An optical in vivo imaging device and method suitable for animal spleen

Abstract

This application discloses an optical in vivo imaging device and method for animal spleen, belonging to the field of biomedical devices and instruments. The imaging device includes a first imaging block and a second imaging block; the first imaging block is provided with a first notch recessed inward from its end face, and the top view projection of the first notch includes a first arc; the second imaging block is provided with a second notch recessed inward from its end face, and the top view projection of the second notch includes a first straight line, a second arc, and a second straight line connected in sequence; the first imaging block is fitted and snapped into the second imaging block so that the first and second notches form a closed imaging window with an adjustable imaging area. This application has a large effective imaging area, enabling full-field, highly stable imaging of the spleen, and 100% observation of all microstructures of the spleen. It can image the marginal areas and white pulp areas located in deeper positions, and can achieve cross-regional tracking observation.

Description

Technical Field

[0001] This application relates to the field of biomedical devices and apparatus, and in particular to an optical in vivo imaging device and method suitable for animal spleen. Background Technology

[0002] The spleen is the largest secondary lymphoid organ in the body, storing approximately one-third of the body's circulating lymphocytes. Therefore, in addition to its functions in hematopoiesis and clearing senescent red blood cells, the spleen also possesses strong immune functions. The spleen is divided into the red pulp and white pulp regions according to function and structure; the area between these two regions is called the marginal zone. The spleen lacks afferent lymphatic vessels; all cells and antigens enter the spleen via the bloodstream, where they are captured and cleared by immune cells in the red pulp region, along with senescent, infected, or dysfunctional red blood cells. The white pulp region of the spleen is a crucial area for initiating adaptive immunity against systemic antigens, containing both T-cell and B-cell regions. The T-cell region mainly contains a high density of T cells, which can interact with antigen-presenting cells to generate cellular immunity. The B-cell region is composed of lymphoid follicles, which, upon antigen stimulation, form germinal centers, generating plasma cells and memory cells, thus producing humoral immunity. Therefore, research on the spleen is of great significance in areas such as vaccine development and the treatment of immune-related diseases.

[0003] The dynamic interactions of various immune cells within the spleen are crucial for antigen presentation, lymphocyte migration, and subsequent immune responses. Visualizing the temporal and spatial dynamics of spleen immune cells is beneficial for vaccine development and the treatment of various immune-related diseases. However, current methods for studying the spleen primarily involve in vitro frozen sections and shaken sections. These in vitro methods cannot fully recreate the dynamic transport processes within the spleen. Therefore, to overcome this limitation, in vivo optical imaging of the spleen in experimental animals is necessary.

[0004] However, the spleen is encased in a dense fibrous capsule composed of collagen, elastic fibers, and smooth muscle, giving it strong resistance to penetration. Furthermore, the white pulp region of the spleen is relatively far from the capsule, mostly located 100μm to 500μm below it. Each spleen has only 1-2 white pulp regions close enough to the capsule for imaging. Existing optical imaging devices for the spleen are circular, resulting in a small effective imaging area. They can only image a localized area of ​​the spleen and cannot perform cross-regional tracking observations, especially struggling to image the deeper peripheral and white pulp regions. Additionally, existing optical imaging devices for the spleen often use glue to fix the spleen to the window, which can easily induce inflammatory responses in mice. Summary of the Invention

[0005] This application provides an optical in vivo imaging device and method suitable for animal spleens, which can solve the problems of existing spleen optical imaging devices having a circular structure, small effective imaging area, and only being able to image a local part of the spleen, unable to achieve cross-regional tracking and observation, especially difficult to image the edge area and white pulp area in deeper locations, and easily causing inflammation in mice.

[0006] To achieve the above objectives, the technical solution of this invention is as follows:

[0007] In a first aspect, embodiments of the present invention provide an optical in vivo imaging device suitable for animal spleen, comprising a first imaging block and a second imaging block;

[0008] The first imaging block is provided with a first notch that is recessed inward from the end face, and the top view projection of the first notch includes a first arc.

[0009] The second imaging block is provided with a second notch that is recessed inward from the end face. The top view projection of the second notch includes a first straight line, a second arc, and a second straight line connected in sequence.

