Rapid animal tissue processing system and method based on physical action to accelerate circulation

Abstract

The present invention relates to a rapid animal tissue processing system and method based on physical acceleration. The tissue is processed using a treatment fluid. The tissue processing system includes a physical acceleration subsystem comprising an ultrasound module and a microwave module. The ultrasound module applies ultrasound to the target object, while the microwave module applies microwaves to the target object, thereby accelerating the diffusion of the treatment fluid through the tissue. The in vivo whole-organ tissue processing solution provided by the present invention, through its rapid and automated nature, provides researchers with simple and feasible whole-organ processing, thereby promoting developments in areas such as organ research, case assessment, and brain mapping.

Description

Technical Field

[0001] The present invention generally relates to a system and method for rapid animal tissue processing based on physical acceleration. Specifically, it relates to a closed-loop control system and method for rapid in vivo whole-organ tissue processing and labeling in animals, based on physical acceleration, such as a rapid in vivo whole-organ tissue processing system based on transcardial perfusion. Background Art

[0002] To fully and deeply understand biological systems, we need to integrate the three-dimensional structural characteristics of intact tissues and organs with their molecular biological characteristics. For example, in the brain, the most complex organ in mammals, molecularly specific neurons and neural connections mediate the animal's processing of external information, thereby completing the complex functions from sensory input to behavioral output.

[0003] Although tissue slicing, thin-section tissue processing, staining, labeling, and imaging are traditional methods that have been widely used in most life science fields such as physiology, pathology, and brain science, it is difficult to restore the complete three-dimensional structure of the entire organ from the cut tissue slices. Furthermore, a large number of slices need to be operated on, which is very time-consuming, labor-intensive, and uneconomical. In addition, it is difficult to ensure sample quality and tissue uniformity.

[0004] To overcome the shortcomings of traditional thin-section imaging methods and obtain complete and continuous structural information of whole organs, researchers have developed various large-volume tissue microscopy tools. For example, optical imaging technologies such as sequential two-photon microscopy and light sheet microscopy directly perform three-dimensional imaging and reconstruction of large tissues; micro-optical-sectioning tomography (MOST) combines ultrathin sectioning technology with fluorescence imaging technology to repeatedly perform cross-sectional imaging and section removal on resin-embedded and hardened tissues, ultimately obtaining a three-dimensional image of the entire tissue. The development of these excellent imaging tools has also made the processing and preparation of large-volume tissue samples on a large scale an urgent need.

[0005] However, due to the limitations of the speed at which molecules can diffuse into dense tissues and their ability to disperse evenly, whole-organ tissue processing and labeling has always been a difficult and time-consuming problem. Whether it is the tissue processing goal of whole-organ transparency or exogenous molecular labeling methods such as whole-organ immunofluorescent staining (IF), it is usually necessary to immerse the tissue in the processing solution for a week or several weeks, and it is difficult to ensure the uniformity and repeatability of the tissue processing effect. Furthermore, most of the passive immersion methods currently used for relatively small tissues such as mice require a long time, which is even more difficult to deal with larger tissues such as primates and even human tissues. The lack of stable, fast and efficient whole-organ tissue processing and labeling technology has severely restricted the application of whole-organ imaging methods, further limiting our comprehensive and systematic understanding of the structure and function of biological organs.

[0006] Existing whole-organ tissue processing and labeling methods mainly rely on passive immersion in tissue processing fluid. However, due to the diffusion rate of molecules in dense biological tissues, the immersion method is time-consuming and it is difficult to ensure the uniformity and repeatability of its effects. Taking the whole-brain processing of mice as an example, immersion staining using antibodies usually takes more than a month. The clearing of whole-brain tissue of mice also usually takes 1-3 weeks, depending on different methods and effect requirements, such as iDisco, CLARITY, SHEILD, CUBIC-X and ScaleS. Moreover, the operation is complicated, cannot be automated, and has poor repeatability. It cannot be applied to scenarios with large data requirements, such as drug screening, case characteristic assessment, database construction, etc.

[0007] CN111458199A discloses a kind of automated tissue transparent system and transparent process of superposition ultrasonic vibration, which superposes ultrasonic vibration in the process of tissue transparentization, utilizes ultrasonic vibration to accelerate transparent reagent to diffuse in tissue. In addition, there are also some other methods that adopt shaking, electric field, ultrasound and high temperature to accelerate the method for tissue block permeability in the immersion process. However, these methods adopt the method of passive immersion, and the target tissue is placed in the working container full of tissue treatment solution, and the purpose of superposition low-power ultrasound is to accelerate the tissue permeability in the immersion process, therefore has the following defectives. On the one hand, it is impossible to effectively solve the problem of tissue treatment effect inhomogeneity, on the other hand, the acceleration effect is limited by the influence of different tissue depth inhomogeneity.

