Low-density biological tissue cryo-embedding method and label-free biological tissue imaging method

Abstract

This invention discloses a method for cryogenic embedding of low-density biological tissue and a label-free imaging method for biological tissue, relating to the field of cryopreservation technology. The method involves first placing the low-density biological tissue in a mold containing embedding medium, placing the container in a liquid nitrogen environment, and then placing the mold in the container for initial cryopreservation. Once the surrounding embedding medium has frozen beyond the edge of the low-density biological tissue, pre-cooled embedding medium is added until it completely covers the surface of the emerging low-density biological tissue, followed by a second cryopreservation process. This method for cryopreservation of low-density biological tissue avoids structural damage caused by the rapid freeze-thaw-rapid freeze process and prevents a clear boundary between the original and newly added cryopreservation solutions. The cryopreservation solutions at the solidified interface exhibit good fusion, and the tissue sample blocks do not dissociate or detach during cutting, significantly improving the cryopreservation embedding effect of low-density biological tissue.

Description

Technical Field

[0001] This invention relates to the field of biological tissue cryopreservation technology, and in particular to a low-density biological tissue cryogenic embedding method and a label-free biological tissue imaging method. Background Technology

[0002] Most existing cryopreservation / embedding techniques for biological tissues involve directly immersing the tissue in a cryopreservation solution (i.e., embedding agent, such as OCT cryopreservation agent) for rapid freezing. However, when conventionally cryopreserving / embedding biological tissues with a density less than or similar to that of the cryopreservation solution, the tissues tend to float rapidly due to the combined effects of buoyancy and rapid freezing. For example, animal lung tissue samples, or samples containing part or all of the lung tissue from various anatomical structures (such as the cardiopulmonary system), have a much lower density than conventional solid organs because lung tissue is mainly composed of alveoli and contains a large amount of air. Therefore, the aforementioned problem easily occurs during embedding, causing this portion of the biological tissue sample to be exposed on the surface of the embedding agent, thus affecting the embedding effect and subsequent cutting results. Summary of the Invention

[0003] To address the shortcomings of existing cryo-embedding techniques for biological tissues when embedding low-density biological tissue samples, this invention provides a low-temperature embedding method for low-density biological tissues and a label-free biological tissue imaging method, specifically implemented through the following techniques.

[0004] A method for low-density biological tissue cryogenic embedding includes the following steps:

[0005] Low-density biological tissue was placed in a mold containing embedding agent, the container was placed in a liquid nitrogen environment, and the mold was placed in the container for initial cryo-embedding treatment.

[0006] Once the surrounding embedding agent has frozen beyond the edge of the low-density biological tissue, pre-cooled embedding agent is added until it completely covers the surface of the emerging low-density biological tissue, and then the freezing and embedding process is repeated.

[0007] The cryogenic embedding method provided by this invention is a specific embedding technique for low-density biological tissues. This method involves selecting a specific moment when the low-density biological tissue has been rapidly frozen until the edges are firmly bonded to the partially solidified embedding agent, and the cryopreservation solution below the liquid surface and inside the mold has not yet frozen. At this point, additional cryopreservation solution is poured onto the surface of the low-density biological tissue exposed outside the embedding agent, allowing the exposed tissue to be re-embedded and covered. Then, rapid freezing continues until the final state. If additional cryopreservation solution is added to the surface of the existing solution after rapid freezing, on the one hand, the exposed tissue will undergo a rapid-freeze-thaw-rapid-freeze process, damaging the tissue structure and morphology; on the other hand, even if the subsequently added embedding agent is partially rapidly frozen, the original rapidly frozen embedding agent and the subsequently added embedding agent will still not be completely integrated, forming a clear boundary line at the boundary. This can lead to easy dissociation and detachment of tissue samples during subsequent cutting processes or re-freezing and embedding, resulting in sample damage. The method described in this application not only solves the problem of tissue floating during cryo-embedding of low-density biological tissue using existing embedding techniques, but also addresses the issue of incomplete fusion of the successively added embedding agents, thereby improving the embedding effect and ensuring subsequent cutting results.

[0008] Furthermore, the embedding agent freezes to a distance of 1.5mm-6mm beyond the edge of the low-density biological tissue.

[0009] Furthermore, the sidewall of the mold is 3mm-10mm away from the edge of the low-density biological tissue.

[0010] Furthermore, the amount of embedding agent used in the initial cryo-embedding process is 50%-75% of the mold volume.

[0011] Furthermore, the total time from placing the low-density biological tissue into the mold containing the embedding agent to starting the initial cryo-embedding process does not exceed 60 seconds.

[0012] Generally, when placing low-density biological tissue into a mold, it is crucial to carefully manage the time spent orienting the tissue. Otherwise, the tissue may float before it has had a chance to freeze, hindering subsequent embedding operations. Therefore, the time between placing the tissue into the mold and initiating the initial cryo-embedding process should be minimized. This time should be 30-60 seconds, and should not exceed 60 seconds.

