A high-frequency ultrasound method and ultrasound system for bulk tissue processing

Abstract

This invention discloses a high-frequency ultrasound method for processing large-volume tissues. The method includes the step of placing biological tissues under high-frequency ultrasound conditions for processing. In the high-frequency ultrasound processing, the ultrasound frequency is 1 MHz, the sound intensity is 0.6 W / cm², and the temperature is 4℃. This invention also includes a high-frequency ultrasound system for processing large-volume tissues, characterized in that the system comprises a sample container or sample carrying platform module, a signal generation module, a power amplification module, an ultrasound transducer module, a temperature control module, and a circulating fluid circuit module. The high-frequency ultrasound method of this invention can be applied to the overall staining, tissue clearing, and resin embedding of large-volume tissues to improve the efficiency of the processing solution penetrating deeper into the tissue, improve the uniformity of internal and external tissue processing, and enhance the applicability of this method to fluorescent samples, thus possessing greater scalability and application value.

Description

Technical Field

[0001] This invention belongs to the field of resin embedding technology, specifically, it relates to a high-frequency ultrasound method and ultrasound system for processing large-volume tissues. Background Technology

[0002] Ultrasonic tissue processing is a promising method for accelerating large-volume tissue processing. Compared to simple static diffusion, ultrasound can promote the transfer of processing solutions within the tissue through mechanical disturbance and local microflow, thus showing potential application value in tissue staining, tissue clearing, and embedding. However, existing ultrasonic tissue processing techniques still have many problems, limiting their application in tissue clearing, staining, and resin embedding. Specifically, existing technologies mainly suffer from the following issues:

[0003] (1) There are problems with frequency selection. Most common existing ultrasound equipment uses low-frequency ultrasound, mainly for ultrasonic cleaning, cell disruption, mixing or material processing. These low-frequency equipment have strong mechanical vibration and severe cavitation effect. Although it is easy to obtain obvious disturbance, when used for large-volume tissue processing, it is easy to cause damage to the tissue surface, structural destruction or even local cavitation, which is difficult to meet the requirements of maintaining the structure for intact tissue processing.

[0004] (2) There are problems with controlling the intensity of the treatment. When the ultrasonic intensity is too low, it is difficult to significantly improve the penetration of the treatment solution into deep tissues; when the ultrasonic intensity is too high, it will increase the risk of tissue damage. Therefore, traditional methods often cannot simultaneously meet the two requirements of "promoting permeability" and "maintaining tissue structure".

[0005] (3) There are problems with temperature control. Ultrasound generates a thermal effect during continuous operation. If there is a lack of effective temperature control, the treatment solution is prone to overheating. For ordinary tissues, this may cause local processing loss of control; for fluorescent samples, it is more likely to cause fluorescence signal attenuation or even inactivation.

[0006] (4) Insufficient in terms of tissue protection and fluorescence compatibility. Large-volume tissue processing not only requires the processing solution to penetrate deep into the tissue, but also requires maintaining the integrity of the tissue structure as much as possible during the processing and avoiding adverse effects on endogenous or exogenous fluorescence. However, most existing ultrasound protocols only focus on processing speed and rarely consider maintaining tissue integrity and fluorescence signal preservation at the same time, thus limiting their applicability in fluorescent sample processing.

[0007] Therefore, there is a need in the field to modify ultrasound technology based on existing technologies so that it can be applied to large-volume tissue processing steps such as tissue clearing, staining, and resin embedding, and can serve as a stable, gentle, and universal solution that also meets the needs of fluorescent sample processing. Summary of the Invention

[0008] In view of the shortcomings of existing technologies and practical needs, the present invention provides a high-frequency ultrasound method and ultrasound system for processing large-volume tissues.

[0009] Specifically, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a high-frequency ultrasound method for processing large-volume tissues, the method comprising the step of placing biological tissues under high-frequency ultrasound conditions for processing; wherein the high-frequency ultrasound processing has an ultrasound frequency of 1 MHz, a sound intensity of 0.6 W / cm², and a temperature of 4 °C.