[0008] In addition, whole-organ tissue processing and labeling of large animals is more difficult and takes longer time. Some basic tissue processing steps, such as cryoprotection and post-fixation, will take exponentially longer in large animals. Sucrose cryoprotection of mouse brain usually takes 3-4 days, rat brain takes nearly a week, and larger animals such as monkeys take even longer. Complex tissue processing processes for large tissues of large animals, such as tissue clearing or immunofluorescence staining, become almost impossible due to the long time required and poor actual results.

[0009] Existing perfusion-based tissue processing and labeling methods (PACT, PARS) that utilize the animal's own blood circulation system lack physical acceleration factors such as ultrasound and microwaves. Effective molecules in the treatment fluid cannot fully cross the blood-brain barrier or other vascular-tissue barriers, resulting in limited effectiveness and still requiring approximately two weeks. Furthermore, due to the location and design of the pipeline recovery, the volume of fluid required for circulation is large, making it uneconomical. The degree of automation is insufficient, and without optimized designs for automated fluid exchange, operations still rely on manual labor and are complex. Furthermore, the necessary real-time monitoring and feedback control to align with the tissue processing objectives are lacking. Summary of the Invention

[0010] To address the shortcomings of existing whole-organ tissue processing and labeling technologies, the present invention aims to provide a fast, efficient, stable, economical, and fully automated in vivo whole-organ tissue processing solution, including closed-loop feedback, multi-channel hardware system design, and organ-specific perfusion ligation surgery.

[0011] Specifically, the present invention provides a rapid animal tissue processing system based on physical action accelerated circulation, wherein the tissue processing is performed using a processing liquid, and the tissue processing system comprises:

[0012] The physical acceleration subsystem includes an ultrasound module and a microwave module. The ultrasound module is used to apply ultrasound to the target object, and the microwave module is used to apply microwaves to the target object, thereby accelerating the diffusion of the treatment liquid in the tissue.

[0013] In one embodiment, the tissue processing is performed by in vivo perfusion using the animal's circulatory system.

[0014] In one embodiment, the ultrasonic module includes an ultrasonic vibrator and a driving board thereof.

[0015] The microwave module includes a high-power transformer, a driving circuit, a magnetron and a microwave probe.

[0016] In one embodiment, the tissue processing system further comprises:

[0017] a pipeline control subsystem for controlling the injection, discharge and / or recycling of the treatment fluid through the circulation pipeline; and / or

[0018] A real-time monitoring and closed-loop feedback control subsystem, which is used to monitor and control the temperature of the target object and the pressure of the treatment fluid in the circulation pipeline;

[0019] In one embodiment, the pipeline control subsystem includes a circulation power module, a channel selection module and a pipeline switching module.

[0020] The circulation power module includes an inflow peristaltic pump located at the liquid inlet end of the circulation pipeline or an outflow peristaltic pump located at the liquid outlet end of the circulation pipeline. The inflow peristaltic pump and the outflow peristaltic pump are preferably multi-channel peristaltic pumps, such as four-channel peristaltic pumps. The pump head of the peristaltic pump clamps the hose in the circulation pipeline, thereby providing power for the circulation pipeline.

[0021] The channel selection module includes one or more channel selection multi-way valves located at the liquid outlet of the inflow peristaltic pump and one or more channel selection multi-way valves located at the liquid inlet of the outflow peristaltic pump, wherein the channel selection multi-way valve is, for example, an electrically controlled three-way valve, and one direction of the multi-way valve is connected to air;

[0022] The pipeline switching module includes one or more loading multi-way valves located at the liquid inlet end of the inlet peristaltic pump, a liquid suction multi-way valve located downstream of the inlet peristaltic pump, and a recovery multi-way valve located upstream of the outflow peristaltic pump. The loading multi-way valve is preferably an electrically controlled loading three-way valve, and the other end of the loading multi-way valve is connected to the waste liquid tank. The liquid suction multi-way valve and the recovery multi-way valve are preferably electrically controlled nine-channel two-position multi-way valves.

[0023] In one embodiment, the real-time monitoring and closed-loop feedback control subsystem includes a temperature sensor for monitoring the temperature of the target object, and an air pressure sensor, such as a micro air pressure sensor, for detecting the pressure of the processing fluid in the monitoring circulation pipeline.

[0024] In one embodiment, the tissue processing system also includes a main controller, which is used to programmably control one or more of the following mechanisms: an ultrasonic module and a microwave module, a motor of a peristaltic pump, an electrically controlled three-way valve of the channel selection module, an electrically controlled loading three-way valve of the pipeline switching module, an electrically controlled nine-channel two-position aspiration multi-way valve and a nine-channel two-position recovery multi-way valve.

[0025] In one embodiment, the tissue treatment is tissue cryoprotection treatment, tissue clearing treatment, or staining treatment, such as immunofluorescence staining.

[0026] In one embodiment, the target object is a whole organ of an animal, such as the whole brain.

[0027] In one embodiment, the target object is placed in an adapter, and ultrasound gel is applied between the adapter and the target object.

[0028] In one embodiment, the adapter includes a single animal processing adapter that physically accelerates closed-loop perfusion of a single animal, a multiple animal processing adapter that physically accelerates closed-loop perfusion of multiple animals, and an ex vivo tissue processing adapter that physically accelerates ex vivo tissue blocks or tissue slices and integrates a liquid circulation chamber.