[0013] Furthermore, before placing the mold in the container for the initial cryo-embedding process, the container is floated in a liquid nitrogen environment for at least 1 minute.

[0014] Furthermore, the method for obtaining the pre-cooled embedding agent is as follows: the embedding agent is placed above the liquid nitrogen surface, and the embedding agent is pre-cooled using liquid nitrogen vapor for 2-4 minutes.

[0015] Furthermore, the embedding agent is located 1-2 cm above the liquid nitrogen surface.

[0016] Preferably, the temperature of the liquid nitrogen vapor is -100℃ to -70℃.

[0017] This invention also provides a rapid optical imaging method for label-free biological tissues, which employs any of the low-density biological tissue cryogenic embedding methods provided by this invention to obtain cryogenically embedded low-density biological tissues; then, the surface layer of the cryogenically embedded low-density biological tissues is imaged; the surface layer is then cut to expose a new surface layer for imaging again; this operation is repeated until the target number of layers is imaged.

[0018] Compared with the prior art, the advantages of the present invention are:

[0019] 1. The method provided by this invention will not cause damage to the tissue structure and morphology of exposed tissues due to the quick-freeze-thaw-quick-freeze process, nor will it create a clear interface between the original cryopreservation solution and the newly added cryopreservation solution; the original cryopreservation solution and the newly added cryopreservation solution have good fusion effect after solidification, and the sample blocks that are quick-frozen twice during cutting will not dissociate and fall off; it significantly improves the cryopreservation and embedding effect of low-density biological tissues.

[0020] 2. Low-density biological tissues obtained using the low-temperature embedding method of the present invention can be used for label-free biological tissue imaging, which can improve the luminescence efficiency of endogenous fluorescent proteins in the tissue and ensure the final high-quality imaging effect. Attached Figure Description

[0021] Figure 1 This is a flowchart of the cryopreservation and embedding method for low-density biological tissues (taking lung tissue as an example) according to the present invention.

[0022] Figure 2 A schematic diagram of the low-density biological tissue cryopreservation and embedding method provided by the present invention;

[0023] Figure 3 The images show the frontal and 45° side views of the cryo-embedded tissues obtained using the embedding methods of Examples 1-3.

[0024] Figure 4 Frontal and 45° side views of cryo-embedded tissues obtained using the embedding method of Comparative Example 2;

[0025] Figure 5A schematic diagram of a rapid optical imaging system for label-free biological tissues;

[0026] Figure 6 This is a schematic diagram of the thermal insulation component.

[0027] Figure 7 Steady-state thermodynamic simulation diagram of the internal environment of the thermal insulation component;

[0028] Figure 8 This diagram shows the temperature changes around the objective lens under different insulation methods.

[0029] Figure 9 This is a schematic diagram of the structure of the low-temperature component;

[0030] Figure 10 Comparison of fluorescence imaging results of brain tissue sections using different objectives;

[0031] Figure 11 Comparison of seismic frequency numerical simulations of the internal structures of different mounting plates to simulate the vibration reduction effect;

[0032] Figure 12 This is a schematic diagram of the structure of the cutting assembly.

[0033] Figure 13 This is a structural diagram of the organization's supporting components.

[0034] In the diagram: 1. Mounting plate; 2. Low-temperature chamber; 3. Two-color light source; 4. Objective lens; 401. Lens; 5. Dichroic mirror; 6. Detector; 7. First pump; 8. Insulation shell; 9. Heat exchange tube; 10. Protective window; 11. Opening; 12. Heating layer; 13. Heating channel; 14. Temperature sensor; 15. Sample stage; 16. Cooling module; 17. Radiator; 18. Second pump; 19. Heat dissipation tube; 20. First base; 21. Clamping device; 22. Tool; 23. Second base; 24. Y / Z axis sliding device. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] The low-density biological tissue cryogenic embedding methods used in the following examples and comparative examples all employ fresh mouse lung organs as tissue samples. Because lung tissue is primarily composed of alveoli and contains a large amount of air, its density is much lower than that of conventional solid organs. During embedding, the tissue is prone to floating in the embedding medium, affecting the embedding quality. In addition to lung organs, the low-density biological tissue of this invention can also be other low-density biological tissues containing air, such as the heart.