[0010] The first imaging block is engaged with the second imaging block so that the first notch and the second notch form a closed imaging window with an adjustable imaging area.

[0011] In conjunction with the first aspect, in one possible implementation, a first card bar is provided on each of the two sides of the first imaging block;

[0012] The first straight line and the second straight line of the second imaging block are respectively provided with a first card slot that is adapted to the first card strip.

[0013] In conjunction with the first aspect, in one possible implementation, the first card strip and the first card slot are fitted with a gap.

[0014] In conjunction with the first aspect, in one possible implementation, the lower sidewalls of both the first and second notches are turned outwards in a direction away from the closed imaging window.

[0015] In conjunction with the first aspect, in one possible implementation, the optical in vivo imaging device for animal spleen also includes at least one fixture;

[0016] The second imaging block has an outer edge stage on its side;

[0017] Each of the aforementioned fasteners includes a fixing post and a fixing strip;

[0018] The fixing post is configured to be fixed to the outer edge platform;

[0019] One end of the fixing strip is fixed to the side wall of the fixing column, and the fixing strip gradually transitions from an upward convex shape to a downward concave shape from the fixed end to the free end.

[0020] In conjunction with the first aspect, in one possible implementation, when there are multiple fasteners, the widths of the multiple fasteners may be the same or different.

[0021] In conjunction with the first aspect, in one possible implementation, the optical in vivo imaging device for animal spleen also includes a fixation plate;

[0022] The fixing plate is provided with a third notch;

[0023] The top view projection of the third notch is rectangular;

[0024] The second imaging block is engaged with the third notch.

[0025] In conjunction with the first aspect, in one possible implementation, a second locking strip is provided on both sides of the second imaging block;

[0026] The third recess has second slots on both sides that are adapted to the second card strip.

[0027] In conjunction with the first aspect, in one possible implementation, the first imaging block and the second imaging block are made of polyamide, and the fixture and the fixing plate are made of titanium.

[0028] Secondly, embodiments of the present invention provide an optical in vivo imaging method suitable for animal spleens, based on the aforementioned optical in vivo imaging device suitable for animal spleens, comprising:

[0029] The skin and peritoneum of the mouse spleen area were cut open to expose the spleen, and the size of the mouse spleen was measured.

[0030] The position of the first imaging block and the second imaging block is determined according to the size of the spleen of the mouse, so that the first notch of the first imaging block and the second notch of the second imaging block form a closed imaging window suitable for placing the spleen, and a glass slide or PDMS film is installed on the top surface of the closed imaging window using glue.

[0031] The skin around the mouse wound is fixed to the lower part of the closed imaging window;

[0032] Optical imaging was performed on the spleen of the mice.

[0033] One or more technical solutions provided in the embodiments of the present invention have at least the following technical effects or advantages:

[0034] The optical in vivo imaging device for animal spleen provided in this invention has a first imaging block with a first recess extending inward from its end face, the top view of which includes a first arc. A second imaging block has a second recess extending inward from its end face, the top view of which includes a first straight line, a second arc, and a second straight line connected sequentially. The first imaging block is fitted into the second imaging block, so that the first and second recesses form a closed imaging window with an adjustable imaging area. The shape of the closed imaging window is well-fitted to the shape of a mouse spleen. The mouse spleen is placed within the closed imaging window, and the lower part of the imaging device is sutured to the skin around the mouse wound. A glass slide or PDMS film is glued to the top surface of the closed imaging window before imaging. This results in a large effective imaging area, enabling full-field, highly stable imaging of the spleen, and allowing for 100% observation of all spleen microstructures. It can image the deeper marginal and white pulp regions and achieve real-time dynamic tracking observation across different areas. Furthermore, because the imaging area of ​​the closed imaging window is adjustable, this imaging device is universally applicable, meeting the requirements for optical in vivo imaging of mice at different developmental stages and of different strains. In addition, by securing the spleen through the closed imaging window, and suturing the lower part of the imaging device to the skin around the mouse's wound, the spleen can be fixed to the closed imaging window without the need for glue, thus preventing inflammatory reactions in the mouse. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The stereoscopic optical in vivo imaging device for animal spleen provided in the embodiments of this application Figure 1 ;