[0029] On the other hand, the present invention provides a method for rapid animal tissue processing based on physical action to accelerate circulation, the method comprising using a processing fluid to perform in vivo perfusion on a target object using the animal circulatory system, and simultaneously applying ultrasound and microwaves to the target object.

[0030] In one embodiment, the target object is a whole organ of an animal, such as the whole brain.

[0031] In one embodiment, the target object is placed in an adapter, and ultrasound gel is applied between the adapter and the target object.

[0032] In one embodiment, the microwave is a pulsed microwave. Preferably, the microwave source is 2 cm above the target object, the emission frequency is 2450 MHz ± 50 MHz, the power is adjustable from 0 to 40 W, and the single working time is 0 to 15 minutes.

[0033] In one embodiment, the ultrasound and the microwave are programmable.

[0034] Beneficial effects

[0035] The present invention addresses the difficulties in whole-organ tissue processing and labeling in vivo, designs an organ-targeted in vivo perfusion scheme with closed-loop feedback accelerated by physical effects, and designs and implements a multi-channel complex hardware system and a matching perfusion ligation surgery and multi-energy processing scheme.

[0036] (1) This technology utilizes the characteristics of the animal circulatory system and combines it with a surgical ligation scheme to perfuse tissue processing fluid into the whole body or targeted organ through the heart and blood vessels, and applies ultrasound and microwave treatment to the target area to accelerate the material exchange in the local blood vessels and the chemical reactions involved in tissue processing, thereby achieving rapid, efficient and automated whole-organ processing.

[0037] (2) This technology has designed a complex multi-channel, multi-liquid piping system and a multi-functional, automatic switching scheme. Through two sets of multi-way valves and corresponding sample pumps and other piping components, it can set up automatic replacement and waste discharge of multiple liquids; at the same time, the system recovers liquids through the chest recovery tube, recovers and recirculates the liquids, and can achieve complex whole-body tissue processing processes using very few multiple liquids, making batch, repetitive, and low-cost whole-organ processing possible.

[0038] (3) The physically accelerated in vivo whole-organ processing and labeling system implemented by this technology is a versatile, efficient, and scalable histological processing device with a wide range of application scenarios:

[0039] a. Preferably, whole-organ clearing of animal tissue in vivo;

[0040] b. Preferably, in vivo whole-organ molecular markers of animal tissues;

[0041] c. Preferably, whole-organ cryopreservation of animal tissue in vivo;

[0042] d. Preferably, large animal (such as non-human primate) tissue processing solution;

[0043] The in vivo whole-organ tissue processing solution provided by this invention aims to provide researchers with simple and feasible whole-organ processing through its rapid and automated characteristics, thereby promoting the development of organ research, case assessment, brain map construction and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 :Schematic diagram of the control principle of the hardware system for the cycle organization processing accelerated by physical action

[0045] Figure 2 :Design diagram of liquid pipeline system for multi-channel parallel processing

[0046] Figure 3 :Cranial circulation-specific animal ligation and perfusion surgery

[0047] Figure 4 :Characteristic curve of air pressure sensor

[0048] Figure 5 :Design diagram of an embodiment of an adapter platform for an in vivo tissue processing and labeling system based on physical acceleration. DETAILED DESCRIPTION

[0049] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0050] The present invention aims to design and implement a closed-loop control system and method based on physical acceleration for rapid in vivo whole-organ tissue processing and labeling in animals. The system primarily comprises a fully automated physical acceleration subsystem, a fully automated pipeline control subsystem, and a real-time monitoring and closed-loop feedback control subsystem. Its key features include: utilizing the animal's circulatory system to achieve fully automated, closed-loop tissue processing through in vivo perfusion combined with ultrasound and microwave physical acceleration. The system boasts a high level of integration, including a fully automated physical acceleration subsystem and pipeline control subsystem. Furthermore, system monitoring via pressure and temperature sensing enables real-time monitoring of tissue conditions and closed-loop feedback control of each module. The system also boasts strong scalability, enabling the design of complex pipeline systems capable of multi-channel parallel processing and multiple fluid switching. Adapters and multi-functional processing solutions are also designed for various tissue processing experiments, along with a comprehensive suite of compatible organ-specific perfusion ligation procedures.

[0051] The following reference Figure 1 Each subsystem is described in detail.

[0052] 1. Fully automatic physics acceleration subsystem

[0053] The fully automatic physical acceleration subsystem (physical action acceleration system) is composed of an ultrasonic module (ultrasonic generation module) and a microwave module (microwave generation module) driven and controlled by single chip computer programming.

