understanding of the present utility model and do not impose any limitations on it. The raw materials and equipment used in the specific embodiments of the present utility model are all known products and can be obtained by purchasing commercially available products.

[0072] Embodiment 1. The device for microscopically in-situ observing the mechanical response of a sample provided by the present utility model

[0073] The schematic diagram of the device for microscopically in-situ observing the mechanical response of a sample provided in this embodiment is shown in Figure 1 and the overall structure diagram is shown in Figure 2 wherein the structure diagram of the sample processing device is shown in Figure 3 and the structure of the device inside the water bath is shown in Figure 4 and the schematic diagram of the process of the second part of the sample undergoing displacement is shown in Figure 5 .

[0074] As shown in Figure 2 the device 1 for microscopically in-situ observing 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 a force to the sample 5. The microscopic observation device 3 is used to in-situ observe the reaction of the sample 5 to the applied force. The sample 5 is located in the liquid 6 in the water bath 4 and within the observation range of the microscopic observation device 3. The sample 5 can be a biological sample. During the mechanical response detection process, due to leaving the original physiological environment, water loss is likely to occur. With this device 1, the sample 5 can be observed while being located in the water bath 4. The water bath 4 is filled with a liquid 6 that can simulate the physiological environment for the sample or keep it active. The sample 5 is immersed in the liquid 6, so as to ensure that during the process of the sample 5 reacting to the applied force, the water content in the sample 5 does not change. The liquid in the water bath can be phosphate buffer solution or physiological saline to ensure that the biological sample does not lose water. The water bath 4 is made of transparent glass material, which can transmit light and has a thickness of about 0.3 cm, so that the water bath 4 can still clearly observe the sample 5 while being within the observation window of the observation device 3.

[0075] As shown in Figures 2 to 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 a 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 photographing of samples in a water bath environment, will not cause damage to the microscope, and has higher shooting clarity.

[0076] As shown in Figure 3, the clamping member 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 under the action of the force-applying member 8, so that the sample 5 deforms. When observing the mechanical response of the sample 5 in situ, it is necessary to control the magnitude of the force applied to the force-applying member 8. However, since the sample 5 is in a water bath environment, when it is subjected to the force applied by the force-applying member 8, it is also easily affected by the resistance of water, and the slight shaking of the water bath will also bring certain forces in different directions. Therefore, it is difficult to accurately control from the magnitude of the applied force. By controlling the displacement distance and displacement direction of the force-applying member, so that the sample also undergoes the same distance of displacement and deformation in a specific direction, this method can more accurately control the force application method each time, and the detection result is also more accurate. The force-applying member 8 in the device 1 of this embodiment can accurately control the displacement distance of the sample 5 by applying a specific magnitude of micro-force to the sample 5.

[0077] Preferably, the fixed end 10 uses a plastic splint 14 and a screw 15 to fix the first part 12 of the sample 5; the movable end 11 uses a magnetic splint 16 to fix the second part 13 of the sample 5. When the upper and lower clips 17 of the magnetic splint 16 approach, they are attracted by magnetism to clamp the sample 5, which can be conveniently opened and closed, so as to adjust the clamping condition of the second part 13 of the sample 5. The fixed end 10 uses a plastic splint 14 and a screw 15 to fix the sample, so as to have a better fixing effect. The movable end 11 uses a more flexible and convenient magnetic splint 16 to fix the sample 5. The magnetic splint 16 is more convenient to open and close. For example, inserting an operating rod between the upper and lower clips 17 of the magnetic splint 16 and gently picking it up can open it, and gently pressing it down can close it, which is convenient for operation and adjustment. Moreover, the magnetic force splint 16 is not easy to damage the sample 5, and it is also convenient to observe and adjust the stress and deformation of the sample 5 at any time to prevent damage to the sample 5.