[0037] Most existing cryopreservation / embedding techniques for biological tissues involve directly immersing the tissue in a cryopreservation solution (i.e., an embedding agent, such as OCT cryopreservation agent) for rapid freezing. However, when conventionally cryopreserving / embedding biological tissues with a density less than or similar to that of the cryopreservation solution, the tissues are subject to the combined effects of buoyancy and rapid freezing, causing them to float rapidly (e.g., ...). Figure 2 -②to Figure 2 -④ (as shown). If no operation is performed at this time, this part of the sample block will be exposed on the surface of the embedding agent, affecting the embedding effect and subsequent cutting effect. If additional cryopreservation solution is added to the surface of the original cryopreservation solution after quick-freezing, on the one hand, the exposed tissue will undergo a quick-freeze-thaw-quick-freeze process, destroying the tissue structure and morphology; on the other hand, even if the additional embedding agent is partially quick-frozen, the original quick-frozen embedding agent and the additional embedding agent will still not be completely integrated, forming a clear boundary line at the boundary. This will cause the second quick-frozen part of the sample block to detach and fall off during the subsequent cutting process, resulting in sample block damage.

[0038] For example Figure 2 The animal lung tissue sample block shown, or a sample block containing part / all of the lung tissue and various anatomical structures (such as the cardiopulmonary system), has a much lower density than conventional solid organs because lung tissue is mainly composed of alveoli and contains a large amount of air. This leads to the aforementioned problems.

[0039] like Figure 1 , 2 As shown, the low-density biological tissue cryogenic embedding method used in the following embodiments and comparative examples is carried out using the following steps:

[0040] S1. Use tin foil to make molds and small boats (i.e. containers). The size of the tin foil molds can be customized according to the size of the tissue samples.

[0041] Generally, the sidewalls of the foil mold are 3-10 mm larger than the sample. In the following examples and comparative examples, the molds are made to a size approximately 5 mm larger than the sample for easy comparison of the embedding effect.

[0042] The length, width, and height of the foil-wrapped boat are determined based on the actual size of the tissue sample. For ease of comparison of embedding effects, the containers in the following examples and comparative cases are made to a size approximately 15mm larger than the mold.

[0043] S2. Pour OCT embedding agent into the mold; then place mouse lung tissue into the mold and adjust the orientation of the lung tissue.

[0044] Generally, the amount of OCT embedding agent added is 50-75% of the mold volume. In the following examples and comparative examples, the amount of OCT embedding agent added is 60% of the mold volume.

[0045] Meanwhile, the small tin foil boat is pre-cooled in liquid nitrogen. The pre-cooling time is generally about 1-2 minutes; in the following examples and comparative examples, the boat was pre-cooled for 2 minutes.

[0046] Generally, the mold, which is at room temperature, is quickly transferred to a small tin foil boat floating on the surface of liquid nitrogen and pre-cooled by the liquid nitrogen. This process should not exceed 60 seconds, otherwise it will affect the subsequent embedding effect.

[0047] S3. Place the mold containing mouse lung tissue into a small tin foil boat to begin the initial cryo-embedding.

[0048] The embedding agent around the tissue freezes until it extends beyond the edge of the low-density biological tissue. The distance between the edge of the embedding agent freezing beyond the tissue and the edge of the tissue itself varies depending on the embodiments and comparative examples.

[0049] S4. Next, add OCT embedding medium pre-cooled by liquid nitrogen vapor into the mold. The amount of embedding medium should be enough to completely cover the tissue surface floating above the embedding medium surface, and then freeze-embed again. After embedding is complete, store the sample at -80℃.

[0050] Optionally, the OCT embedding agent is pre-cooled with liquid nitrogen vapor for 2-4 minutes. For ease of comparison, the following examples and comparative examples all use a pre-cooling time of 3 minutes.

[0051] Generally, the re-freezing and embedding time is about 8-15 minutes. For ease of comparison, the embedding process in the following examples and comparative examples is uniformly 12 minutes.

[0052] It should be noted that the term "low-density biological tissue" is relative to the cryopreservation solution, referring to tissues whose density is lower or not significantly different from that of the cryopreservation solution in all or part of the tissue portion. Therefore, based on the density of different cryopreservation solutions, in addition to lung tissue, other low-density biological tissues such as the combined heart and lung organs may also exist that can be cryopreserved and embedded using the above methods.

[0053] When using low-density biological tissue to select mouse lung organs, the mouse's trachea needs to be ligated before the lung is harvested by opening the chest to prevent the lung from collapsing, thus ensuring the shape of the lung.

[0054] Example 1

[0055] The low-density biological tissue low-temperature embedding method provided in this embodiment is based on the above method. Specifically, in step S3, the embedding agent is frozen and extends beyond the edge of the tissue by 5 mm from the edge of the tissue itself. Example 2

[0056] The low-density biological tissue low-temperature embedding method provided in this embodiment is based on the above method. Specifically, in step S3, the embedding agent is frozen and extends beyond the edge of the tissue by a distance of 1.5 mm from the edge of the tissue itself. Example 3

[0057] The low-density biological tissue low-temperature embedding method provided in this embodiment is based on the above method. Specifically, in step S3, the embedding agent is frozen and extends beyond the edge of the tissue by a distance of 6 mm from the edge of the tissue itself. Comparative Example 1

[0058] The low-density biological tissue low-temperature embedding method provided in this embodiment is based on the above method. Specifically, in step S3, the embedding agent is frozen and extends beyond the edge of the tissue by 0.5 mm from the edge of the tissue itself. Comparative Example 2

[0059] The low-density biological tissue low-temperature embedding method provided in this embodiment is based on the above method. Specifically, in step S3, the embedding agent is frozen and extends beyond the edge of the tissue by a distance of 7 mm from the edge of the tissue itself.