[0011] In one or more embodiments, the large volume tissue is mouse brain tissue.

[0012] Secondly, the present invention provides the application of the high-frequency ultrasound method in the whole-body staining of biological tissues, wherein the application is: while placing the whole biological tissue in the staining solution, the high-frequency ultrasound treatment is applied to improve the permeability efficiency of the dye inside the biological tissue and the uniformity of staining.

[0013] In one or more embodiments, the staining solution is eosin or hematoxylin.

[0014] Thirdly, the present invention provides the application of the high-frequency ultrasound method in the transparentization of biological tissues, wherein the application is: applying the high-frequency ultrasound treatment during the transparentization process of biological tissues to improve the transparentization efficiency of biological tissues and improve the consistency of internal and external treatment of biological tissues.

[0015] In one or more embodiments, the degreasing system in the biological tissue transparency is DCM:TB=2:1.

[0016] Fourthly, the present invention provides the application of the high-frequency ultrasound method in resin embedding or blackening embedding of biological tissues, wherein the application is: applying the high-frequency ultrasound treatment during the resin embedding or blackening embedding of biological tissues to promote the penetration and distribution of resin and blackening agent inside the biological tissues.

[0017] In one or more embodiments, the resin used in the resin embedding is BMA; the blackening agent in the blackening embedding is 0.6% SBB.

[0018] Fifthly, the present invention provides a high-frequency ultrasound system for processing large volumes of tissue, the system comprising a sample container or sample carrying platform module, a signal generation module, a power amplification module, an ultrasound transducer module, a temperature control module, and a circulating fluid circuit module;

[0019] The sample container or sample carrying platform module is used to place large-volume biological tissue samples;

[0020] The signal generation module and power amplification module are used to output high-frequency ultrasonic signals at a preset frequency;

[0021] The ultrasonic transducer module is used to convert electrical signals into ultrasonic vibrations and apply them to the treatment fluid.

[0022] The temperature control module and the circulating fluid circuit module are used to control the temperature of the processing environment, especially to suppress the accumulation of thermal effects in the processing of fluorescent samples.

[0023] In one or more embodiments, the ultrasonic frequency is set to 1 MHz in the power amplification module, the ultrasonic intensity is set to 0.6 W / cm² in the ultrasonic transducer module, and the temperature is set to 4°C in the temperature control module.

[0024] Compared with existing static diffusion methods, low-frequency ultrasonic equipment, and other physical-aided processing methods, the present invention has the following advantages:

[0025] (1) This invention takes "high-frequency ultrasound to promote tissue permeability" as the main technology, which can more effectively solve the problem that the treatment solution is difficult to penetrate deep into the tissue during the treatment of large volume tissue. Therefore, compared with the traditional static diffusion method, the treatment efficiency is higher and the uniformity is better.

[0026] (2) The present invention uses high-frequency ultrasound instead of low-frequency ultrasound, which can reduce the risk of tissue damage while promoting the penetration of the treatment solution. Therefore, it is more suitable for processing intact tissue samples than the traditional low-frequency ultrasound method.

[0027] (3) The present invention combines temperature control and high-frequency ultrasound, making it not only suitable for ordinary tissue processing, but also for fluorescently labeled sample processing, thus having better fluorescence compatibility than existing ultrasound methods.

[0028] (4) This invention is not limited to a single step, but can be applied to multiple pretreatment steps such as tissue clearing, overall staining and resin embedding, thus having greater scalability and application value.

[0029] (5) This invention combines high-frequency ultrasound, temperature control, liquid circulation and sample carrying module, which promotes the permeation of the treatment liquid into the tissue, reduces the risk of tissue damage and improves the applicability to fluorescent samples. Attached Figure Description

[0030] Figure 1These are comparison images of tissue and cell integrity imaging effects at different ultrasound frequencies. (AC) No ultrasound imaging effect; (DF) 40 kHz ultrasound imaging effect; (GI) 1 MHz ultrasound imaging effect; Scale bar: (A, D, G) 1500 μm; (B, E, H) 200 μm; (C, F, I) 20 μm.