[0078] Such as Figure 2 And 4, a slide rail 18 is provided below the mobile end 11, and the mobile end 11 can be displaced along the slide rail 18 under the action of the force - applying component 8; 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 displace 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 second part 13 of the sample 5 on 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 the sample 5 is in the water bath 4, the mobile end 11 of the clamping component 7 and the force - applying end 19 of the force - applying component 8 also need to be in the water bath 4, while the control end 20 of the force - applying component 8 needs to be outside the water bath 4. In order to accurately control the displacement of the force - applying end 19, the control end 20 needs to extend into the water bath 4 from outside the water bath 4 through a certain connection method, so as to be connected to the force - applying end 19 and apply force to it to accurately control its displacement distance. The control end 20 accurately controls the displacement of the force - applying end 19 by the advancing distance of the rocker 21.

[0079] Preferably, the thickness of the sample 5 is less than 500 um, and the size is less than 2x2 cm; the liquid in the water bath 4 is phosphate - buffered solution. In order to enable the sample 5 in the water bath 4 to be clearly observed at the microscope observation window, the size of the sample 5 must be strictly controlled within an appropriate range. Preferably, the length and width dimensions of the sample 5 are 1 - 2 cm, and the thickness is 100 - 200 um. The sample 5 is in the shape of a thin sheet.

[0080] The method for microscopic in - situ observation of the mechanical response of a sample using the device provided in this embodiment includes the following steps:

[0081] (1) Fix the sample 5 in the clamping component 7;

[0082] (2) Set the moving distance of the sample at the control end 20 of the force - applying component 8;

[0083] (3) Turn on the camera mode of the optical microscope 9;

[0084] (4) Turn on the motor of the force - applying component 8, and the force - applying component 8 drives the sample 5 to deform, and in - situ record the changes of the sample 5 under the force - bearing state;

[0085] (5) Use software to analyze the mechanical response of the sample 5. The in - situ mechanical response video of the sample 5 can be analyzed by taking screenshots frame by frame. The analysis software includes DIC (Digital Image Correlation Analysis) or PIV (Particle Image Velocimetry Analysis).

[0086] Example 2: Observe the deformation of ligament tissue under micro - force

[0087] This embodiment uses the device for microscopic in-situ observation of the mechanical response of samples provided in Embodiment 1 to observe the deformation of ligament tissue under micro-force. The main process is as follows:

[0088] 1. Select a fresh porcine ligament-bone sample slice with length and width dimensions of 1 - 2 cm and a thickness of 100 - 200 μm. Sample pretreatment: Stain it with the fluorescent dye sulfonated Cy3-NHS (MultiFluor Bio), and sulfonated Cy3-NHS contains NHS active ester.

[0089] 2. Place the ligament sample on the stage of the optical microscope and the sample clamping system of the device of the present utility model, ensuring that the sample and the stepping motor applying the force are on the same horizontal line. The liquid in the water bath is phosphate buffer (the composition is 137 mM sodium chloride, 2.7 mM potassium chloride, 8 mM disodium hydrogen phosphate, and 2 mM potassium dihydrogen phosphate).

[0090] 3. Apply 5 displacements of known magnitudes (the lengths corresponding to 4%, 8%, 12%, 16%, and 18% strain of the sample) to the ligament end of the ligament-bone sample through the stepping motor.

[0091] 4. In-situ observation: Use the high-resolution microscope equipped with the device to observe the deformation of the ligament sample during the force application process in real time. The microscope lens is 20x.

[0092] 5. Data recording: Record the deformation process of the sample, including the magnitude, rate, and recovery of local deformation, and save it in the form of a video.

[0093] 6. Analysis: Use PIV to analyze the mechanical response of the sample and calculate the local deformation characteristics of the ligament tissue.

[0094] The results of the local displacement and Lagrangian strain data of the sample obtained by PIV analysis are shown in Figure 6 , and it can be seen that: when the strain is less than or equal to 12%, the ligament area far from the bone end deforms first. After the strain reaches 16%, the ligament-bone interface area begins to deform, proving that there is a protection mechanism for hysteretic deformation in this interface area.