[0060] Most existing cryopreservation / embedding techniques for biological tissues involve directly immersing the tissue in a cryopreservation solution (i.e., an embedding agent, such as OCT cryopreservation agent) for rapid freezing. However, when conventionally cryopreserving / embedding biological tissues with a density less than or similar to that of the cryopreservation solution, the tissues are subject to the combined effects of buoyancy and rapid freezing, causing them to float rapidly (e.g., ...). Figure 2-② to 2-④). If no operation is performed at this time, this part of the biological tissue sample will be exposed on the surface of the embedding agent, affecting the embedding effect and subsequent cutting effect. If additional cryopreservation solution is added to the surface of the original cryopreservation solution after quick-freezing, on the one hand, the exposed tissue will undergo a quick-freeze-thaw-quick-freeze process, destroying the tissue structure and morphology; on the other hand, even if the additional embedding agent is partially quick-frozen, the original quick-frozen embedding agent and the additional embedding agent will still not be completely integrated, and a clear boundary line will be formed at the boundary. This will make the sample block that has been quick-frozen a second time very easy to detach and fall off during the subsequent cutting process, causing sample damage.

[0061] A comparison of the embedding methods in Examples 1-3 and Comparative Examples 1 and 2 revealed that the embedding method in Examples 1-3, such as... Figure 3 As shown, it can not only solve the problem of low-density biological tissue floating up and affecting the embedding effect, but also solve the problem that the embedding agent cannot fully fuse during the two quick-freezing processes, forming a clear boundary line at the boundary.

[0062] In Comparative Example 1, the embedding method failed to secure the biological tissue edge because the embedding agent froze and penetrated too shallowly beyond the tissue margin. Consequently, the biological tissue easily floated back up after the second addition of embedding agent, negating the purpose of secondary embedding. In Comparative Example 2, the embedding method... Figure 4 As shown, because the embedding agent froze and went too deep beyond the edge of the tissue, it affected the fusion of the two embedding agents at the boundary line, resulting in a clear boundary line around the embedded biological tissue and a small amount of separation and detachment after sectioning.

[0063] This invention also proposes a label-free biological tissue imaging method, including the aforementioned cryogenic embedding method to obtain low-density biological tissue after cryo-embedding, and further comprising: imaging the surface layer of the cryo-embedded low-density biological tissue, then cutting the surface layer to expose a new surface layer for imaging again, until imaging of the target number of layers is completed. After obtaining images of all layers, rapid imaging of label-free biological tissue can be achieved through data processing.

[0064] Application example: Sections of embedded low-density biological tissue prepared in Example 1 and rapid optical imaging experiments.

[0065] Low-density biological tissue obtained by embedding using the method of Example 1 was selected, and such tissue was selected as... Figure 5 The system shown performs rapid optical imaging of label-free biological tissues. The system includes a mounting plate 1, a cryogenic chamber 2, an imaging component, an insulation component, a cryogenic component, a control component, a tissue support component, and a tissue cutting component.

[0066] like Figure 5As shown, the imaging assembly includes a dichroic light source 3, an objective lens 4, a dichroic mirror 5, and a detector 6; the dichroic light source 3, objective lens 4, dichroic mirror 5, and detector 6 are all fixed on the mounting plate 2; the dichroic light source 3 faces the incident end of the dichroic mirror 5, the objective lens 4 faces the reflecting end of the dichroic mirror 5, and the detector 6 faces the transmitting end of the dichroic mirror 5.

[0067] The imaging component is used for rapid optical scanning imaging of the upper surface layer of the sample block. To improve the weak signal acquisition capability and obtain higher resolution imaging, this system employs a high-performance objective lens. During operation, the optical path and working principle of the imaging component are as follows: the laser emitted from the dual-color light source is expanded and compressed in one dimension to form a line spot, which is reflected after entering the dichroic mirror and reaches the objective lens, where it is focused to form an illumination spot. This illumination spot is projected onto the cut surface of the sample block for line scanning imaging. The light excites various endogenous fluorescent proteins in the surface tissue of the sample block, and the resulting signal light is efficiently collected by the objective lens and re-entered into the dichroic mirror for transmission; finally, it is acquired by the detector.

[0068] Optionally, the detector is a device for collecting light emitted by the excitation of endogenous fluorescent proteins in the sample block, and scientific cameras such as sCMOS are generally selected.