[0037] Figure 2 The stereoscopic optical in vivo imaging device for animal spleen provided in the embodiments of this application Figure 2 ;

[0038] Figure 3 The stereoscopic optical in vivo imaging device for animal spleen provided in the embodiments of this application Figure 3 ;

[0039] Figure 4 A perspective view of the first imaging block provided in an embodiment of this application;

[0040] Figure 5 This is a front view of the first imaging block provided in an embodiment of this application;

[0041] Figure 6 Right view of the first imaging block provided in an embodiment of this application;

[0042] Figure 7 A top view of the first imaging block provided in an embodiment of this application;

[0043] Figure 8 A perspective view of the second imaging block provided in the embodiments of this application;

[0044] Figure 9 This is a front view of the second imaging block provided in an embodiment of this application;

[0045] Figure 10 This is a right view of the second imaging block provided in an embodiment of this application;

[0046] Figure 11 This is a top view of the second imaging block provided in an embodiment of this application;

[0047] Figure 12 A perspective view of the fastener provided in the embodiments of this application;

[0048] Figure 13 A perspective view of the fixing plate provided in the embodiments of this application;

[0049] Figure 14 Image A shows a fluorescently stained frozen section of a mouse spleen with B-cell fluorescence; Image B shows a frequency distribution histogram of the distance from the edge of the white pulp of the spleen to the capsule at the imaging site.

[0050] Figure 15 An optical section image of a C57 mouse spleen obtained by fluorescence confocal imaging using an optical in vivo imaging device suitable for animal spleens according to embodiments of this application;

[0051] Figure 16 The image shows a cell tracking image of a mouse spleen with B-cell fluorescence obtained by fluorescence confocal imaging using an optical in vivo imaging device suitable for animal spleen according to an embodiment of this application.

[0052] Figure 17 Long-term images of splenic vessels in C57 mice obtained by fluorescence confocal imaging using an optical in vivo imaging device suitable for animal spleens according to embodiments of this application.

[0053] Icons: 1-First imaging block; 11-First notch; 12-First locking strip; 2-Second imaging block; 21-Second notch; 22-First slot; 23-Outer edge platform; 3-Fixing component; 31-Fixing post; 32-Fixing strip; 4-Screw; 5-Fixing plate; 51-Third notch; 52-Second slot. Detailed Implementation

[0054] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] In the description of the embodiments of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the embodiments of the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. The terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Furthermore, the terms "installed," "connected," and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of the present invention according to the specific circumstances.

[0056] Please refer to Figures 1-3 As shown, this embodiment of the invention provides an optical in vivo imaging device suitable for animal spleen, including a first imaging block 1 and a second imaging block 2.

[0057] like Figures 4-7 As shown, the first imaging block 1 is provided with a first notch 11 recessed inward from its end face, and the top view projection of the first notch 11 includes a first arc. For example, the first arc provided in this embodiment is a semicircle. Wherein, the top view... Figure 1 and Figure 2 The direction shown is from top to bottom.

[0058] like Figures 8-11 As shown, the second imaging block 2 is provided with a second notch 21 that is recessed inward from its end face. The top view projection of the second notch 21 includes a first straight line, a second arc, and a second straight line connected in sequence. For example, the second arc provided in this embodiment is a semicircle.

[0059] The first imaging block 1 is engaged with the second imaging block 2, so that the first notch 11 and the second notch 21 form a closed imaging window with an adjustable imaging area. This closed imaging window is as follows: Figure 1The placement cavity formed between the first notch 11 and the second notch 21 shown. The width between the outer walls of the first imaging block 1 is adapted to the width of the inner wall of the second notch 21 of the second imaging block 2.