[0095] Embodiment 3. Influence of sample pretreatment on the analysis results of microscopic in-situ observation of samples in water bath

[0096] This embodiment uses the device provided in Embodiment 1 and observes the ligament samples (from pigs) in-situ in water bath according to the method provided in Embodiment 2. The following several staining agents are used for the pretreatment of the samples respectively:

[0097] 1. As in Example 2, the fluorescent dye sulfonated Cy3-NHS active ester (MultiFluorBio) contains NHS active ester;

[0098] 2. Type I collagen antibody (diluted 1:200, catalog number ab88147, Abcam) and fluorescent secondary antibody Goat Anti-Mouse IgG H&L (diluted 1:400, Alexa 488, catalog number ab150113, Abcam), which does not contain NHS active ester, specifically labels type I collagen, the most abundant component in tissues, by immunofluorescence;

[0099] 3. Cy3 carboxylic acid (MultiFluorBio), which does not contain NHS active 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, connecting the fluorescent dye to the protein molecule;

[0100] 4. Cy3-NHS active ester contains NHS active ester, but its effect is also not as good as that of 1. The water solubility of Cy3-NHS active ester is relatively low, so an organic co-solvent needs to be used in the aqueous-phase labeling reaction system.

[0101] The samples stained with the above three dyes were observed in a water bath by microscopic in-situ observation, and the clarity, fluorescence intensity, signal-to-noise ratio, and color saturation of the images taken by the optical microscope were investigated. The detection method for clarity was to calculate the Laplacian sharpness of the image using OpenCV, and the detection methods for fluorescence intensity, signal-to-noise ratio, and color saturation were to quantify them through ImageJ software, and the results were compared with the clear static pictures (without the water bath) of the samples and normalized. The detection results are shown in Table 1.

[0102] Table 1. Influence of different dyes on the analysis results

[0103]

[0104] It can be seen from Table 1 that when using a dye containing NHS active ester, the quality of the taken images is significantly better than that of the dye without NHS active ester. The reason may be that NHS active ester can react with amino groups in the sample. The samples prepared by this reaction principle are more suitable for taking pictures of samples in a water bath, and the color development effect is better during the water bath process, which helps to improve the shooting clarity; at the same time, although the first and fourth dyes both contain NHS active ester, compared with the first one, the image quality of the fourth one still decreases. The reason may be that the water solubility of the fourth dye is not as good as that of the first one, thus affecting its fluorescence labeling efficiency. Therefore, the most preferred dye is the first one.

[0105] Example 4. Influence of Phosphate Buffer Solution on the Analysis Results of Samples in the Microscopic In-Situ Observation Water Bath

[0106] In this example, the device provided in Example 1 was used, and the method provided in Example 2 was followed to observe ligament samples (from pigs) in the microscopic in-situ water bath. The liquid in the water bath was respectively phosphate buffer solution, pure water, physiological saline (the buffer concentration was the same as that in Example 1). The clarity, fluorescence intensity, signal-to-noise ratio, and color saturation of the images taken when observing the mechanical response of the samples in the microscopic in-situ water bath were investigated. The detection results are shown in Table 2.

[0107] Table 2. Influence of Different Staining Agents on the Analysis Results

[0108]

[0109] It can be seen from Table 2 that when the samples are immersed in different liquids, it also has different effects on the quality of the images taken when observing the mechanical response of the samples in the microscopic in-situ water bath. The most preferred one is the phosphate buffer solution. The reason may be that biological samples can better maintain their biological activities in the phosphate buffer solution, so it is more conducive to microscopic observation.

[0110] Example 5. Influence of Sample Size on the Analysis Results of Samples in the Microscopic In-Situ Observation Water Bath

[0111] In this example, the device provided in Example 1 was used, and the method provided in Example 2 was followed to observe the following several types of samples in the microscopic in-situ water bath. The sizes of the samples were respectively as follows:

[0112] 1. Ligament sample, with length and width dimensions of 1 - 2 cm and thickness of 100 - 200 μm;

[0113] 2. Ligament sample, with length and width dimensions of 2 - 3 cm and thickness of 200 - 300 μm;

[0114] 3. Ligament sample, with length and width dimensions of 3 - 4 cm and thickness of 300 - 400 μm;

[0115] 4. Ligament sample, with length and width dimensions of 0 - 1 cm and thickness of 50 - 90 μm.