[0031] Figure 2 These are comparison images of eosin staining results under ultrasound with different power; where (A) 0 W / cm2; (B) 0.2 W / cm2; (C) 0.4 W / cm2; (D) 0.6 W / cm2; scale bar: 1000μm.

[0032] Figure 3 This is a diagram showing the effect of low-temperature ultrasonic transparency in Example 2.

[0033] Figure 4 These are images showing the fluorescence retention effect after ultrasound-mediated transparency in Example 2; (A) fluorescence comparison before and after treatment; scale bar: 20 μm; (B) quantitative comparison of fluorescence retention rate, n = 20 brain slices, error bars represent standard error, and significant differences are expressed as p-values. .

[0034] Figure 5 These are the blackening embedding experiment results of the ultrasound group and the control group in Example 3; where (A) is the coronal section imaging image of the control group; (B, C) are progressively magnified views of the selected area in A; (D) is the coronal section imaging image of the ultrasound group; (E, F) are progressively magnified views of the selected area in D; and (G) is the quantified background fluorescence signal intensity map. n = 20 fMOST imaging sections, error bars represent standard error, and significant differences are expressed as p-values. Scale: (A, D) 500 μm; (B, E) 50 μm; (C, F) 20 μm.

[0035] Figure 6 The results are fMOST imaging of the whole brain of Thy1-GFP-M mice after ultrasound blackening and embedding in Example 3; (A) Three-dimensional visualization of GFP fluorescence in the whole brain of mice; (B) Coronal image of GFP channel; (C) Coronal image of PI channel; (DE) Gradual magnification of the area selected in B; (FG) Gradual magnification of the area selected in C; Scale bar: (B, C) 1000μm; (D, F) 200μm; (E, G) 40μm. Detailed Implementation

[0036] This invention proposes a high-frequency ultrasound tissue processing method and system to promote the permeability of large-volume tissues. The core idea is to abandon the traditional low-frequency, highly destructive, and temperature-difficult-to-control ultrasound processing method and instead adopt high-frequency ultrasound parameters suitable for large-volume tissue processing. It combines high-frequency ultrasound, temperature control, liquid circulation, and sample support structure to promote the permeability of the processing solution into the tissue while reducing the risk of tissue damage and improving the applicability of the method to fluorescent samples.

[0037] Example

[0038] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0039] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0040] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value in between may be selected. Unless otherwise stated, all percentages mentioned in this invention are mass percentages. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all those that can be purchased through legitimate channels.

[0042] Example 1

[0043] A high-frequency ultrasound method for processing large volumes of tissue includes the following steps:

[0044] (1) Sample loading and contact with processing solution

[0045] Large-volume tissue samples to be processed are placed in a sample container or sample support structure, immersing the sample in the processing solution. Acoustic coupling is then established between the sample and the high-frequency ultrasound transducer through the liquid medium. This step creates an optimal environment for subsequent high-frequency ultrasound to promote tissue permeability.

[0046] (2) Applying high-frequency ultrasound to promote tissue permeability

[0047] The ultrasonic transducer is driven by a signal generation module and a power amplification module to apply high-frequency ultrasound to the sample in the treatment solution. The high-frequency ultrasound generates mechanical disturbance and local mass transfer enhancement in the treatment solution, thereby promoting the diffusion and penetration of treatment solution molecules into the deeper tissues.