[0060] The optical in vivo imaging device for animal spleen provided in this embodiment of the invention features a first imaging block 1 with a first recess 11 extending inward from its end face. The top view projection of the first recess 11 includes a first arc. A second imaging block 2 has a second recess 21 extending inward from its end face. The top view projection of the second recess 21 includes a first straight line, a second arc, and a second straight line connected sequentially. The first imaging block 1 is fitted into the second imaging block 2, allowing the first recess 11 and the second recess 21 to form a closed imaging window with an adjustable imaging area. The shape of the closed imaging window is well-suited to the shape of a mouse spleen. The mouse spleen is placed within the closed imaging window, and the lower part of the imaging device is sutured to the skin around the mouse wound. A glass slide or PDMS film is glued to the top surface of the closed imaging window before imaging. This results in a large effective imaging area, enabling full-field, highly stable imaging of the spleen and allowing for 100% observation of all spleen microstructures. It can image the deeper marginal and white pulp regions and achieve real-time dynamic tracking observation across different areas. Furthermore, because the imaging area of ​​the closed imaging window is adjustable, this imaging device is universally applicable, meeting the requirements for optical in vivo imaging of mice at different developmental stages and of different strains. In addition, by securing the spleen through the closed imaging window, and suturing the lower part of the imaging device to the skin around the mouse's wound, the spleen can be fixed to the closed imaging window without the need for glue, thus preventing inflammatory reactions in the mouse.

[0061] like Figures 4-7 As shown, a first locking strip 12 is provided on each of the two sides of the first imaging block 1. Figure 8 and Figure 10 As shown, the surfaces of the first straight line and the second straight line of the second imaging block 2 are respectively provided with first card slots 22 that are adapted to the first card strip 12.

[0062] In practical use of the optical in vivo imaging device for animal spleen according to the embodiments of this application, the first locking strips 12 on both sides of the first imaging block 1 are slid into the first locking slots 22 of the second imaging block 2, thereby engaging the first imaging block 1 with the second imaging block 2. The overlap length of the first imaging block 1 and the second imaging block 2 can be adjusted by sliding the first locking strips 12 into different positions of the first locking slots 22, thus adjusting the imaging area of ​​the closed imaging window formed by the first notch 11 and the second notch 21. The length of the imaging window can be flexibly selected within the range of 10mm to 18mm to accommodate spleens of different sizes. After the first imaging block 1 and the second imaging block 2 are engaged, the top surface of the closed imaging window is composed of two semicircles at both ends and a rectangle in the middle, which better conforms to the shape of the spleen, increases the effective imaging area, and provides full field of view, high stability, and 100% imaging of all microstructures within the spleen.

[0063] Of course, a card slot can also be provided on each side of the first imaging block 1, and card strips adapted to the card strips can be provided on the first straight line and the second straight line of the second imaging block 2.

[0064] Furthermore, the radius of the circle containing the first arc and the radius of the circle containing the second arc can be the same or different, but the curvature is the same, thus the first notch 11 and the second notch 21 form a closed imaging window with an adjustable imaging area. Figures 1-3 As shown in the figure, this application embodiment provides a structural schematic diagram in which the radius of the circle containing the first arc and the radius of the circle containing the second arc are inconsistent.

[0065] Optionally, the first locking strip 12 and the first locking slot 22 are fitted with a clearance, thereby facilitating the quick and easy insertion of the first locking strip 12 into the first locking slot 22. During subsequent optical imaging research, a glass slide or PDMS film will be mounted on the top surface of the closed imaging window using adhesive. This ensures that even with the clearance between the first locking strip 12 and the first locking slot 22, when the first locking strip 12 is inserted into the second locking slot 52 to form a closed imaging window, the first imaging block 1 will not slip off the second imaging block 2.

[0066] Of course, the first card slot 12 and the first card groove 22 can also be interference-fitted.

[0067] like Figure 4 , Figures 6-10 The lower sidewalls of the first notch 11 and the second notch 21 are both turned outwards in the direction away from the closed imaging window, so that the inner sidewall of the closed imaging window formed by the first notch 11 and the second notch 21 fits the shape of the spleen better.

[0068] Furthermore, the outer walls of the closed imaging windows formed by the first notch 11 and the second notch 21 are both everted, facilitating subsequent suturing and fixation of the skin around the mouse wound to the imaging device, making the process convenient and quick. Specifically, in actual imaging tests, the skin and peritoneum of the mouse's spleen region are first cut open, creating a roughly elliptical window. A ring of sutures is added around the skin and peritoneum surrounding the window. The lower everted portions of the first imaging block 1 and the second imaging block 2 are placed inside the mouse's body, positioned below the ring of sutures, thus fixing the skin to the recessed outer walls of the everted portions of the first notch 11 and the second notch 21. The sutures are then tightened, thus fixing the imaging device to the skin—a purse-string suture fixation method. Fixing the imaging window to the skin around the mouse's spleen region with sutures isolates the intra-abdominal environment from the external environment, preventing infection in the experimental animals.

