Negative pressure isolation living body microscope experiment box

Abstract

This invention provides a negative pressure isolation live microscope experimental box. The experimental box, through its closed negative pressure design, ensures the biosafety of experiments with pathogens of BSL-2 level and above, meeting the requirements of the safety committee review. At the same time, its innovative box-type integrated window structure eliminates the vibration torque caused by animal movement in traditional suspended windows from a mechanical design perspective, providing unparalleled stability for high-precision imaging. Ultimately, it makes it possible to safely and stably conduct live microscopic studies of highly pathogenic infection models in a conventional laboratory.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a negative pressure isolation in vivo microscope experimental box. Background Technology

[0002] In existing research, in vivo microscopy has been applied to observe the dynamics of immune cells, tumor cell development, neural network mapping, and even to assist in precision medicine. By combining it with more advanced fluorescent labeling technologies, novel laser scanning imaging equipment, and artificial intelligence-assisted image analysis technologies, the research fields applicable to in vivo microscopy are constantly expanding.

[0003] However, live microscopy technology still faces many challenges in practical applications, especially in terms of application areas. It frequently encounters unstable factors such as image jitter caused by the breathing or heartbeat of laboratory animals, affecting the imaging focal plane. Furthermore, since most microscopes suitable for experimental use are generally stored in conventional laboratories, they cannot be used for infectious experiments involving live animals, or their operation in biosafety laboratories is cumbersome and cannot guarantee efficient equipment utilization.

[0004] Therefore, there is an urgent need in this field to develop a safe, stable and easy-to-operate experimental device that would enable further research on the dynamic interaction between infectious pathogens and host immune cells in conventional laboratories. Summary of the Invention

[0005] The present invention aims to develop a safe, stable and easy-to-operate experimental device that enables further research on the dynamic interaction between infectious pathogens and host immune cells in conventional laboratories, specifically relating to a negative pressure isolation in vivo microscope experimental box.

[0006] In a first aspect of the present invention, a negative pressure isolation in vivo microscope experimental box is provided, the negative pressure isolation in vivo microscope experimental box is provided with a negative pressure in vivo microscope window, the negative pressure in vivo microscope window is disposed on the top surface 8 of the experimental box, and the negative pressure in vivo microscope window includes a slide support recess 2 and an observation area;

[0007] The slide-supporting recess 2 is used to support the cover glass, thereby forming a semi-airtight or fully airtight space with the cover glass, the slide-supporting recess 2 and the observation area.

[0008] The microscope viewing window cavity includes an animal tissue fixation cavity 5 and a body fluid containment cavity 3. The upper parts of the two chambers are connected to each other, and the bottom is separated by an annular structure 4 that is lower than the bottom surface of the slide.

[0009] The animal tissue fixation cavity 5 is provided with an outlet of an airway 6, through which air is drawn from the microscope viewing window cavity to reduce the pressure inside the microscope viewing window cavity. The airway 6 is located inside the top surface 8 of the experimental box.

[0010] In another preferred embodiment, the pressure inside the microscope window cavity is lower than the external pressure, thereby forming a negative pressure region; preferably, the pressure difference between the inside and outside pressure of the microscope window cavity is >15 inHg.

[0011] In another preferred embodiment, the side 10 of the negative pressure isolation in vivo microscope experimental box is also provided with an airflow control system; wherein, the airflow control system includes a gas inlet end, a gas exhaust end and a port 6-1 of the airway 6;

[0012] The air inlet and exhaust end enable bidirectional airflow. Both ports 1-1 and 1-2 can be used as air inlets or exhaust outlets. One port acts as an air inlet to allow external gas to enter the experimental box, while the other port acts as an exhaust outlet to expel the gas inside the box. By switching the air inlet and exhaust functions of the two ports, unidirectional airflows in opposite directions can be formed inside the experimental box.

[0013] In another preferred embodiment, the airflow control system establishes a stable unidirectional airflow for ≥3 hours.

[0014] In another preferred embodiment, the airflow control system maintains a negative pressure of -35Pa to -15Pa between the box and the open space outside.