[0048] This invention uses 1 MHz as the high-frequency ultrasound frequency. The reason for choosing this frequency is that if the frequency is too low, the cavitation and mechanical effects will be too strong, easily causing tissue damage; if the frequency is too high, the ultrasound attenuation in the medium will increase significantly, which is not conducive to deep treatment of large volumes of tissue; 1 MHz achieves a good balance between promoting tissue permeability and reducing tissue damage. Experimental results are as follows... Figure 1 As shown, significant damage to brain tissue was observed at 40 kHz, while no significant damage to tissues and cells was observed at 1 MHz. Therefore, the present invention preferably uses 1 MHz high-frequency ultrasound.

[0049] (3) Controlling the intensity of ultrasound

[0050] This invention uses an acoustic intensity parameter of 0.6 W / cm². This intensity was chosen because: when the acoustic intensity is too low, the ultrasound's effect on tissue permeability is insufficient, making it difficult for the treatment solution to effectively penetrate deep regions; when the acoustic intensity is too high, it easily increases the risk of tissue damage. In experimental verification, 0.6 W / cm² has shown good deep penetration, and no significant tissue damage was observed at 1 MHz.

[0051] like Figure 2 The complete mouse brain eosin staining experiment showed that among the four acoustic intensity gradients of 0, 0.2, 0.4 and 0.6 W / cm², the 0.6 W / cm² group showed the best staining effect, and the dye could reach deep into the tissue.

[0052] (4) Temperature control

[0053] During high-frequency ultrasound treatment, the ambient temperature is maintained via a temperature control module and a circulating fluid circuit module. For routine tissue processing steps, the temperature can be set according to the type of processing solution and process requirements; for fluorescent sample processing steps, this invention is performed at a low temperature of 4 °C.

[0054] The reasons for choosing low-temperature conditions are: increased temperature will accelerate the inactivation of fluorescent proteins or fluorescence decay; low temperature is beneficial to maintaining the stability of fluorescence signals; high-frequency ultrasound can compensate for the problem of slowed reagent diffusion under low-temperature conditions, thereby achieving the unity of "maintaining fluorescence at low temperature" and "improving processing efficiency".

[0055] Experiments show that the fluorescence retention effect at 4 ℃ is significantly better than that at high temperature, indicating that the present invention prefers a low-temperature high-frequency ultrasound scheme in the processing of fluorescent samples.

[0056] Application Example 1: Applied to whole-body staining

[0057] The effect of high-frequency ultrasound on the penetration of staining in intact mouse brain was verified using 0.5% eosin solution, specifically including the following steps:

[0058] (1) Take intact mouse brain tissue that has been fixed with 4% paraformaldehyde and thoroughly rinsed with PBS as the staining sample.

[0059] (2) Place the complete mouse brain sample in a sample container and immerse the sample completely in 0.5% eosin staining solution. The amount of staining solution used is 40 mL / brain.

[0060] (3) Start the high-frequency ultrasound system and drive the ultrasound transducer through the signal generation module and power amplification module so that the intact mouse brain sample in the treatment liquid is in a high-frequency ultrasound environment.

[0061] (4) The ultrasonic frequency was set to 1 MHz, the sound intensity to 0.6 W / cm², and the staining time to 6 h. During the treatment, the sample was kept completely submerged, and the acoustic coupling between the ultrasonic transducer and the tissue sample was achieved through a liquid medium.

[0062] (5) After staining, the intact mouse brain sample was removed from the staining solution and rinsed with PBS to remove residual dye from the tissue surface.

[0063] (6) Perform coronal sectioning and imaging observation on the processed complete mouse brain samples to compare the distribution of dye in the surface and deep regions of the tissue.

[0064] (7) The results showed that, compared with the conventional treatment without high-frequency ultrasound, the distribution range of eosin dye in the intact mouse brain was wider after high-frequency ultrasound treatment, the degree of internal staining was improved, and the difference in staining inside and outside the tissue was reduced. This indicates that high-frequency ultrasound treatment at 1 MHz and 0.6 W / cm² can promote the penetration of dye into the deep part of large-volume tissues and improve the overall staining uniformity.