[0069] like Figure 1 , Figure 3 and Figure 12 As shown, the optical in vivo imaging device for animal spleen also includes at least one fixing element 3. Figure 1 As shown, an outer edge stage 23 is provided on the side of the second imaging block 2. The outer edge stage 23 is the part that extends outward from the side of the second imaging block 2.

[0070] Each fastener 3 includes a fixing post 31 and a fixing strip 32, which are integrally formed. The fixing post 31 is configured to be fixed to the outer edge platform 23. Specifically, the outer edge platform 23 is provided with a plurality of first internal threaded holes (e.g., Figure 1 and Figure 2 The diagram shows three first internal threaded holes on the outer edge stage 23 and a second internal threaded hole on the fixing post 31. The fixing post 31 is fixed to the outer edge stage 23 by using an M1.6 screw 4, the first internal threaded holes, and the second internal threaded holes, thereby fixing the fixing member 3 to the second imaging block 2. The number of fixing strips 32 and their horizontal position can be flexibly determined according to the size of the spleen. Furthermore, the vertical height of the fixing strips 32 and the distance from the top surface of the imaging window can be adjusted by controlling the number of rotations of the M1.6 screw 4, thus meeting the imaging needs of mouse spleens at different developmental stages, strains, and body sizes.

[0071] One end of the fixing strip 32 is fixed to the side wall of the fixing post 31 (e.g.) Figure 12As shown, the fixing strip 32 and the fixing post 31 are integrally formed. The fixing strip 32 gradually transitions from a convex shape to a concave shape from the fixed end to the free end, and the fixing member 3 serves to support the spleen. In actual imaging measurements, because the spleen has a long strip structure with a flat outer surface suitable for close contact with the glass slide for imaging, and a slightly convex inner surface, the spleen has a shape that is thin at both sides and slightly thicker in the middle. The fixing post 31 of the fixing member 3 is fixed to the outer edge platform 23, and the fixing strip 32 of the fixing post 31 is located below the imaging window. The concave shape at the distal end of the fixing strip 32 conforms to the physiological curvature of the inner surface of the spleen, which can increase the contact area between the fixing strip 32 and the spleen. The fixing strip 32 firmly supports the spleen, further enabling the imaging device to stabilize and support the spleen well without glue, and allowing the outer side of the spleen to be attached to the closed imaging window and the upper surface to be attached to the glass slide or PDMS film.

[0072] Furthermore, by setting the number of fixation pieces 3 and adjusting the horizontal position of multiple fixation strips 32, the spleen is supported at multiple points, allowing it to adhere to the imaging window. The fixation strips 32 transition from a convex shape to a concave shape from the fixed end to the free end, conforming to the physiological curvature of the spleen's inner surface. The concave arc shape at their distal ends matches the shape of the spleen, providing better support and increasing stability during imaging, while also avoiding the inflammatory effects of using biological adhesives to fix the spleen. The fixation pieces 3 can be freely and flexibly disassembled, replaced, and their distance from the top surface of the closed imaging window can be adjusted, allowing for full-field, highly stable imaging of the spleen in mice at different developmental stages and from different strains.

[0073] The imaging device provided in this application embodiment employs a first imaging block 1 and a second imaging block 2 that can be interlocked. Based on the spleen size of the mouse in a specific experiment, the size of the closed imaging window is adjusted by changing the overlap length of the first imaging block 1 and the second imaging block 2, allowing the length of the imaging window to be flexibly adjusted within the range of 10mm to 18mm. A slide or PDMS film is placed on the top surface of the imaging window using bio-adhesive. The spleen is then lifted, with a fixing strip 32 positioned between the mouse and the spleen. Furthermore, the height, number, and horizontal position of the fixing strips 32 of the fixing device 3 can also be adjusted to support the spleen, ensuring that the outer surface of the spleen is flush with the top surface of the closed imaging window and close to the slide or PDMS film. This allows for imaging of spleens of different sizes, making it suitable for mice of different strains and developmental stages.