[0015] In another preferred embodiment, the observation area is further provided with a toothed protrusion 7 and an annular structure 4. The toothed protrusion 7 is composed of multiple alternating high and low regions, wherein the high regions are at the same height as the bearing recess 2, and the low regions are at a lower height than the bearing recess 2; the annular structure is at the same height as the bearing recess 2.

[0016] In another preferred embodiment, when the coverslip is placed on the support recess 2, the toothed protrusion 7 and the coverslip form a semi-enclosed space that allows liquid to pass through but prevents tissue from penetrating.

[0017] In another preferred embodiment, during the observation process, bodily fluids or tissue fluids flow through the lower region of the toothed protrusion into the space between the toothed protrusion and the annular structure, thereby avoiding affecting the observation effect of the viewing window.

[0018] In another preferred embodiment, the negative pressure isolation in vivo microscope experimental box is provided with a cover 9 on the front, and the cover is sealed to the experimental box.

[0019] In another preferred embodiment, the lid 9 is tightly connected to the experimental box by magnetic attraction, snap fastener, hinge, sealant, etc.

[0020] In another preferred embodiment, magnets or magnetic strips are provided on each side of the front of the negative pressure isolation in vivo microscope experimental box and sealed with sealant.

[0021] In another preferred embodiment, there is a flexible tube placement between the lid 9 and the experimental box, so that an indwelling needle can be placed when the animal is inside the experimental box.

[0022] In another preferred embodiment, the negative pressure isolation in vivo microscope experimental box can also be equipped with an air filtration device, which filters the gas entering and exiting the experimental box.

[0023] In another preferred embodiment, the air filtration device filters the gas entering the air intake.

[0024] In another preferred embodiment, the air filtration device filters the gas at the exhaust end.

[0025] In another preferred embodiment, the air filtration device may be a conventional commercially available filtration device.

[0026] In another preferred embodiment, the air filtration device is a high-efficiency air filter, a commercially available high-efficiency filter membrane, etc.

[0027] In a second aspect of the invention, a method for in vivo microscopic imaging of animals for non-diagnostic or therapeutic purposes is provided, the method comprising the steps of:

[0028] (1) Preparation of the viewing window for a negative pressure in vivo microscope:

[0029] Place the coverslip coated with sealant on the slide support recess 2 and the toothed protrusion 7 to form a sealed microscope window cavity. Open the cover 9 on the front of the negative pressure isolation live microscope experimental box and place the test animal whose target tissue has been surgically exposed. Close the cover 9 to seal the experimental box.

[0030] The airflow control system is activated, and air is drawn from the microscope window cavity through the air passage 6 to create a negative pressure environment in which the pressure inside the microscope window cavity is lower than the external pressure, thereby adsorbing and fixing the target tissue in the fixation cavity 5.

[0031] (2) Image acquisition: The negative pressure in vivo microscope window is placed under the in vivo microscope, and a vascular marker dye is injected through the tail vein. After the dye circulates, a stable field of view is determined under the microscope, and image data is acquired.

[0032] In another preferred embodiment, the movement of the animal tissue on the coverslip is controlled to be ≤10μm / h in the XY axis direction and ≤10μm / h in the Z axis direction.

[0033] In another preferred embodiment, the method further includes: controlling the airflow within the negative pressure isolation in vivo microscope experimental chamber, wherein the airflow control is achieved through an airflow control system, and the airflow control method includes the following steps:

[0034] In the airflow control system, external gas enters the experimental chamber through the inlet 1-1, and gas inside the experimental chamber is discharged through the exhaust 1-2, creating a unidirectional airflow from front to back within the chamber; or

[0035] External gas enters the experimental box through the inlet 1-2, and the gas inside the experimental box is discharged through the exhaust 1-1, creating a unidirectional airflow from front to back inside the box.

[0036] In another preferred embodiment, the gas at the inlet and outlet ends is free of detectable pathogens after passing through an air filtration device.

[0037] In another preferred embodiment, an indwelling needle is inserted into the animal before the test. Preferably, the indwelling needle is a tail vein indwelling needle.

[0038] In another preferred embodiment, during the testing process, the tested animal is given fluid supplementation via the indwelling needle.

[0039] In another preferred embodiment, the supplemental liquid is an anesthetic.

[0040] In another preferred embodiment, the gas at the inlet and outlet ends, after passing through an air filtration device, contains no more than 1,000 pathogenic bacteria or fungal particles per cubic meter of gas.