[0054] The ultrasound module consists of an ultrasonic transducer and its driver board. The transducer frequency is 28kHz / 40kHz. The driver board has a programmable output current of 0-1A and an adjustable single-shot continuous operating time of 0-999s. The driver board also supports Modbus RTU communication to receive commands from the main control board. The main controller issues commands according to a specific timing sequence, rewriting the driver board's register data, such as power, start / stop, and single-shot continuous operating time, to achieve pulsed ultrasound delivery to tissue samples. The timing is self-adjusted via a temperature sensor. The specific temperature feedback adjustment strategy is described below in the real-time monitoring and closed-loop feedback control section. The ultrasonic transducer is connected to a head or body adapter and placed on a table for animal placement. Ultrasound gel is applied between the adapter and the tissue to ensure that the ultrasound waves fully penetrate the animal's body tissue. Preferably, for the purpose of clearing mouse brain tissue, the output current of the ultrasonic driver board is 0.15A, and the pulsed ultrasound cycle timing is 30 seconds on and 40 seconds off to maintain the target tissue temperature within the range of 34-36°C.

[0055] The microwave module consists of a high-power transformer, a drive circuit, a magnetron, and a microwave probe. The probe is the part that applies the generated microwaves to the target tissue. Ideally, the probe is suspended approximately 2 cm above the target tissue and transmits pulsed sinusoidal microwaves at a frequency of 2450 MHz ± 50 MHz, with an adjustable power of 0-40 W and a single operating time of 0-15 minutes. The pulsed microwaves are programmable via the main control board and automatically shut down using a temperature sensor.

[0056] Ultrasonic vibrators, microwave probes and tissue temperature sensors are integrated into different types of adapters (i.e., corresponding to Figure 1 Adapters include single animal processing adapters that physically accelerate closed-loop perfusion of a single animal, multi-animal processing adapters that physically accelerate closed-loop perfusion of four animals, and ex vivo tissue processing adapters that physically accelerate ex vivo tissue blocks or tissue slices and integrate a liquid circulation chamber. Adapters can also be adjusted to meet different experimental needs. For example, see Figure 5 , which shows a design diagram of an example of an adapter station for an in vivo tissue processing and marking system based on physical acceleration according to one embodiment of the present invention.

[0057] 2. Fully automatic pipeline control subsystem

[0058] The liquid pipeline control subsystem (liquid pipeline control system) consists of a circulation power module, a channel selection module and a pipeline switching module. It can fully automatically perform sequencing, timing, speed, quantity and channel-specific perfusion of up to eight liquids and four channels, and control the discharge and recovery of liquids according to the program.

[0059] Preferably, the circulation power module is composed of two four-channel peristaltic pumps, one at the liquid inlet and one at the liquid outlet. The pump head of the peristaltic pump clamps the hose in the circulation pipeline, thereby providing a power source for liquid circulation for the entire pipeline system. The motor behind the pump head is driven by the corresponding driver board, which can receive RS485 instructions from the main control board, and then change the data in the data registers such as speed, direction, start and stop, thereby changing the working state. The speed of the peristaltic pump is adjustable from 0 to 99.9 rpm, with a step of 0.1 rpm. Combined with a hose of a specific diameter, it can achieve precise control of the liquid flow rate and flow in the pipeline.

[0060] Preferably, the channel selection module consists of six electrically controlled three-way valves. The channel selection three-way valve at the liquid inlet end and the three-way valve at the liquid outlet end of the corresponding channel are synchronously controlled by a relay receiving commands from the main controller to select the channel. It should be noted that when a single channel is enabled, channel 1 is enabled by default. The remaining three channels are each connected to an electrically controlled channel selection three-way valve (channel tees, CT), one of which is connected to the inlet peristaltic pump and the other is connected to air. When the two liquid pipelines are connected, the channel is in an open state; when the inlet pump end pipeline is connected to the air and disconnected from the multi-directional valve end pipeline, the peristaltic pump will only cause the pump tube to inhale air, so the channel is in a closed state. The four channels then enter the pump head of the four-channel inlet peristaltic pump in parallel, running at the same speed to ensure its power stability and synchronization. The channel selection principle and implementation method at the liquid outlet end are exactly the same as those at the liquid inlet end.

[0061] Preferably, the pipeline switching module consists of 4 electronically controlled three-way valves and 2 nine-channel two-position multi-way valves, which are mainly designed for complex liquid perfusion strategies and liquid replacement operations. Figure 2 After the pump exits the inlet, each of the four channels is connected to an electronically controlled three-way loading valve (LT), one end connected to the animal's inlet and the other to a waste tank. Relays receive commands from the main controller to synchronize loading and perfusion control during fluid switching. This function ensures that residual fluid from the previous fluid in the line and air trapped between the next fluid and the aspiration reversing valve are completely expelled during fluid changes, ensuring fluid continuity on both sides of the loading tee. After the animal's circulation, the fluid is collected by the outlet tube and fed in parallel to the pump head of a four-channel outflow peristaltic pump to achieve stable fluid delivery. At the inlet, an electronically controlled nine-channel two-position aspiration multiway valve switches the eight fluids (solutions 1-8) toward the perfusion direction, subsequently dividing them into four channels. The four channels then merge and flow into an electronically controlled nine-channel two-position recovery multiway valve, which distributes the recovered fluid into corresponding liquid bottles or waste tanks, completing the fluid recovery cycle or discharge. It should be noted that seven of the eight liquids can be recycled, while one cannot be recycled (solution 1) and can only be discharged. It is usually loaded with balanced phosphate solution and other low-cost liquids used to clean the animal circulatory system.