[0074] Furthermore, when there are multiple fasteners 3, the widths of the multiple fasteners 3 may be the same or different. This allows for the flexible use of fasteners 3 of different widths according to imaging needs.

[0075] like Figure 3 and Figure 13As shown, the optical in vivo imaging device for animal spleen also includes a fixing plate 5. The fixing plate 5 can be rectangular as shown. The fixing plate 5 has a third recess 51. The top view projection of the third recess 51 is rectangular. The second imaging block 2 is fitted and snapped into the third recess 51. Second locking strips are provided on both sides of the second imaging block 2 (of course, the two sides of the second imaging block 2 can also serve as second locking strips). Figure 13 As shown, the two sides of the third notch 51 are provided with second slots 52 that are adapted to the second card strip.

[0076] In actual use, the second locking strips on both sides of the second imaging block 2 are slid into the second locking slot 52 of the fixing plate 5, so that the second imaging block 2 can be engaged with the third notch 51, thereby fixing the second imaging block 2 to the fixing plate 5. The fixing plate 5 is then fixed to the imaging stage, which facilitates the imaging of the closed imaging window formed by the first imaging block 1 and the second imaging block 2 under different types of confocal optical microscopes.

[0077] Furthermore, the bottom of the third notch 51 is also provided with a third slot that connects to the second slot 52. When the second imaging block 2 is engaged with the third notch 51, the side of the second imaging block 2 that is away from the first imaging block 1 can be engaged in the third slot, thereby making the second imaging block 2 more securely fixed to the fixing plate 5.

[0078] Optionally, the first imaging block 1 and the second imaging block 2 are made of polyamide, and the fixing member 3 and the fixing plate 5 are made of titanium. Both polyamide and titanium are biocompatible materials. When the fixing member 3 is fixed to the second imaging block 2 by the M1.6 screw 4, the M1.6 screw 4 is also made of titanium alloy.

[0079] In practice, mice weigh only about 20g, which is relatively light. If all components were made of titanium, the imaging device would be too heavy relative to the mouse's weight, making it inconvenient for the mouse to carry and impacting its subsequent life. Polyamide, on the other hand, is lightweight. Making the first imaging block 1 and the second imaging block 2 from polyamide reduces the overall weight of the imaging device. The fixture 3 has a small structure and is made of titanium, which can be manufactured using 3D printing technology to achieve the required precision. The fixture plate 5, also made of titanium, improves its load-bearing capacity and strength. The M1.6 screw 4 comes into contact with the mouse's abdominal cavity during use; therefore, it is also made of biocompatible titanium alloy, minimizing damage to the mouse. Furthermore, titanium alloy M1.6 screws are readily available.

[0080] An example is given illustrating the dimensions of an optical in vivo imaging device for animal spleen according to an embodiment of this application.

[0081] The first imaging block 1 has an overall width of 9.6 mm, a length of 5 mm, and a height of 2.9 mm. The top surface is 1.4 mm thick. The upper part of the first notch 11 is a semicircle with a radius of 3 mm. The first locking strip 12 has a height of 0.5 mm and a width of 0.8 mm. The side wall of the first imaging block 1 has a height of 1.5 mm, and the outer diameter of the bottom surface is 9.3 mm.

[0082] The second imaging block 2 is 17mm wide, 15mm long, and 2.9mm high. Its top surface is 1.4mm thick. The upper part of the second recess 21 consists of a rectangle 15mm long and 8mm wide, and a semicircle with a radius of 4mm. Each side wall of the rectangle has a first slot 22, 8mm long, 0.8mm wide, and 0.7mm high, for engaging with the first locking strip 12 of the first imaging block 1. One side wall of the second imaging block 2 is 4mm wide and has three first internal threaded holes that engage with M1.6 screws 4, with a 4mm distance between adjacent holes. The other side wall of the second imaging block 2 is 2mm wide. The bottom surface of the inner side wall of the second imaging block 2 is 9.5mm wide, fitting snugly against the first imaging block 1. After the first imaging block 1 and the second imaging block 2 are fitted together, the length of the closed imaging window can be freely selected within the range of 10mm to 18mm.