[0041] The air filtration device described in 1-1 is a filter membrane superimposed activated carbon adsorption column system.

[0042] In another preferred embodiment, the pore diameter of the filter membrane is 0.2-0.3 micrometers.

[0043] In another preferred embodiment, the airway 6 remains clean and unblocked when the negative pressure isolation in vivo microscope experimental box operates intermittently or continuously for ≥45 hours.

[0044] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0045] Figure 1 A view of the negative pressure isolation in vivo microscope experimental kit of the present invention is shown, wherein Figure 1 In the diagram, A represents the front view of the experimental kit. Figure 1 Figure B in the diagram is a side view of the experimental kit. Figure 1 C in the diagram represents the top view (top view) of the experimental box. Figure 1 Figure D in the diagram is a three-dimensional view of the experimental kit;

[0046] Figure 2The negative pressure in vivo microscope window view of the present invention is shown, wherein Figure 2 In this context, A represents the top front view (top view) of the window. Figure 2 In the diagram, B represents a cross-sectional view of the top surface of the window, where the filled diagonal lines represent solid parts, and the blank areas represent cavities or holes. Figure 2 In this context, C represents the front view of the window viewed from inside the box.

[0047] Figure 3 This shows a schematic diagram of the airflow control system on the side of the negative pressure isolation in vivo microscope experimental box of the present invention;

[0048] Figure 4 The image shows a front view of the negative pressure isolation in vivo microscope experimental box of the present invention and its matching sealing lid, wherein, Figure 4 Figure A in the diagram is a front view of the experimental kit. Figure 4 Figure B shows the matching size lid;

[0049] Figure 5 A cross-sectional view showing the negative pressure in vivo microscope window view of the present invention;

[0050] Figure 6 This illustrates the XY axis displacement of mouse kidney surface tissue during imaging in an embodiment of the present invention;

[0051] Figure 7 The movement characteristics of the sphere in the microvessels on the surface of the mouse kidney were shown;

[0052] Figure 8 The study showed the aggregation of globules in the microvessels on the surface of mouse kidneys. Detailed Implementation

[0053] Through extensive and in-depth research and numerous experimental screenings, the inventors have unexpectedly developed a negative pressure isolation in vivo microscope experimental box for the first time. This negative pressure isolation in vivo microscope experimental box, through its closed negative pressure design, ensures biosafety for experiments with pathogens of BSL-2 level and above, meeting the requirements of the safety committee review. Simultaneously, its innovative box-type integrated window structure mechanically eliminates the vibration torque caused by animal movement in traditional suspended windows, providing unparalleled stability for high-precision imaging. Ultimately, this makes it possible to safely and stably conduct in vivo microscopic studies of highly pathogenic infection models in conventional laboratories. Based on this, the inventors completed this invention.

[0054] Terminology Explanation

[0055] Optical microscopy technique

[0056] In optical microscopy experiments, mouse in vivo microscopy refers to a technique that allows for real-time observation of living tissues or cells without damaging their function or appearance. Building upon the advantages of modern high-resolution optics, this technique further meets the specific needs of real-time research into various phenomena occurring in living animals, playing an irreplaceable role in revealing cell dynamics, animal tissue system development, and pathological changes associated with specific infections.

[0057] This invention provides a negative pressure isolation in vivo microscope experimental box, aiming to solve the safety and stability bottlenecks in in vivo microscopy experiments using highly pathogenic infection models. In this invention, the built-in negative pressure isolation system effectively prevents pathogen leakage, enabling in vivo imaging experiments with pathogens of BSL-2 and above to be safely conducted in conventional laboratories and successfully pass biosafety committee review. Furthermore, this invention adopts a box-type integrated window design, using an optimized mechanical structure to provide overall support for the window and the animal, fundamentally eliminating the vibration torque and physiological vibration interference of traditional installation methods, ensuring a highly stable imaging field of view.

[0058] The negative pressure isolation in vivo microscope experimental box of the present invention fully retains the real-time, high-resolution observation capability of in vivo microscopy technology, providing a safe and reliable cutting-edge platform for studying key biomedical issues such as infection pathology and cell dynamics in real physiological environments.