[0069] like Figure 6 As shown, the heat preservation assembly includes a first pump 7, a heat preservation shell 8, a heat exchange pipe 9, and a protective window 10. The heat preservation shell 8 has an opening 11, and the protective window 10 is provided on the opening 11. The lens 401 of the objective lens 4 is located inside the heat preservation shell 8, and the lens 401 faces the opening 11. A heating layer 12 is provided on the inner side of the heat preservation shell 8. A heating channel 13 is provided in the heating layer 12. The inlet and outlet ends of the heating channel 13 are respectively connected to the first pump 7 through the heat exchange pipe 9. The heating channel 13 is filled with heat exchange liquid.

[0070] Objective lenses are generally assembled from materials and structures such as metal, glass, and adhesives. If directly exposed to low-temperature environments, the inconsistent coefficients of thermal expansion of different materials can lead to dimensional mismatches in the internal assembly due to thermal expansion and contraction. This can cause changes in the focal plane of the objective lens, and in severe cases, loosening of the lens element, ultimately damaging the objective lens. The high-performance objective lens used in this specific embodiment has parameters of 20x magnification, 0.7NA, and a 1.6mm working distance. Theoretically, higher-performance objectives result in better image quality. However, higher-performance objectives are more susceptible to the effects of low-temperature environments due to the rapidly shortening working distance. Therefore, current technology can only apply objectives with a 7.6mm working distance to low-temperature environments. The imaging system of this application can apply objectives with a 1.6mm working distance in a -50°C environment, providing a larger stereo angle and significantly improving the collection efficiency of weak signals. However, high-performance objectives are significantly affected by temperature; therefore, preventing the effects of low-temperature environments on the objectives is crucial. To address this issue, this system incorporates a thermal insulation component on the exterior of the objective lens.

[0071] The function of the heating channel and heat exchange tube is to absorb heat from the ambient temperature environment using the heat exchange fluid, and actively and rapidly transfer it to the vicinity of the lens through the heating layer, thus accelerating the maintenance of a stable, temperature-fluctuation-free environment near the lens. Therefore, the heating channel can be either enclosed within the heating layer or relatively open; neither design affects the function of the heating layer. Similarly, the insulation component can be sealed inside the objective lens or only the lens portion, or it can be semi-open; these configurations do not significantly affect the insulation effect on the environment near the lens.

[0072] Optionally, in the insulation components, the heat exchange fluid serves to exchange heat; the commonly used heat exchange fluid is pure water; other liquids that can achieve the same or similar function can also be used. The insulation shell is generally composed of materials with insulation properties, such as polyurethane insulation foam; to maintain structural strength, a rigid outer shell structure (metal or plastic) is also provided outside the insulation shell. Protective windows are generally composed of glass with good light transmittance and low-temperature resistance, such as ultra-clear glass, high borosilicate glass, and fused silica glass.

[0073] When this system is working, the working process of the insulation component is as follows: the heat exchange liquid in the inner wall of the heating chamber is circulated to the outside room temperature environment through the heat exchange pipe by the first pump. After the heat exchange liquid is heated by the outside room temperature environment, it flows back to the heating channel of the heating layer. The insulation shell composed of insulation material outside the heating layer can isolate it from the low temperature environment of the second box. Together, they ensure that the temperature of the inner wall is maintained at a certain value and avoid affecting the low temperature uniformity and stability of the low temperature environment.

[0074] It should be noted that the actual insulation effect of the insulation component is determined by the flow rate of the first pump, the inner diameter of the heating channel and heat exchange tube, and the specific heat capacity of the heat exchange fluid. When designing the insulation component, the dimensions and tolerances of the protective window installation position must be strictly guaranteed by the manufacturing materials and processing technology to ensure compatibility with high-performance objectives with a working distance of only 1.6mm. The operation of the first pump is controlled by the control component.

[0075] like Figure 7 As shown, steady-state thermodynamic simulation calculations were performed on the temperature environment around the objective lens of this application. With a room temperature of 22℃, and the temperature inside the cryogenic chamber at -37.5℃, the insulation component of this application can effectively maintain the objective lens temperature at 21.3℃. Furthermore, since the insulation component of this application uses a heat exchange fluid and heat exchange tube circulation method, utilizing the external room temperature environment to heat the heat exchange fluid, the temperature around the objective lens can be stably controlled. Compared to directly heating and insulating the objective lens, this avoids large fluctuations in temperature control, which is beneficial for maintaining a stable working environment for the objective lens.