[0083] The outer diameter of the fixing block of the fixing member 3 is 3mm, the second internal thread hole matches the M1.6 screw 4, the fixing strip 32 is 6mm long, 1.5mm or 2.5mm wide, and 1mm thick, and the distance between the concave shape at the far end and the fixing post 31 is 4mm.

[0084] The M1.6 screw has an overall length of 5mm and a thread length of 4mm.

[0085] The fixing plate 5 is 55mm long, 48mm wide, and 2.6mm high. The second slots 52 on both sides of the third notch 51 are 1.6mm high and are used to fix the second imaging block 2 to facilitate imaging under a confocal microscope.

[0086] Another embodiment of the present invention provides an optical in vivo imaging method suitable for animal spleen, based on the above-described optical in vivo imaging device suitable for animal spleen, including the following steps.

[0087] The skin and peritoneum of the mouse spleen region were cut open to expose the spleen, and the size of the mouse spleen was measured. Specifically, ophthalmic scissors were used to cut open the skin and peritoneum of the mouse spleen region to expose the spleen, and the size of the mouse spleen, including length, width, and thickness, was measured using vernier calipers.

[0088] The position of the first imaging block 1 and the second imaging block 2 is determined according to the size of the mouse spleen (i.e., the length of the closed imaging window is determined), so that the first notch 11 of the first imaging block 1 and the second notch 21 of the second imaging block 2 form a closed imaging window suitable for placing the spleen, and a glass slide or PDMS film is installed on the top surface of the closed imaging window using glue.

[0089] When the spleen is supported by the fixation member 3, the steps also include: determining the width, number and horizontal position of the fixing strip 32 of the fixation member 3 according to the size of the mouse's spleen, and connecting the two through the second internal thread hole of the fixing post 31 of the fixation member 3 and the first internal thread hole of the second imaging block 2 by means of M1.6 screw 4.

[0090] The spleen of the mouse is lifted and placed above the fixation bar 32. The height of the fixation bar 32 is adjusted so that the outer surface of the spleen is in close contact with the glass slide or PDMS membrane.

[0091] The skin around the mouse wound was fixed to the lower part of the closed imaging window. Specifically, the skin around the wound was fixed to the lower outer wall of the closed imaging window using a purse-string suture technique to create a closed environment.

[0092] Optical imaging studies were conducted on the spleen of mice. Specifically, optical imaging studies of the mouse spleen were carried out using confocal scanning imaging, two-photon imaging, and other techniques. The research content included, but was not limited to: dynamically analyzing the heterogeneous transport process of polymeric carriers with different properties in the spleen and the dynamic interactions of various cells in the spleen under normal or infected conditions.

[0093] Figure 14 Figure A shows a fluorescently stained frozen section of a mouse spleen with B-cell fluorescence imaging; the white arrow indicates the outer surface of the spleen, i.e., the imaging plane. Figure B is a histogram showing the frequency distribution of the distance from the edge of the white pulp of a single spleen to the capsule at the imaging site. As can be seen from the figures, the white pulp of the spleen is mostly distributed at a depth of 100μm-500μm from the capsule. Within a single spleen, very little white pulp below 100μm is suitable for fluorescence confocal microscopy imaging, making it difficult to observe the white pulp region using existing optical imaging devices for the spleen. However, the in vivo optical imaging device for animal spleens provided in this invention increases the effective imaging area, allowing imaging of the entire spleen and ensuring that suitable white pulp for imaging can be found. This minimizes the number of mice used in the experiment, fulfilling the "3R principle" of animal experiments.

[0094] Figure 15 Optical slice images of the spleen of C57 mice obtained by fluorescence confocal imaging using an optical in vivo imaging device suitable for animal spleens according to embodiments of this application. Figure 15The left side shows a panoramic view of the spleen, with the white pulp region contained within the white box. The right side shows a magnified image of a specific area of ​​the panoramic view, with the white pulp region within the yellow dotted line. As can be seen from the figure, by using the imaging device of this embodiment, a good and complete fluorescence image of the spleen can be obtained, and all the microstructures of the spleen, as well as the immune cells and white pulp region within the spleen, can be clearly observed.