[0059] Negative pressure isolation in vivo microscope experimental box

[0060] In this invention, the negative pressure isolation in vivo microscope experimental box is provided with a negative pressure in vivo microscope window, which is located on the top surface 8 of the experimental box. The negative pressure in vivo microscope window includes a slide support recess 2, a toothed protrusion 7, and an annular structure 4.

[0061] The slide support structure is used to support the cover glass slide. The support recess 2 and the toothed protrusion 7 have the same height. When the cover glass slide is placed on the support recess 2 and the toothed protrusion 7, a closed space microscope window cavity is formed.

[0062] The microscope viewing window cavity includes an animal tissue fixation cavity 5 and a body fluid containment cavity 3. The upper parts of the two chambers are connected to each other, and the bottom is separated by an annular structure 4 that is lower than the bottom surface of the slide.

[0063] The animal tissue fixation cavity 5 is provided with an outlet of an airway 6, through which air is drawn from the microscope viewing window cavity to reduce the pressure inside the microscope viewing window cavity. The airway 6 is located inside the top surface 8 of the experimental box.

[0064] Preferably, the microscope window cavity does not leak air when the pressure inside is more than 15 inHg lower than the external pressure.

[0065] In this invention, the side 10 of the negative pressure isolation live microscope experimental box is also provided with an airflow control system; wherein, the airflow control system includes a gas inlet end 1-1, a gas exhaust end 1-2 and a port of the air passage 6. In the airflow control system, external gas enters the experimental box through the inlet end 1-1, and the gas inside the experimental box is discharged through the exhaust end 1-2, creating a unidirectional airflow from front to back inside the box.

[0066] Preferably, the airflow control system establishes a stable unidirectional airflow for ≥3 hours.

[0067] Preferably, the airflow control system maintains a negative pressure of -35Pa to -15Pa between the inside of the box and the open space outside.

[0068] In this invention, the front of the negative pressure isolation in vivo microscope experimental box is provided with a cover 9, and the cover is sealed to the experimental box.

[0069] Preferably, the lid 9 is tightly connected to the experimental box by magnetic attraction, snap fastener, hinge, sealant, etc.

[0070] Preferably, the negative pressure isolation in vivo microscope experimental box has magnets or magnetic strips on each side of its front side and is sealed with sealant.

[0071] In this invention, the negative pressure isolation in vivo microscope experimental box is also equipped with an air filtration device, which filters the gas entering and exiting the experimental box.

[0072] Preferably, the air filtration device filters the gas entering the air intake.

[0073] Preferably, the air filtration device filters the gas discharged from the exhaust end.

[0074] Preferably, the gas at the inlet and outlet ends does not contain detectable pathogens after passing through the air filtration device.

[0075] Preferably, the gas at the inlet and outlet ends contains no more than 1,000 pathogenic bacteria or fungal particles per cubic meter of gas after passing through the air filtration device.

[0076] Preferably, the air filtration device is a filter membrane superimposed with an activated carbon adsorption column system.

[0077] Preferably, the pore diameter of the filter membrane is 0.2-0.3 micrometers.

[0078] Mouse in vivo microscopy

[0079] The present invention also provides a mouse in vivo microscopy technique, the method comprising the following steps:

[0080] (1) Preparation of the viewing window for a negative pressure in vivo microscope:

[0081] Place the coverslip coated with sealant on the slide support recess 2 and the toothed protrusion 7 to form a sealed microscope window cavity. Open the cover 9 on the front of the negative pressure isolation live microscope experimental box and put in the test animal whose target tissue has been surgically exposed and whose tail vein indwelling needle has been inserted. Close the cover 9 to seal the experimental box.

[0082] The airflow control system is activated, and air is drawn from the microscope window cavity through the air passage 6 to create a negative pressure environment in which the pressure inside the microscope window cavity is lower than the external pressure, thereby adsorbing and fixing the target tissue in the fixation cavity 5.

[0083] (2) Image acquisition: The negative pressure in vivo microscope window is placed under the in vivo microscope, and a vascular marker dye is injected through the tail vein. After the dye circulates, a stable field of view is determined under the microscope, and image data is acquired.