[0076] Three sets of control experiments were conducted under the conditions of room temperature 22℃ and low-temperature chamber -37.5℃ (i.e., the system set temperature). Figure 8 As shown. The "Complete Protection Group" (indicated by dashed lines) uses the insulation component of this application to circulate heat exchange and maintain the objective lens temperature, effectively and stably controlling the objective lens periphery temperature at approximately room temperature (22°C). The "No Circulation Protection Group" (indicated by dashed lines) does not utilize room temperature circulation control; under this non-circulation insulation condition, insufficient power occurs, resulting in a lower objective lens periphery temperature than room temperature. The "Heating Wire Protection Group" (indicated by solid lines) uses a heating wire or other heating structure to directly heat and maintain the objective lens periphery. This method requires the use of a temperature sensor for temperature control, making sensitive real-time adjustment difficult. Furthermore, direct heating leads to faster temperature rise, resulting in larger temperature fluctuations that affect the objective lens's stability. The insulation component of this application requires no complex adjustments; it achieves stable objective lens temperature control near room temperature simply by exchanging heat with the heat exchange fluid at room temperature.

[0077] In order to maintain a low temperature environment of -40~-20℃ inside the cryogenic chamber and improve the luminescence efficiency of endogenous fluorescent proteins in tissues, a cryogenic component was constructed.

[0078] like Figure 5 As shown, the low-temperature chamber 2 is a sealed and insulated structure. The low-temperature component is located inside the low-temperature chamber 2 and is used to cool the internal environment of the low-temperature chamber 2. A temperature sensor 14 is provided inside the low-temperature chamber 2, and the control component is electrically connected to the temperature sensor 14.

[0079] like Figure 5 As shown, the tissue cutting assembly is fixed on the low-temperature chamber 2 and located on one side of the objective lens 4; the tissue support assembly is provided with a sample stage 15; the sample stage 15 is controlled to move along the y and z axes by the control assembly; the tissue support assembly slides linearly between the objective lens 4 and the tissue cutting assembly.

[0080] To enable the alternating process of cutting and imaging the sample block, the tissue support assembly is used to hold the sample block to be imaged and sliced. To facilitate rapid imaging and slicing of the sample block, the tissue support assembly must also have independent x, y, and z-axis movement capabilities. The tissue cutting assembly is responsible for slicing the sample block. Here, the x-axis represents linear sliding between the objective lens and the tissue cutting assembly, the y-axis represents the direction of approaching and moving away from the inner wall of the cryogenic chamber, and the z-axis represents vertical lifting and lowering.

[0081] The control components play a comprehensive role in ensuring the smooth and normal operation of the entire system. They not only regulate the normal operation of the insulation and cryogenic components based on real-time monitoring data from temperature sensors, achieving closed-loop PID control, but also control the back-and-forth movement and fine-tuning of the tissue support components between the objective lens and the tissue cutting components.

[0082] It should be noted that the tissue cutting component of this system is used to cut embedded cryopreserved sample blocks. Considering the requirements for high structural rigidity and wear resistance, the cutting components are typically made of materials with high Young's modulus and high Mohs hardness. For example, the clamping device uses TC4 titanium alloy, and the custom-made cutting tools are made of tungsten carbide. TC4 titanium alloy has a Young's modulus of 110 GPa, higher than most metal materials used to fabricate flexible mechanisms. Tungsten carbide has a Young's modulus of approximately 550 GPa and a Mohs hardness of approximately 8.5-9, far exceeding other commonly used metals or non-metals for cutting tools, except for diamond.

[0083] Optionally, the cutting assembly is designed as a gantry structure and directly mounted to the inner wall of the cryogenic chamber. This internal fixing method ensures significantly higher rigidity compared to external bracket support. The cutting tool can be customized to the required dimensions, taking into account material properties, to achieve the necessary structural rigidity and wear resistance.

[0084] In some embodiments, a sliding groove is provided on the air-floating vibration isolation platform under the low-temperature chamber 2. The sliding groove is arranged along the straight direction of the tissue cutting assembly and the objective lens 4, and the tissue bearing assembly slides along the sliding groove.

[0085] In some embodiments, such as Figure 9As shown, the cryogenic assembly includes a refrigeration module 16, a radiator 17, a second pump 18, and a heat dissipation pipe 19; the refrigeration end of the refrigeration module 16 is located on the inner wall of the cryogenic chamber 2, the radiator 17 is connected to the heat dissipation end of the refrigeration module 16, the radiator 17 is connected to the second pump 18 through the heat dissipation pipe 19, and the heat dissipation pipe 19 is filled with heat exchange fluid.

[0086] To improve the luminescence efficiency of endogenous fluorescent proteins in tissue blocks, this system requires maintaining the tissue blocks at a low temperature. This is because as the temperature decreases, the molecular velocity slows down, the probability of intermolecular collisions decreases, and the occurrence of non-radiative transitions is reduced, ultimately improving fluorescence efficiency. Therefore, this system features a specially designed cryogenic component that creates a low-temperature environment throughout the cryogenic chamber. The control component monitors the real-time temperature inside the cryogenic chamber using temperature sensors to adjust the cooling effect of the cryogenic component, achieving a control accuracy of ±0.05℃, ensuring that the cryogenic chamber remains at a constant low temperature, down to -50℃.