[0065] Application Example 2: Applied to tissue clearing, an organic dehydration / degreasing system and a refractive index matching system were used. A DCM:TB ratio of 2:1 was employed for degreasing, followed by a refractive index matching step to improve sample transparency while maintaining fluorescence. The specific steps included:

[0066] (1) Take intact mouse brain tissue that has been fixed with 4% paraformaldehyde and thoroughly rinsed with PBS as the sample to be transparentized.

[0067] (2) Place the complete mouse brain sample in the sample container and immerse it completely in the treatment solution.

[0068] (3) Start the high-frequency ultrasound system, set the ultrasound frequency to 1 MHz and the sound intensity to 0.6 W / cm².

[0069] (4) Start the temperature control module and the circulating liquid circuit module to maintain the processing environment temperature at 4℃.

[0070] (5) Place the sample in a 50% ethanol aqueous solution for 1 h to perform preliminary dehydration.

[0071] (6) Place the sample in a 75% ethanol aqueous solution for 1 h and continue to perform gradient dehydration.

[0072] (7) Place the sample in 100% ethanol for 1 h.

[0073] (8) The sample was placed in 100% ethanol again for 0.5 h to further remove water from the tissue.

[0074] (9) The dehydrated sample was placed in a mixed degreasing system of DCM:TB=2:1 for 36 h for the first degreasing treatment.

[0075] (10) Replace with a new DCM:TB=2:1 mixed degreasing system and treat again for 36 h for a second degreasing treatment.

[0076] (11) After degreasing, the sample was placed in 100% ethanol for 0.5 h.

[0077] (12) The sample was placed in 75% ethanol aqueous solution for 1 h and then subjected to gradient rehydration.

[0078] (13) Place the sample in a 50% ethanol aqueous solution for 1 h and continue rehydration.

[0079] (14) Rinse the sample in PBS for 1 h to remove residual organic solvents.

[0080] (15) Place the sample in EasyIndex refractive index matching solution for 2 days to complete the refractive index matching.

[0081] (16) All the above steps were performed under the conditions of 1 MHz, 0.6 W / cm², and 4℃ with high frequency ultrasound assistance.

[0082] (17) After processing, observe the transparency effect of the sample.

[0083] (18) For fluorescently labeled samples, a relatively clear GFP fluorescence signal can still be observed after the transparentization process, indicating that the method can maintain the intrinsic fluorescence signal well while promoting the penetration of the transparentization reagent.

[0084] Sample transparency effect Figure 3 As shown, after cryo-ultrasound treatment and refractive index matching, the overall transparency of the intact mouse brain sample was significantly improved, and the background grid lines could be clearly observed through the sample, indicating reduced light scattering within the tissue and a smaller difference in refractive index between the inside and outside of the tissue. The overall morphology of the sample remained relatively intact, with no obvious breakage or severe deformation observed, indicating that this cryo-ultrasound transparency process can achieve good transparency results while maintaining the tissue morphology.

[0085] To further evaluate the effect of this cryogenic ultrasound clearing technique on endogenous fluorescence signals, this embodiment uses Thy1-GFP mouse brain samples for verification. After cryogenic ultrasound clearing, the GFP fluorescence signal was observed using a confocal microscope, and the fluorescence intensity before and after treatment was quantitatively analyzed. The results are as follows: Figure 4 As shown, even after clearing, the sample still exhibits relatively clear GFP fluorescence signals, and the outlines of neuronal cell bodies and some fiber structures remain relatively clear, indicating that this process can effectively preserve endogenous GFP fluorescence signals while improving tissue transparency. The quantitative results of fluorescence retention rate are shown below. Figure 4 As shown in Figure B, the GFP fluorescence retention rate remained at a high level after low-temperature ultrasonic clearing treatment. This result indicates that combining a low-temperature condition of 4°C with ultrasound assistance can promote the penetration of the clearing agent into the intact mouse brain while reducing fluorescence signal attenuation, thus helping to achieve a balance between clearing effect and fluorescence retention.