[0084] Preferably, airflow control can also be applied within the negative pressure isolation in vivo microscope experimental chamber. This airflow control is achieved through an airflow control system, and the method for airflow control includes the following steps:

[0085] In the airflow control system, external gas enters the experimental box through the inlet 1-1, and gas inside the experimental box is discharged through the exhaust 1-2, creating a unidirectional airflow from front to back inside the box.

[0086] Preferably, the movement of the animal tissue on the coverslip is controlled within ≤10μm / h in the XY axis direction and ≤10μm / h in the Z axis direction.

[0087] Preferably, under the condition that the negative pressure isolation in vivo microscope experimental box operates intermittently or continuously for ≥45 hours, the airway 6 can remain clean and unblocked.

[0088] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0089] (1) This invention ensures that pathogens in the experimental box will not leak into the external environment through negative pressure isolation and high-efficiency filtration system, thus ensuring the safety of experimental operation and environmental safety, and making it possible to conduct live microscopy experiments on infectious pathogens of BSL-2 level or above in a conventional laboratory.

[0090] (2) The present invention integrates the live microscope window and the main body of the experimental box into an integrated structure. By shortening the lever arm and providing rigid support, the vibration and torque sway caused by animal activity in traditional external windows are fundamentally eliminated, thereby obtaining a highly stable imaging image.

[0091] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0092] Example 1: Negative pressure isolation in vivo microscope experimental box of the present invention

[0093] The present invention provides a negative pressure isolation live microscope experimental box, which is provided with a negative pressure live microscope window. The negative pressure live microscope window is located on the top surface 8 of the experimental box. The negative pressure live microscope window includes a glass slide support recess 2, a toothed protrusion 7 and an annular structure 4.

[0094] The slide support structure is used to support the cover glass slide. The support recess 2 and the toothed protrusion 7 have the same height. When the cover glass slide is placed on the support recess 2 and the toothed protrusion 7, a closed space microscope window cavity is formed.

[0095] The microscope viewing window cavity includes an animal tissue fixation cavity 5 and a body fluid containment cavity 3. The upper parts of the two chambers are connected to each other, and the bottom is separated by an annular structure 4 that is lower than the bottom surface of the slide.

[0096] The animal tissue fixation cavity 5 is provided with an outlet of airway 6, through which air is drawn from the microscope window cavity to reduce the pressure inside the microscope window cavity. The airway 6 is located inside the top surface 8 of the experimental box.

[0097] The animal tissue fixation cavity 5 is provided with an outlet for an airway 6, through which air is pumped into the microscope viewing window cavity to >15 inHg without leakage. The airway 6 is located inside the top surface 8 of the experimental box. A view of the negative pressure isolation in vivo microscope experimental box of the present invention is shown below. Figure 1 As shown, the negative pressure in vivo microscope window view of the present invention is as follows: Figure 2 As shown, the cross-sectional view of the microscope window is as follows: Figure 5 As shown.

[0098] The side 10 of the negative pressure isolation in vivo microscope experimental box is also equipped with an airflow control system. This airflow control system includes a gas inlet 1-1, a gas outlet 1-2, and a port 6-1 of the airway 6. In this airflow control system, external gas enters the experimental box through the inlet 1-1, and gas inside the experimental box is discharged through the outlet 1-2, creating a unidirectional airflow from front to back within the box. The airway 6 maintains a negative pressure of -35Pa to -15Pa between the inside of the box and the open space outside. Figure 3 As shown.

[0099] The negative pressure isolation in vivo microscope experimental box has a lid 9 on its front. The lid and the front of the negative pressure isolation in vivo microscope experimental box fit together to seal, ensuring that air inside and outside the box cannot circulate. The lid is the same size as the isolation box. Before the experiment, it is necessary to... Figure 4 In the experiment, magnets that attract each other are installed at positions A-1 to A-4 of A and B-1 to B-4 respectively. After the experiment begins, before installing the lid, additional sealant needs to be applied to each side, and then the lid is attracted to the corresponding position to ensure a seal.

[0100] The negative pressure isolation live microscope experimental box is also equipped with an air filtration device. The air filtration device filters the gas entering and exiting the experimental box. After passing through the air filtration device, the gas at the inlet and outlet ends contains no more than 1,000 pathogenic bacteria or fungal particles per cubic meter of gas.