[0087] Figure 10 Image a shows the results of imaging an unlabeled brain tissue slice using an objective lens with a magnification of 20x, a numerical aperture of 0.45, and a working distance of 7.6mm at a low temperature of -20℃. Figure 6 Image b shows the imaging results of unlabeled brain tissue sections using the objective lens of this application at a low temperature of -20°C. Without labeling, imaging relies solely on the tissue's own endogenous fluorescence signal. Compared to the fluorescence signal emitted by fluorescently labeled brain tissue sections, the endogenous fluorescence signal is weaker and more difficult to collect. Figure 10 The comparison shows that, when faced with the same unlabeled biological samples, the imaging results of this application can achieve better results, with higher image brightness, contrast and resolution.

[0088] Optionally, in the low-temperature components, the refrigeration module can be a thermoelectric cooler. Thermoelectric coolers achieve refrigeration using the Peltier effect, utilizing the PN junction of semiconductor materials to form a thermocouple pair. The entire unit consists of a hot end and a cold end, and the refrigeration process does not generate any vibration, thus ensuring the stability of the refrigeration environment.

[0089] Specifically, when the thermoelectric cooler is working, its cold end face is tightly bonded to the inner wall of the cryogenic chamber via thermally conductive silicone grease, and its hot end face is tightly bonded to the radiator (water-cooled type). A second pump uses circulating water to remove heat, ensuring the hot end temperature of the cooler remains within the normal range. A temperature sensor measures the temperature inside the cryogenic chamber and feeds it back to the control module. If the measured temperature differs from the set temperature, the control module automatically adjusts the power output to the thermoelectric cooler based on the pre-set PID parameters and program. The control module also includes a computer with built-in host computer software, allowing the control module to feed parameters back to the computer. If the set PID parameters are unsuitable, they can be modified and adjusted via software. Experimental testing shows that the closed-loop temperature control method of this invention can achieve a control accuracy of ±0.05℃ in cryogenic environments, with a minimum temperature of -50℃.

[0090] In this system, the mounting plate includes a mounting surface and a non-mounting surface that are arranged opposite to each other. The imaging component is fixed on the mounting surface, and the non-mounting surface has several cavities. A support rib (i.e., a support member) is formed between two adjacent cavities. The positions of the cavities correspond one-to-one with the positions of the optical elements in the imaging component.

[0091] For a component, the first natural frequency is positively correlated with its stiffness and negatively correlated with its mass. The following formula principle should be followed when designing the cavity of the mounting plate: ,in, This represents the first-order natural frequency of the mounting plate. Indicates the rigidity of the mounting plate. This refers to the mass of the mounting plate. Therefore, minimizing the weight of the mounting plate while ensuring its structural stiffness will increase its first natural frequency. A higher first natural frequency results in better seismic resistance.

[0092] This application reduces the weight of the mounting plate by creating multiple cavities on the non-mounting surface of the mounting plate, and makes the positions of the cavities correspond one-to-one with the positions of the optical elements fixed in the mounting surface. Then, the four supporting ribs on the periphery of a cavity can be used to improve the structural rigidity of the area where the optical element is fixed, ultimately achieving the minimum weight of the mounting plate while ensuring rigidity.

[0093] like Figure 11 As shown, Figure 11 a is a mounting plate without any cavities, and its first natural frequency is 210.03Hz; Figure 11 b is in Figure 11 The mounting plate of a has several cavity designs inside, and its first-order modal frequency is 335.53Hz. This shows that the groove design of this application can effectively improve the first-order natural frequency of the mounting plate. Figure 11 c is in Figure 11Based on a, a simulated detector 6 mass block was added, with a first-order mode frequency of 138.33Hz. Figure 11 d is in Figure 11 Based on b, a mass block of analog detector is added, with a first-order modal frequency of 200.51 Hz. In the imaging system of this application, the overall mass of the mounting plate and detector accounts for as much as 98%, while other optical components account for nearly 2%. Therefore, by... Figure 11 c and Figure 11 Adding a detector mass block to d can effectively simulate the effect of the entire imaging assembly on the first-order natural frequency of the mounting plate. Figure 11 a and Figure 11 The comparison between c shows that adding the imaging component directly to the solid mounting plate will reduce its first-order natural frequency from 210.03Hz to 138.33Hz, while the cavity design can increase the first-order natural frequency to 200.51Hz.

[0094] This system sets the interior of the first enclosure to contain several cavities. Through seismic model simulation, it has been confirmed that it can avoid the frequencies of interference generated by the external environment and the operation of some components and equipment in the system, and has a significant seismic resistance and vibration reduction effect, eliminating the equipment resonance problem.

[0095] In some embodiments, a temperature sensor may also be provided on the heating layer 12 of the insulation component to monitor the insulation effect of the insulation component and the temperature near the objective lens.