[0086] Application Example 3: Applied to resin embedding or blackening embedding

[0087] An encapsulation system consisting of resin monomers, crosslinking agents, initiators, and blackening agents, using BMA resin and 0.6% SBB blackening agent, is employed to improve blackening uniformity and enhance background suppression. The specific steps include:

[0088] (1) Take intact mouse brain tissue that has been fixed with 4% paraformaldehyde and thoroughly rinsed with PBS as the sample to be embedded.

[0089] (2) Place the complete mouse brain sample in the sample container and immerse it completely in the treatment solution.

[0090] (3) Start the high-frequency ultrasound system, set the ultrasound frequency to 1 MHz and the sound intensity to 0.6 W / cm².

[0091] (4) Start the temperature control module and the circulating liquid circuit module to maintain the processing environment temperature at 4℃.

[0092] (5) Place the sample in 50% ethanol for 1 h to perform preliminary dehydration.

[0093] (6) Place the sample in 75% ethanol for 1 h to continue dehydration.

[0094] (7) Place the sample in 95% ethanol for 1 h.

[0095] (8) Place the sample in 100% ethanol for 1 h.

[0096] (9) The sample was placed in 100% ethanol again for 1 h to further remove moisture from the tissue.

[0097] (10) The dehydrated sample was placed in 50% BMA for 2 h to perform preliminary resin permeation.

[0098] (11) Place the sample in 75% BMA for 2 h and continue the resin gradient permeation.

[0099] (12) The sample was placed in 100% BMA for 12 h to allow the BMA resin monomer to penetrate further into the tissue.

[0100] (13) The sample was placed in a 100% BMA system containing 0.6% SBB for 8 h to undergo blackening and penetration treatment.

[0101] (14) High-frequency ultrasound of 1 MHz and 0.6 W / cm² was applied during the above dehydration, resin penetration and blackening penetration processes to promote the penetration of BMA resin system and SBB blackening agent into the deep part of intact brain tissue.

[0102] (15) After the blackening penetration is completed, the sample is transferred into a 100% BMA polymerization system containing 0.6% SBB.

[0103] (16) The polymerization system is slowly heated from 25°C to 38°C and kept for 6 hours to promote the initial polymerization of the resin system.

[0104] (17) The temperature was then slowly increased from 38°C to 40°C and held for 6 h to promote further cross-linking and curing of the resin system.

[0105] (18) After polymerization, blackened resin-embedded samples are obtained.

[0106] (19) Fix the blackened resin-embedded sample onto the sample stage of the fMOST system.

[0107] (20) Before local or continuous imaging with fMOST, the blackened resin-embedded samples are stained with propidium iodide in real time. Propidium iodide is abbreviated as PI, which can bind to nuclear DNA and generate a red fluorescent signal to label nuclear structures.

[0108] (21) Start the fMOST system to perform continuous cutting and fluorescence imaging of the sample. During the imaging process, the system removes the surface tissue of the sample layer by layer and acquires multi-channel fluorescence images of the newly exposed tissue plane.

[0109] (22) The endogenous fluorescence signal was observed through the GFP channel, the nuclear labeling signal was observed through the PI channel, and the background fluorescence intensity was statistically analyzed to evaluate the background suppression effect, nuclear imaging contrast and continuous imaging adaptability of the blackened embedded samples.

[0110] (23) The results showed that after high-frequency ultrasound-assisted blackening and embedding treatment, the SBB blackening agent and BMA resin system penetrated more fully in the intact mouse brain, and the degree of tissue blackening was higher and the distribution was more uniform. At the same time, BMA polymerization can provide mechanical support for the tissue, SBB can reduce tissue background autofluorescence, and PI real-time staining can mark the cell nuclear structure, thereby improving the sample fit for subsequent fMOST continuous cutting, multi-channel fluorescence imaging and three-dimensional reconstruction.