[0062] 3. Real-time monitoring and closed-loop feedback control

[0063] This system utilizes various design features to enable real-time monitoring of tissue conditions and closed-loop feedback control, maintaining the target tissue in the most appropriate state for the intended treatment. Temperature and pressure are particularly important in the perfusion equipment designed for this system. Maintaining an appropriate reaction temperature ensures the rates of various biochemical reactions during tissue processing and also plays a key role in protecting signaling molecules within the tissue, such as fluorescence. Real-time monitoring of the hydraulic pressure within the circulation system can mitigate efficiency variations caused by individual differences (such as differences in circulatory systems due to varying vascular elasticity caused by the animal's age) or surgical procedures (such as the degree of ligation or the location of the cardiac injection needle insertion port). It also prevents excessive or insufficient flow rates from affecting the tiny blood vessels within the target tissue, potentially leading to rupture or collapse of the vessel ends.

[0064] Preferably, a temperature sensor is used to monitor the target tissue temperature. The temperature sensor serves as a peripheral to the main controller, and the sensor data is directly read and calculated by the main controller. Based on the acquired tissue temperature and the designed program logic, the main controller issues instructions to the driver boards of the ultrasound and microwave modules via RTU communication to adjust the power and cycle timing of the ultrasound and microwave modules, implementing closed-loop feedback control to stabilize the temperature within the target range.

[0065] Preferably, a micro air pressure sensor is used to monitor the liquid pressure in the pipeline. A buffer bottle is set between the liquid inlet pump and the animal. The upper end of the buffer bottle is connected to the air pressure sensor through a hose with an air column, and the lower end and the side inlet are connected to the circulating liquid pipeline. Through the dynamic balance of air pressure and hydraulic pressure at the gas-liquid interface in the buffer bottle, the hydraulic pressure in the circulation system is converted into air pressure for quantitative and effective monitoring. Most preferably, the sensor characteristic curve needs to be calibrated according to the ideal gas state equation. For example, in one embodiment, the pressure sensor characteristic curve measurement fitting result diagram can be seen in Figure 4 As a peripheral device of the main controller, the miniature air pressure sensor communicates with the main controller via I2C. Based on the current pipeline hydraulic pressure value and the designed program logic, the main controller issues instructions to the peristaltic pump driver board via RS485 communication, adjusting the peristaltic pump speed and implementing closed-loop feedback control, thereby stabilizing the system pipeline hydraulic pressure within the target range.

[0066] 4. Multi-channel and liquid switching process design

[0067] This system can realize multi-channel parallel processing according to user needs, and designs a complete set of liquid exchange pipeline switching process solutions for different processing targets.

[0068] In order to avoid introducing bubbles during the liquid replacement process and to completely drain the previous liquid, preferably, a complete and specific liquid pipeline control system workflow embodiment is as follows:

[0069] S1. Set the three-way channel selection valve so that channels ① and ② are open (CT2, 3, 5, and 6 are connected to air) and use two channels for perfusion.

[0070] S2. The suction multi-way valve connects the liquid 1 container and the perfusion end pipeline.

[0071] S3. The inflow peristaltic pump operates until liquid 1 is discharged from channels ① and ② at the infusion end, and the pipeline is filled with liquid.

[0072] S4. Stop the inflow peristaltic pump and establish circulation.

[0073] S5. The recovery multi-way valve connects the perfusion end pipeline and the waste liquid tank.

[0074] S6. Start the inlet peristaltic pump and the outlet peristaltic pump to perfuse and discharge liquid 1.

[0075] S7. The aspiration multi-way valve is switched to connect the Liquid 2 container and the filling end pipeline. Then, the loading three-way valve is switched to connect the aspiration end pipeline and the waste liquid tank. The residual Liquid 1 and Liquid 2 in the pipeline and the air in the aspiration multi-way valve are discharged into the waste liquid tank until Liquid 2 reaches the loading three-way valve.

[0076] S8. Load the three-way valve to connect the suction end pipeline and the perfusion end pipeline. Liquid 2 enters the perfusion end and is discharged into the waste liquid tank until the residual liquid 1 in the pipeline and animal circulation is drained.

[0077] S9. The recovery multi-way valve is reversed, connecting the liquid suction end pipeline and the liquid 2 container to recover liquid 2, forming a closed loop.

[0078] …

[0079] In the above embodiment, we have achieved non-recycling perfusion of liquid 1, discharge of the portion of liquid 2 that may have initially mixed with liquid 1, and subsequent recovery of liquid 2. By controlling the pump and valve program, more diverse recovery strategies can be designed to ensure the rational and full utilization of liquids.

[0080] In addition, for different tissue processing targets, this system has designed corresponding processing processes and overall fluid replacement plans, which are described below with three examples.