[0096] In some embodiments, such as Figure 12 As shown, the tissue cutting assembly includes a first base 20, a clamping device 21, and a cutting tool 22. The first base 20 is fixedly connected to the low-temperature chamber 2 or designed as an integral part thereof. The clamping device 21 is fixed on the first base 20 and clamps the cutting tool 22.

[0097] In some embodiments, such as Figure 13 As shown, the tissue support assembly also includes a second base 23, the sample stage 15 is located on the second base 23, and the sample stage 15 includes a y / z axis sliding device 24; the second base 23 is provided with a heat insulation structure.

[0098] Alternatively, the thermal insulation structure serves to prevent heat from being conducted to the sample stage, ensuring a low temperature on the stage, and also meeting the cutting force requirements of the tissue cutting components. Therefore, the material used to fabricate the tissue support components must have a high Young's modulus and low thermal conductivity. Considering the distance between the two parts of the component and the sample, the second base, since it is in direct contact with the low-temperature sample, needs to prioritize thermal insulation while maintaining high rigidity. A non-metallic material with a high Young's modulus and low thermal conductivity, such as POM, can be used. The bottom of the second base is in indirect contact with the low-temperature sample, requiring high rigidity while maintaining thermal insulation. Therefore, a metallic material with a high Young's modulus and low thermal conductivity, such as TC4 titanium alloy, can be used.

[0099] Alternatively, in addition to using insulating materials, the insulation structure can be designed as a hollow truss structure, which can reduce the heat conduction area while ensuring structural rigidity. The dimensional proportions of the sample stage and the insulation structure can be optimized and determined based on the results of repeated experiments / simulation calculations.

[0100] In some embodiments, both the mounting plate 1 and the cryogenic chamber 2 are fixed on the air-floating vibration isolation platform.

[0101] The specific steps for using the above-mentioned rapid optical imaging system for label-free biological tissues are as follows: Based on the suitable operating temperature of the objective lens and the temperature required for the experiment, the control component adjusts the insulation component; the control component adjusts the low-temperature component to pre-cool the tissue support component and the tissue cutting component; and the imaging component is adjusted.

[0102] The embedded cryopreserved biological tissue is fixed on the tissue support assembly. Initially, the biological tissue is positioned directly below the objective lens. After the imaging assembly takes an image, the control assembly controls the tissue support assembly to move towards the tissue cutting assembly, so that the tissue cutting assembly completes the first sectioning of the biological tissue.

[0103] The control component controls the tissue support component to move towards the objective lens, and the imaging component completes the second image capture; the control component controls the tissue support component to move towards the tissue cutting component, so that the tissue cutting component completes the second sectioning of the biological tissue; the image capture and sectioning process is repeated in this way.

[0104] As the sample block becomes thinner with each slice, the height of the sample stage needs to be fine-tuned using control components to ensure stable slicing results.

[0105] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for low-density biological tissue cryo-embedding, characterized by, The method comprises the following steps: placing the low-density biological tissue into a mold containing embedding agent, placing a container in a liquid nitrogen environment, and placing the mold into the container for primary freeze-embedding treatment; the time taken for placing the low-density biological tissue into the mold and starting the primary freeze-embedding treatment is no more than 60 s in total; when the surrounding embedding agent freezes to a distance of 1.5 mm-6 mm beyond the edge of the low-density biological tissue, adding pre-cooled embedding agent until the surface of the low-density biological tissue is completely covered, and performing secondary freeze-embedding treatment.

2. The method according to claim 1, wherein The distance between the side wall of the mold and the edge of the low-density biological tissue is 3 mm-10 mm.

3. The method according to claim 1, wherein the low-density biological tissue cryoembedding method is characterized by, The amount of embedding agent used in the primary freeze-embedding treatment is 50%-75% of the volume of the mold.

4. The method according to claim 1, wherein Before placing the mold into the container for primary freeze-embedding treatment, the container is floated in a liquid nitrogen environment for at least 1 min.

5. The method according to claim 1, wherein The method for obtaining the pre-cooled embedding agent is as follows: placing the embedding agent above the liquid level of the liquid nitrogen, and pre-cooling the embedding agent using liquid nitrogen vapor for 2 min-4 min.

6. The method according to claim 5, wherein the low-density biological tissue cryoembedding method is characterized by, The embedding agent is located 1 cm-2 cm above the liquid level of the liquid nitrogen.

7. The method according to claim 5 or 6, wherein the low-density biological tissue cryoembedding method is characterized by, The temperature of the liquid nitrogen vapor is -100℃ to -70℃.

8. A label-free bio-tissue imaging method, characterized by, The low-density biological tissue is obtained by using any one of the low-density biological tissue cryo-embedding methods described in items 1-6, and then the surface layer of the freeze-embedded low-density biological tissue is imaged; the surface layer is cut to expose a new surface layer, and the new surface layer is imaged again; the operation is repeated until the imaging of the target number of layers is completed.

Images