[0101] Example 2: Observation of the kinematic characteristics of polystyrene (PS) spheres in microvessels on the surface of mouse kidneys using the in vivo microscope experimental kit of the present invention.

[0102] In this experiment, the in vivo microscope experimental kit of the present invention was used to observe the surface of mouse kidneys. The specific operation is as follows:

[0103] A 1cm incision was made parallel to the lower edge of the left rib in the mouse to expose the left kidney, 4mm below the rib. After connecting the aforementioned in vivo microscope experimental box to negative pressure, the mouse's left kidney was suctioned and fixed to the viewing window. A tail vein catheter was then inserted to pre-build the complete experimental platform for fluid resuscitation. At the start of the experiment, 50μl of Tomato Lectin-Dylight488 was injected via the tail vein to mark blood vessels. After the dye circulated with the mouse's circulatory system for 30 minutes, a stable and clear field of view was sought. Once the field of view was determined, the bright blood vessel signal was used as a positional reference, and continuous imaging was performed at 30s / frame for one hour, monitoring the tissue displacement in the XY plane and the vertical direction of the Z axis during the experiment. Results are as follows: Figure 6 As shown in the figure, using this experimental system, the tissue displacement along the XY axis is less than 10 μm per hour and the displacement along the Z axis is less than 10 μm per hour during imaging. This system can meet the design expectations and specific experimental needs.

[0104] In addition, another mouse was prepared using the same surgical procedure. 50 μl of Tomato Lectin-Dylight647 was injected via the tail vein to mark blood vessels. After the dye circulated in the mouse's circulatory system for 30 minutes, a stable and clear field of view was sought. Microscopy was used to image at 130 ms / frame. A small ball was injected 10 seconds after the start of imaging, and the motion characteristics of the ball were recorded. The results are as follows: Figure 7 As shown and Figure 8 As shown.

[0105] As shown in the figures, the imaging system constructed based on the negative pressure isolation in vivo microscope experimental kit described in this invention can achieve stable and clear observation of microscopic movements within living tissues. In the experiment, 50 μl of Tomato Lectin-Dylight 647 was injected into the tail vein to label blood vessels. After the dye circulated for 30 minutes, the movement of the small ball after injection was recorded at a high-speed imaging rate of 130 ms / frame. The results show that the system can clearly and continuously capture the trajectory and instantaneous pauses of the small ball within the blood vessels, demonstrating its excellent dynamic imaging capability and temporal resolution in a living environment.

[0106] Further observation revealed a significant aggregation phenomenon at the point where the spheres stopped moving within the blood vessel. Given that the spheres used had a diameter of 1 μm, this aggregation indicates the presence of structures or cells within the blood vessel capable of effectively blocking the passage of the spheres, and their size must be greater than 1 μm. This discovery not only verifies the reliability of this experimental system in observing and analyzing microscopic motion characteristics but also provides intuitive experimental evidence for understanding the physical barriers to substance transport within blood vessels, demonstrating the promising application prospects of the negative pressure isolation in vivo microscope experimental box of this invention in in vivo microcirculation research.

[0107] discuss

[0108] Current in vivo microscope window products only have a single window function, and users need to manually match the equipment required to fix the window. Since many high-performance microscopes capable of performing in vivo microscopy are placed in conventional laboratories, and the existing in vivo microscope systems mentioned above do not have isolation and purification systems, experiments involving infectious pathogens of BSL-2 level or higher cannot be conducted if existing window systems are used for in vivo microscopy experiments.

[0109] Meanwhile, existing viewing windows mainly use pins and screws to rigidly fix user-supplied iron frames, vises, and other equipment and viewing window arms, which will generate a large lever arm. This may cause significant vibration in the field of vision due to the movement of experimental animals, which is very detrimental to observation.

[0110] This invention provides an integrated box-type negative pressure isolation system. This device integrates the in vivo microscope window and the negative pressure isolation system within a sealed box, completely eliminating the vibration torque caused by traditional fixation methods and ensuring imaging stability. Furthermore, it provides a high level of biosafety protection, making it possible to safely conduct in vivo microscopic studies of highly pathogenic pathogens in conventional laboratories.