[0081] 1) Example of overall scheme for cryoprotection treatment of mouse brain tissue:

[0082] The most preferred procedure involves 1 hour of fixation with paraformaldehyde solution, 30 minutes of perfusion with cryoprotectant solution 1 (1% sodium deoxycholate, 15% trehalose, 10% ethylene glycol, 4% paraformaldehyde, 0.01M pH 7.2-7.4 phosphate buffer, 0.1% MW 27,000 alcoholysis 89% polyvinyl alcohol), and 30 minutes of perfusion with cryoprotectant solution 2 (35% trehalose, 10% ethylene glycol, 0.01M pH 7.2-7.4 phosphate buffer), for a total of 2 hours. The mouse brain is then removed and immersed in cryoprotectant solution 2 for 4 hours or overnight before freezing. This process, which originally took 4 days, now takes approximately 6 hours. Approximately 20 mL of each solution is required.

[0083] 2) Example of overall protocol for mouse whole brain clearing:

[0084] The most preferred procedure is a 1-hour fixation with paraformaldehyde solution, a 10-minute equilibrated phosphate solution, a 3-hour perfusion with clearing solution 1 (12% w / v sodium deoxycholate, 10% N,N-diisopropylethanolamine, 3% lauramidopropylhydroxysulfobetaine), a 6-hour perfusion with clearing solution 2 (12% w / v sodium deoxycholate, 10% N,N-diisopropylethanol, 25% lauramidopropylhydroxysulfobetaine, 16% thiourea), and a 2-hour perfusion with refractive index matching solution. The mouse brain is removed and placed in the refractive index matching solution. The original procedure took several weeks, but now takes approximately 12 hours. For optimal clearing results, the clearing time can be increased to approximately one day. Approximately 20 mL of each solution is required.

[0085] 3) Example of overall protocol for whole-brain immunofluorescence staining of mice:

[0086] Most preferably, the usual procedure is 1 hour of paraformaldehyde solution fixation, 10 minutes of balanced phosphate solution washing, 30 minutes of antigen protection / blood-brain barrier permeability enhancing solution, 4 hours of primary antibody, 30 minutes of balanced phosphate solution, 2 hours of secondary antibody, and 30 minutes of balanced phosphate solution. The original process took several weeks, but now it takes less than 8 hours. After staining, you can continue to pour in cryoprotectant solution 2 (35% trehalose, 10% ethylene glycol, 0.01M pH = 7.2-7.4 phosphate buffer) for cryoprotection or perform a whole-brain tissue clearing process. Each antibody requires 10-20mL of working solution, which is much less than the amount used for immunofluorescence staining of whole-brain sections.

[0087] 5. Organ Targeted Treatment Perfusion Ligation Surgery

[0088] The surgery is generally divided into two stages: first, pre-fixing the circulatory system by perfusion, and then achieving targeted tissue perfusion and delivery by ligating the corresponding blood vessels near the heart. The specific process of the surgery is described below using the example of brain tissue clearing in mice. Figure 3 :

[0089] S1. Anesthetize the mouse and fix it on a flat board. Make a transverse incision below the sternum to open the abdominal cavity. Cut the diaphragm upward along the ventral side and cut the ribs from both sides to the clavicle to expose the heart.

[0090] S2. Insert the needle into the left ventricle of the heart, make an opening in the right atrium, and infuse balanced phosphate-heparin solution.

[0091] S3. Change the perfusion fluid to paraformaldehyde-heparin solution to initially fix the mouse's entire body and blood vessels to prevent the collapse of the capillary ends of the circulatory system.

[0092] S4. Remove the perfusion needle, remove the mouse, and carefully flush the mouse's chest and abdominal cavities with balanced phosphate-heparin solution until no visible blood or blood clots are seen.

[0093] S5. Re-fix the mouse on the flat plate, move the left lung to the left above the heart, expose the descending aorta, inferior vena cava, pulmonary vein and pulmonary artery on the dorsal side of the chest cavity, use a blunt-tipped suture needle to pass through the aorta, close to the dorsal side of the chest cavity, and insert the needle into the right chest cavity at the branches of the left superior vena cava and inferior vena cava. Move all lung lobes to the lower side of the ligature and the heart on the upper side, tie with a ligature, and then use hemostatic forceps / hemostatic clips to clamp the distal end, thorax and clavicle.

[0094] S6. Hold the mouse by the hand and use a metal tube to cover the rubber tube. Puncture the mouse's back to connect the rubber tube to the mouse's chest cavity and the external air. Then place the mouse flat on the designed adaptive surgical bed and insert the rubber tube into the recovery tube of the adapter as a liquid recovery tube.

[0095] S7. Insert the inflow needle into the left ventricle through the original needle hole and secure it. Before insertion, make sure there are no bubbles in the tubing and check whether there is air in the ventricle. You can squeeze the heart to expel the air. Then, secure the mouse's head in an ultrasound tank filled with ultrasound gel and secure the microwave probe above the mouse's head.