[0116] The above 4 types of samples with different sizes were observed in the microscopic in-situ water bath, and the clarity, fluorescence intensity, signal-to-noise ratio, and color saturation of the images taken by the optical microscope were investigated. The detection results are shown in Table 3.

[0117] Table 3. Influence of Different Sample Sizes on the Analysis Results

[0118]

[0119] As can be seen from Table 3, the size of the sample will significantly affect the quality of the images taken during the microscopic in-situ observation of the mechanical response in the water bath. When the sample size is too large, it will be difficult for the microscope to accurately focus. When the sample size is too small, a higher requirement for shooting clarity will lead to a decrease in image quality. Therefore, the most preferred sample size is 1-2 cm in length and width and 100-200 um in thickness.

[0120] Example 6, Selection of the Optical Microscope Lens

[0121] In this example, the device provided in Example 1 was used, and the following several samples were observed in-situ in the water bath according to the method provided in Example 2. Among them, the optical microscope was statically used at 10x, 20x, 50x, and 100x respectively. The samples were observed in-situ in the water bath using different lenses, and the clarity, fluorescence intensity, signal-to-noise ratio, and color saturation of the images taken by the optical microscope were investigated. The test results are shown in Table 4.

[0122] Table 4. Influence of Different Lenses on the Analysis Results

[0123]

[0124] As can be seen from Table 4, the selection of the lens will significantly affect the quality of the images taken during the microscopic in-situ observation of the mechanical response in the water bath. The reason may be that the focusing effects of different lenses are different, and there are also obvious differences in the image effects during shooting in the water bath. The 10X lens has a large field of view, but limited resolution and clarity; although the 50X and 100X lenses have high resolution, their focusing ranges are too narrow, and it is extremely easy to defocus, making it difficult to meet the observation requirements. Therefore, the most preferred lens is 20x.

[0125] Although the present utility model is disclosed as above, the present utility model is not limited thereto. For example, it can be extended according to its application scope in medicine. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present utility model. Therefore, the protection scope of the present utility model should be subject to the scope defined by the claims.

Claims

1. An apparatus for micro-in-situ 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 a force to the sample; the microscopic observation device is used to in-situ observe the reaction of the sample to the applied force; the sample is located in the liquid in the water bath and within the observation range of the microscopic observation device.

2. The device according to 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 a force to the sample.

3. The device according to claim 2, characterized in that, The microscopic observation device is an optical microscope.

4. The device according to claim 3, characterized in that, 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 displace under the action of the force applying component, so that the sample is deformed.

5. The device according to claim 4, characterized in that, The fixed end uses a splint and screws to fix the first part of the sample; the movable end uses a magnetic splint to fix the second part of the sample.

6. The device according to claim 5, characterized in that, The magnetic splint has upper and lower clips. When the upper and lower clips approach, they are attracted by magnetism to clamp the sample, and can be conveniently opened and closed to adjust the clamping condition of the second part of the sample.

7. The device according to claim 6, characterized in that, A slide rail is provided below the movable end, and the movable end can displace along the slide rail under the action of the force applying component.

8. The device according to claim 7, characterized in that, The force applying component includes a force applying end and a control end; the force applying end is connected to the movable end to drive the movable end to displace together; the control end is used to control the displacement distance of the force applying end, so as to control the displacement distance of the second part of the sample at the movable end.

9. The device according to claim 8, wherein, The thickness of the sample is less than 500um, and the size is less than 2x2cm; the liquid in the water bath is phosphate buffer solution; the sample needs to be fluorescently stained, and the fluorescent staining dye contains NHS active ester.

10. The device according to claim 9, characterized in that, The lens of the optical microscope is 10 - 100x.

Images