[0095] Figure 16 Cell tracking images of mouse spleens obtained by fluorescence confocal imaging using an optical in vivo imaging device suitable for animal spleens according to embodiments of this application. Figure 16 Time-lapse imaging of spleen B cells after intravenous injection of nanoparticles clearly tracks the migration trajectory of B cells within the spleen, and the window remains highly stable throughout the imaging process. White arrows indicate B cells in motion.

[0096] Figure 17 Long-term images of splenic vessels in C57 mice obtained by fluorescence confocal imaging using an optical in vivo imaging device suitable for animal spleens according to embodiments of this application. Figure 17 Imaging of blood vessels within the spleen revealed that the window remained highly stable even after intravenous injection of nanoparticles and during subsequent imaging processes.

[0097] The imaging devices and surgical instruments used in all the above steps were disinfected with alcohol or sterilized by ultraviolet irradiation, and the experimental mice were anesthetized with abdominal anesthesia or gas anesthesia during the surgery and imaging process.

[0098] In summary, the imaging device of this application embodiment, through the design of a matching first imaging block 1 and a second imaging block 2 to adjust the length of the closed imaging window within the range of 10mm to 18mm, and the height-adjustable fixing member 3, enables the imaging device to perform full-field, highly stable imaging of the spleen, and allows for 100% observation of all microstructures of the spleen. Furthermore, the imaging device is universally applicable, meeting the imaging needs of mice of different strains and developmental stages. The multi-point support provided by multiple fixing strips 32 avoids the use of biological glue to fix the spleen, greatly reducing inflammation during the imaging process.

[0099] The various embodiments in this specification are described in a progressive manner. For the same or similar parts between the various embodiments, please refer to each other. Each embodiment focuses on describing the differences from other embodiments.

[0100] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. An optical in vivo imaging device suitable for animal spleen, characterized in that, It includes a first imaging block, a second imaging block, and at least one fixing element; The first imaging block is provided with a first notch that is recessed inward from the end face, and the top view projection of the first notch includes a first arc. The second imaging block is provided with a second notch that is recessed inward from the end face. The top view projection of the second notch includes a first straight line, a second arc, and a second straight line connected in sequence. The first imaging block is engaged with the second imaging block so that the first notch and the second notch form a closed imaging window with an adjustable imaging area; The second imaging block has an outer edge stage on its side; Each of the aforementioned fasteners includes a fixing post and a fixing strip; The fixing post is configured to be fixed to the outer edge platform; One end of the fixing strip is fixed to the side wall of the fixing column, and the fixing strip gradually transitions from an upward convex shape to a downward concave shape from the fixed end to the free end.

2. The optical in vivo imaging device for animal spleen according to claim 1, characterized in that, A first locking bar is provided on each of the two sides of the first imaging block; The first straight line and the second straight line of the second imaging block are respectively provided with a first card slot that is adapted to the first card strip.

3. The optical in vivo imaging device for animal spleen according to claim 2, characterized in that, The first card strip and the first card slot are fitted with a clearance.

4. The optical in vivo imaging device for animal spleen according to claim 1, characterized in that, The lower sidewalls of both the first and second notches are turned outwards in a direction away from the closed imaging window.

5. The optical in vivo imaging device for animal spleen according to claim 1, characterized in that, When there are multiple fasteners, the widths of the multiple fasteners may be the same or different.

6. The optical in vivo imaging device for animal spleen according to claim 1, characterized in that, It also includes a fixing plate; The fixing plate is provided with a third notch; The top view projection of the third notch is rectangular; The second imaging block is engaged with the third notch.

7. The optical in vivo imaging device for animal spleen according to claim 6, characterized in that, The second imaging block has a second locking strip on both sides; The third recess has second slots on both sides that are adapted to the second card strip.

8. The optical in vivo imaging device for animal spleen according to claim 6, characterized in that, The first imaging block and the second imaging block are made of polyamide, and the fixing member and the fixing plate are made of titanium.

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