[0111] The results of the blackening embedding experiment in the ultrasound group and the control group are shown in the figure below. Figure 5 As shown, in the static control group, the background fluorescence was strong in some local images, the contrast between the cell nuclear signal and the background was low, and some areas had obvious non-specific background signals, indicating that the penetration and background inhibition of SBB in the tissue under conventional static conditions were still insufficient. In contrast, the background fluorescence in the ultrasound-assisted group was reduced, the outline of PI-labeled cell nuclei was clearer, and the distinction between the cell nuclear signal and the background was improved, indicating that ultrasound-assisted treatment helps to enhance the penetration and effect of SBB in the tissue, thereby improving the contrast of microscopic imaging. The quantitative results of background fluorescence intensity are shown below. Figure 5 As shown in Figure G, the average background fluorescence intensity of the static control group was approximately 1402 arbitrary units (AU), with significant fluctuations between different sections, indicating uneven background signal suppression. The average background fluorescence intensity of the ultrasound-assisted group was approximately 949 AU, a reduction of about 32% compared to the static control group, with relatively smaller fluctuations between different sections. These results indicate that ultrasound-assisted blackening embedding can reduce the background fluorescence intensity of samples and improve the consistency of background signals between different sections.

[0112] This embodiment also preprocesses the acquired raw image data, including destripping, fluorescence intensity homogenization correction, and inter-slice registration, to reduce the impact of strip artifacts, local intensity fluctuations, and inter-slice positional deviations on subsequent analysis during imaging. Subsequently, the registered two-dimensional image sequence is reconstructed using the three-dimensional visualization software Amira to generate a complete three-dimensional model of the mouse brain. Based on the above ultrasound-assisted melanin embedding scheme, this study achieved continuous fMOST coronal imaging and three-dimensional reconstruction of the whole brain of Thy1-GFP-M mice. Related results are as follows: Figure 6 As shown, the three-dimensional visualization of the mouse whole brain revealed a relatively continuous distribution of fluorescence signals in the coronal, sagittal, and horizontal planes, indicating that the overall structure of the sample remained relatively intact and the spatial continuity of the signal was good. The GFP channel coronal image showed that the overall outline of the brain tissue was clear, and the main brain regions and fiber tracts could be well distinguished, with low background stray signal. Figure 6 -B). Further magnification reveals that the boundaries of the hippocampus and other layered structures are relatively clear, the cell layers are distributed in bands, and the signals of some individual neuron cell bodies can be distinguished, indicating that this sample exhibits good structural preservation and spatial resolution during continuous cutting and imaging. The coronal image of the PI channel shows that the tissue outline is intact and the nuclear staining signal distribution is relatively uniform. Figure 6 -C). The magnified partial image shows relatively dense and distinguishable cell nuclear signals, indicating that the sample maintained good tissue morphology and microstructure visualization during blackening embedding and continuous imaging. These results demonstrate that intact mouse brain samples treated with ultrasound-assisted blackening embedding can support fMOST whole-brain continuous ablation, multi-channel fluorescence imaging, and three-dimensional reconstruction.

[0113] The above embodiments and application examples demonstrate that the present invention has significant advantages over the prior art, specifically in the following aspects:

[0114] Regarding organizational integrity, the comparative experiment in Example 1 shows that ( Figure 1 At 40 kHz, significant cavitation, fragmentation, and cell damage were observed in the brain tissue, while the treatment solution temperature increased from 25 ℃ to 45 ℃ within 10 min. However, at 1 MHz, no significant tissue or cell damage was observed. These results demonstrate that the high-frequency ultrasound used in this invention can significantly reduce the risk of tissue damage commonly seen in traditional low-frequency ultrasound treatment.