[0096] S8. Start the designed program, begin the automatic fluid exchange cycle, and perform fully automatic brain tissue clearing treatment.

[0097] Optimally, the S1 incisions should be made not too dorsally to maintain a more intact dorsal thoracic structure and facilitate subsequent fluid recovery from the thoracic cavity. Bleeding should be minimized during this procedure, and a balanced phosphate-heparin solution should be used to flush the bleeding area and prevent clots.

[0098] Most preferably, the perfusion rate in S2 is 20 mL / min for 3-5 min, during which balanced phosphate-heparin solution is used to continue to clean the chest cavity to avoid blood accumulation until the outflowing fluid is clear.

[0099] Most preferably, the perfusion rate in S3 is 15 mL / min and the duration is 5 min.

[0100] Most preferably, in S5, the pulmonary artery, pulmonary vein, descending aorta and inferior vena cava are tied into a bundle, which is then lifted and clamped with a hemostat at the distal end near the ligature; a second hemostat is used to clamp the upper end of the ventral thorax, and two hemostatic clamps are used to clamp the skin and ribs near the clavicle.

[0101] In addition to the head circulation-specific ligation surgery suitable for brain treatment in the above embodiments, similar pulmonary and subclavian ligation surgeries can also be performed to simultaneously treat the brain and abdominal organs; the pulmonary arteries and veins, and the superior and inferior vena cava are ligated outside the aorta, and the proximal end of the ligation is cut open to recover fluid from the chest cavity through the inferior vena cava. At the same time, the subcutaneous tissue of the chest and abdominal cavity is ligated, and all upper limb and head circulations are locked in the neck to perform targeted circulation of abdominal organs.

[0102] The main purpose of ligating the pulmonary blood vessels is to prevent the perfusion fluid from passing through the lungs to the trachea and then being lost from the mouth and nose. The main purpose of ligating the subclavian thorax is to prevent the perfusion fluid from being lost from the vascular ports of the chest cavity opening.

[0103] The present invention addresses the difficulties in whole-organ tissue processing and labeling in vivo, designs an organ-targeted in vivo perfusion scheme with closed-loop feedback accelerated by physical effects, and designs and implements a multi-channel complex hardware system and a matching perfusion ligation surgery and multi-energy processing scheme.

[0104] Compared with the prior art, it has achieved at least the following improvements.

[0105] 1. Physical action accelerates the in vivo tissue processing circulatory system

[0106] This system, for the first time, utilizes a combination of ultrasound and microwaves to accelerate the physical effects of tissue processing during in vivo perfusion. On the one hand, in vivo perfusion treatment fully utilizes the rich terminal network of the animal's own circulatory system to deliver tissue processing fluid deeply to every corner of the targeted organ, optimizing tissue treatment results and avoiding uneven treatment caused by immersion. On the other hand, this physical effect greatly promotes the exchange of intravascular fluid to the extravascular space, increasing the rate of chemical reactions during tissue processing and significantly reducing the time required to process the entire organ.

[0107] 2. Automated multi-channel pipeline control subsystem

[0108] The multi-channel piping control system in this technology achieves fully automatic liquid replacement and recycling of multiple liquids in multiple channels through a circulation power module, a channel selection module, and a pipeline switching module. For example, a multi-channel peristaltic pump and a matching external control module are used to control the selective perfusion of liquids in multiple channels and provide them with stable and synchronous power; an electrically controlled two-position multi-way valve is used to achieve liquid replacement, a three-way valve for channel selection connected to air before the pump inlet is used to control channel selection, and a three-way valve directly connected to the waste liquid tank is used to discharge gas during liquid replacement, thereby achieving liquid circulation or waste discharge that matches the treatment purpose.

[0109] 3. Real-time monitoring and closed-loop feedback of in vivo perfusion

[0110] Furthermore, to minimize variations between perfused organs or individuals and achieve optimal perfusion results, this system is the first to implement automated, real-time feedback monitoring of pipeline fluid pressure and tissue processing temperature. For example, based on the dynamic balance of gas-liquid pressure within the pipeline, the pressure within the perfusion pipeline is calculated in real time using a pressure sensor module and the HX710 chip. RS485 communication is used to change the peristaltic pump speed, achieving closed-loop feedback control of pipeline pressure. A temperature sensor is used to obtain the target tissue temperature in real time, and RS485 communication is used to change the physical power of ultrasound and microwaves, achieving closed-loop feedback control of tissue temperature.

[0111] 4. Organ-specific perfusion ligation and puncture surgery

[0112] This system has been designed with a complete closed-loop perfusion surgical protocol to match in vivo tissue processing of the target organ. Its characteristics include: perfusion of the target organ through single ligation of the inferior vena cava, pulmonary artery and vein, and aorta (cerebral circulation ligation, abdominal circulation not ligated), combined with clamping of the subcutaneous, thoracic, head, and limb areas; rapid blood removal using phosphate-buffered heparin solution and paraformaldehyde pre-fixation to establish the subsequent circulation system; and dorsal puncture using a recovery tube with a self-designed adapter to penetrate the thorax and recover circulating fluid within the chest cavity.