[0111] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A negative pressure isolation in vivo microscope experimental box, characterized in that, The negative pressure isolation live microscope experimental box is provided with a negative pressure live microscope window, which is located on the top surface (8) of the experimental box. The negative pressure live microscope window includes a slide support stage (2) and an observation area. The slide support recess (2) is used to support the cover glass, thereby forming a semi-airtight or fully airtight space with the cover glass, the slide support recess (2) and the observation area. The microscope viewing window cavity includes an animal tissue fixation cavity (5) and a body fluid containment cavity (3). The upper parts of the two cavities are connected to each other, and the bottom is separated by an annular structure (4) that is lower than the bottom surface of the slide. The animal tissue fixation cavity (5) is provided with an outlet of an airway (6), through which air is drawn from the microscope window cavity to reduce the pressure inside the microscope window cavity. The airway (6) is located inside the top surface (8) of the experimental box.

2. The experimental kit as described in claim 1, characterized in that, The pressure inside the microscope window cavity is lower than the external pressure, thus forming a negative pressure region; preferably, the pressure difference between the inside and outside of the microscope window cavity is >15 inHg.

3. The experimental kit as described in claim 1, characterized in that, The side (10) of the negative pressure isolation live microscope experimental box is also provided with an airflow control system; wherein, the airflow control system includes a gas inlet end, a gas exhaust end and a port (6-1) of the airway (6); The air inlet and exhaust end enable bidirectional airflow. Both ports (1-1) and (1-2) can be used as air inlets or exhaust outlets. One port acts as an air inlet to allow external gas to enter the experimental box, while the other port acts as an exhaust outlet to expel the gas inside the box. By switching the air inlet and exhaust functions of the two ports, unidirectional airflows in opposite directions can be formed inside the experimental box.

4. The experimental kit as described in claim 1, characterized in that, The observation area is also provided with a toothed protrusion (7) and an annular structure (4). The toothed protrusion (7) is composed of multiple alternating high and low areas, wherein the high area is at the same height as the bearing recess (2), and the low area is at a lower height than the bearing recess (2); the annular structure is at the same height as the bearing recess (2).

5. The experimental kit as described in claim 1, characterized in that, The negative pressure isolation live microscope experimental box has a cover (9) on the front, and the cover is sealed to the experimental box.

6. The experimental kit as described in claim 1, characterized in that, The negative pressure isolation in vivo microscope experimental box can also be equipped with an air filtration device, which filters the gas entering and exiting the experimental box.

7. A method for in vivo microscopic imaging of animals for non-diagnostic or therapeutic purposes, characterized in that, The method includes the following steps: (1) Preparation of the viewing window for a negative pressure in vivo microscope: Place the coverslip coated with sealant on the slide support recess (2) and toothed protrusion (7) to form a sealed microscope window cavity. Open the lid (9) on the front of the negative pressure isolation live microscope experimental box and put in the test animal whose target tissue has been surgically exposed. Close the lid (9) to seal the experimental box. The airflow control system is activated, and air is drawn from the microscope window cavity through the air passage (6) to form a negative pressure environment in which the pressure inside the microscope window cavity is lower than the external pressure, thereby adsorbing and fixing the target tissue in the fixation cavity (5). (2) Image acquisition: The negative pressure in vivo microscope window is placed under the in vivo microscope, and a vascular marker dye is injected through the tail vein. After the dye circulates, a stable field of view is determined under the microscope, and image data is acquired.

8. The method as described in claim 7, characterized in that, The movement of the animal tissue on the coverslip is controlled to be ≤10μm / h in the XY axis direction and ≤10μm / h in the Z axis direction.

9. The method as described in claim 7, characterized in that, The method further includes: controlling the airflow within the negative pressure isolation in vivo microscope experimental box, wherein the airflow control is achieved through an airflow control system, and the airflow control method includes the following steps: In the airflow control system, external gas enters the experimental chamber through the inlet (1-1), and the gas inside the experimental chamber is discharged through the exhaust (1-2), creating a unidirectional airflow from front to back within the chamber; or External gas enters the experimental box through the inlet (1-2), and the gas inside the experimental box is discharged through the exhaust (1-1), creating a unidirectional airflow from front to back inside the box.

10. The method as described in claim 9, characterized in that, The air filtration device is a system of filter membrane superimposed with activated carbon adsorption column.

Images