[0115] Regarding the effect on promoting tissue permeability, acoustic intensity was screened using intact mouse brain eosin staining as a model. The results showed that ( Figure 2Among the four acoustic intensity gradients of 0, 0.2, 0.4, and 0.6 W / cm², the 0.6 W / cm² group showed the best staining effect, and the dye was able to reach deep into the tissue. This indicates that the preferred parameters of the present invention can significantly improve the permeability and penetration efficiency of the treatment solution into large-volume tissues.

[0116] Regarding fluorescence signal retention, the low-temperature fluorescence retention experiment in the basic data shows that the fluorescence retention effect at 4°C is significantly better than that at higher temperatures; further low-temperature ultrasound long-term treatment experiments show that after 14 days of treatment, the fluorescence retention rate is still about 84%. These results indicate that the present invention not only promotes tissue permeability but also has good compatibility with fluorescence signal retention.

[0117] In the application of tissue transparency, the overall transparency of the sample is significantly improved after low-temperature high-frequency ultrasound treatment. At the same time, the sample can still maintain a strong and stable fluorescence signal after treatment, indicating that the present invention can simultaneously achieve the two goals of "promoting the permeability of the treatment solution" and "maintaining the fluorescence signal" in tissue transparency treatment.

[0118] In resin embedding and blackening embedding applications, samples treated with high-frequency ultrasound can achieve more uniform blackening and more stable subsequent imaging, and support continuous imaging at the whole brain scale, indicating that the present invention also has significant application value in the field of resin embedding.

[0119] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A high frequency ultrasound method for bulk tissue treatment, characterized by, The method includes the step of treating biological tissue under high-frequency ultrasound conditions; in the high-frequency ultrasound treatment, the ultrasound frequency is 1 MHz, the sound intensity is 0.6 W / cm², and the temperature is 4℃.

2. The high-frequency ultrasound method of claim 1, wherein, The large volume of tissue is mouse brain tissue.

3. Use of the high-frequency ultrasound method according to claim 1 for bulk staining of biological tissue, characterized in that, The high-frequency ultrasound treatment is applied while the intact biological tissue is placed in the staining solution to improve the permeability of the dye inside the biological tissue and the uniformity of staining.

4. Use according to claim 3, wherein the compound is ###0002### The staining solution is eosin or hematoxylin.

5. Use of the high-frequency ultrasound method according to claim 1 for the transparentization of biological tissue, characterized in that, Applying the high-frequency ultrasound treatment during the transparentization process of biological tissues improves the transparency efficiency of biological tissues and enhances the consistency of internal and external treatments.

6. The application according to claim 5, characterized in that, The fat removal system used in the biological tissue transparency process is DCM:TB = 2:

1.

7. The application of the high-frequency ultrasound method according to claim 1 in resin embedding or blackening embedding of biological tissues, characterized in that, Applying the high-frequency ultrasound treatment during the resin embedding or blackening embedding of biological tissue promotes the penetration and distribution of resin and blackening agent within the biological tissue.

8. The application according to claim 7, characterized in that, The resin used in the resin embedding is BMA; the blackening agent in the blackening embedding is 0.6% SBB.

9. A high-frequency ultrasound system for processing large volumes of tissue, characterized in that, The system includes a sample container or sample carrying platform module, a signal generation module, a power amplification module, an ultrasonic transducer module, a temperature control module, and a circulating fluid circuit module. The sample container or sample carrying platform module is used to place large-volume biological tissue samples; The signal generation module and power amplification module are used to output high-frequency ultrasonic signals at a preset frequency; The ultrasonic transducer module is used to convert electrical signals into ultrasonic vibrations and apply them to the treatment fluid. The temperature control module and the circulating fluid circuit module are used to control the temperature of the processing environment, especially to suppress the accumulation of thermal effects in the processing of fluorescent samples.

10. The high-frequency ultrasound system as described in claim 9, characterized in that, The power amplification module sets the ultrasonic frequency to 1 MHz, the ultrasonic transducer module sets the ultrasonic intensity to 0.6 W / cm², and the temperature control module sets the temperature to 4℃.

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