[0113] 5. Various tissue treatment solutions

[0114] This system design realizes a versatile, efficient, and highly scalable histological processing device. Its characteristics include: wide applicability to various tissue processing scenarios, such as tissue clearing, cryoprotection, and staining, and its matching design enables a variety of tissue processing solutions. The present invention can rapidly fix, cryoprotect, perform immunofluorescence staining, and clear tissue, reducing processing time from days or weeks to hours or half a week.

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A rapid animal tissue processing system based on physical action accelerated circulation, characterized in that: The tissue processing is performed by perfusing the target object in vivo using a treatment solution through the animal circulatory system. The tissue processing is tissue cryoprotection processing, tissue clearing processing, or staining and marking processing. The target object is the whole brain of the animal. The tissue processing system includes: a physical acceleration subsystem, the subsystem comprising an ultrasound module and a microwave module, the ultrasound module being used to apply ultrasound to the target object, the microwave module being used to apply microwaves to the target object, thereby accelerating the diffusion of the treatment fluid in the tissue, and a pipeline control subsystem, the subsystem being used to control the injection, discharge and / or recovery of the treatment fluid through the circulation pipeline; The pipeline control subsystem includes a circulation power module, a channel selection module and a pipeline switching module. The circulation power module includes an inflow peristaltic pump located at the liquid inlet end of the circulation pipeline or an outflow peristaltic pump located at the liquid outlet end of the circulation pipeline. The inflow peristaltic pump and the outflow peristaltic pump are multi-channel peristaltic pumps. The pump head of the peristaltic pump clamps the hose in the circulation pipeline, thereby providing power for the circulation pipeline. The channel selection module includes one or more channel selection multi-way valves located at the liquid outlet end of the inflow peristaltic pump and one or more channel selection multi-way valves located at the liquid inlet end of the outflow peristaltic pump, and one direction of the multi-way valve is connected to air; The pipeline switching module includes one or more loading multi-way valves located at the liquid inlet end of the inflow peristaltic pump, a liquid suction multi-way valve located downstream of the inflow peristaltic pump, and a recovery multi-way valve located upstream of the outflow peristaltic pump. The other end of the loading multi-way valve is connected to the waste liquid tank. The ultrasound and the microwave are programmable and controllable. The ultrasound is pulsed ultrasound, and the microwave is pulsed microwave. The target object is placed in the adapter, and ultrasound gel is coated between the adapter and the target object.

2. The tissue processing system according to claim 1, wherein: The ultrasonic module includes an ultrasonic vibrator and a driving board thereof. The microwave module includes a high-power transformer, a driving circuit, a magnetron and a microwave probe.

3. The tissue processing system according to claim 1, wherein: The tissue processing system further comprises: A real-time monitoring and closed-loop feedback control subsystem is used to monitor and control the temperature of the target object and the pressure of the processing fluid in the circulation pipeline.

4. The tissue processing system according to claim 1, wherein: The pipeline control subsystem includes a circulation power module, a channel selection module and a pipeline switching module. The inlet peristaltic pump and the outlet peristaltic pump in the circulation power module are four-channel peristaltic pumps; The channel selection multi-way valve in the channel selection module is an electrically controlled three-way valve; The loading multi-way valve in the pipeline switching module is an electrically controlled loading three-way valve, and the liquid suction multi-way valve and the recovery multi-way valve are electrically controlled nine-channel two-position multi-way valves.

5. The tissue processing system according to claim 3, wherein: The real-time monitoring and closed-loop feedback control subsystem includes a temperature sensor for monitoring the temperature of the target object and a pressure sensor for detecting the pressure of the processing liquid in the monitoring circulation pipeline.

6. The tissue processing system according to claim 5, characterized in that The air pressure sensor is a miniature air pressure sensor.

7. The tissue processing system according to any one of claims 1 to 6, characterized in that: The tissue processing system also includes a main controller, which is used to programmably control one or more of the following mechanisms: an ultrasonic module and a microwave module, a motor of a peristaltic pump, an electrically controlled three-way valve of the channel selection module, an electrically controlled loading three-way valve of the pipeline switching module, an electrically controlled nine-channel two-position aspiration multi-way valve and a nine-channel two-position recovery multi-way valve.

8. The tissue processing system according to claim 1, wherein: The tissues were processed for immunofluorescence staining.

9. The tissue processing system according to claim 1, wherein: The adapters include a single animal processing adapter that physically accelerates closed-loop perfusion of a single animal, a multi-animal processing adapter that physically accelerates closed-loop perfusion of multiple animals, and an ex vivo tissue processing adapter that physically accelerates ex vivo tissue blocks or tissue slices and integrates a liquid circulation chamber.

10. The tissue processing system according to claim 1, wherein: The microwave source is 2 cm above the target object, with an emission frequency of 2450 MHz ± 50 MHz, an adjustable power of 0-40 W, and a single working time of 0-15 minutes.

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