Optical living body imaging device and method suitable for animal spleen

Abstract

The invention discloses an optical living body imaging device and method suitable for animal spleen, and belongs to the technical field of biomedical apparatus and instruments. The imaging device comprises a first imaging block and a second imaging block; the first imaging block body is provided with a first notch which is recessed inwards from the end face, and the overlook projection of the first notch comprises a first arc line; the second imaging block body is provided with a second notch which is recessed inwards from the end face. The overlook projection of the second notch comprises a first straight line, a second arc line and a second straight line which are connected in sequence. The first imaging block is clamped to the second imaging block in a matched mode so that the first notch and the second notch can form a closed imaging window with the adjustable imaging area. The effective imaging area is large, full-view and high-stability imaging can be carried out on the spleen, all microstructures of the spleen can be observed 100%, imaging can be carried out on an edge region and a white marrow region at a deep position, and cross-region tracking observation can be achieved.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical devices and instruments, and in particular to an optical in vivo imaging device and method suitable for animal spleen. Background Art

[0002] The spleen is the largest secondary lymphoid organ in the body, storing about one-third of the body's circulating lymphocytes. Therefore, in addition to its role in hematopoiesis and removal of aging red blood cells, the spleen also has a strong immune function. The spleen is divided into the red pulp area and the white pulp area according to its function and structure, and the area between these two areas is called the marginal zone. The spleen lacks afferent lymphatic vessels, and all cells and antigens enter the spleen through the blood. Foreign pathogens and aging, infected or dysfunctional red blood cells are captured and removed by immune cells in the red pulp area of ​​the spleen. The white pulp area of ​​the spleen is an important area for initiating adaptive immunity against systemic antigens, and contains T cell areas and B cell areas. The T cell area mainly gathers high-density T cells, which can interact with antigen-presenting cells to produce cellular immunity. The B cell area is composed of lymphoid follicles, which form germinal centers after being stimulated by antigens, generate plasma cells and memory cells, and produce humoral immunity. Therefore, research on the spleen is of great significance in the fields of vaccine development and treatment of immune-related diseases.

[0003] The dynamic interactions of various immune cells in the spleen are related to antigen presentation, lymphocyte migration, and subsequent immune responses. Visualization of the temporal and spatial dynamics of splenic immune cells is conducive to deepening the development of vaccines and the treatment of various immune-related diseases. However, the current methods for studying the spleen are mainly in vitro frozen sections, shaking sections, etc. These in vitro methods cannot completely restore the dynamic transport process in the spleen. Therefore, in order to make up for this deficiency, it is necessary to perform optical imaging of the spleen of experimental animals at the in vivo level.

[0004] However, the spleen is wrapped in a dense fibrous capsule composed of collagen, elastic fibers and smooth muscles, which has strong anti-penetration ability. In addition, the white pulp area of ​​the spleen is far away from the capsule, mostly distributed 100μm to 500μm below the capsule. Each spleen has only 1 to 2 white pulps that are close enough to the capsule and suitable for imaging. The existing spleen optical imaging device has a circular structure with a small effective imaging area. It can only image a part of the spleen and cannot perform cross-region tracking observation, especially for the marginal area and white pulp area in a deeper position. In addition, the existing spleen optical imaging device often uses glue to fix the spleen on the window, which is easy to cause inflammatory response in mice. Summary of the invention

[0005] The embodiments of the present application provide an optical in vivo imaging device and method suitable for animal spleens, which can solve the problems that the existing spleen optical imaging devices have a circular structure, a small effective imaging area, can only image a part of the spleen, and cannot perform cross-region tracking observation, especially the marginal area and white pulp area in a deeper position are difficult to image, and are prone to cause inflammation in mice.

[0006] In order to achieve the above object, the technical solution of the embodiment of the present invention is:

[0007] In a first aspect, an embodiment of the present invention provides 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 recess which is recessed inwardly from the end surface, and the top projection of the first recess includes a first arc line;

[0009] The second imaging block is provided with a second notch which is indented inwardly from the end surface, and the top projection of the second notch includes a first straight line, a second arc line and a second straight line which are connected in sequence;

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

[0011] In combination with the first aspect, in a possible implementation manner, a first clamping strip is respectively provided on both sides of the first imaging block;

[0012] The first straight line of the second imaging block and the surface where the second straight line is located are respectively provided with a first clamping groove matched with the first clamping strip.

[0013] In combination with the first aspect, in a possible implementation manner, the first card strip and the first card slot are gap-matched.

[0014] In combination with the first aspect, in a possible implementation manner, side walls at the lower portions of the first recess and the second recess are both turned outward in a direction away from the closed imaging window.

[0015] In combination with the first aspect, in a possible implementation, the optical in vivo imaging device for animal spleen further includes at least one fixing member;

[0016] The side surface of the second imaging block is provided with an outer edge platform;

[0017] Each of the fixing members comprises a fixing column and a fixing bar;

[0018] The fixing column 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 in a direction from the fixing end to the free end.

[0020] In combination with the first aspect, in a possible implementation manner, when there are multiple fixing members, widths of the multiple fixing members are consistent or inconsistent.

[0021] In combination with the first aspect, in a possible implementation, the optical in vivo imaging device for animal spleen further includes a fixing plate;

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

[0023] The top projection of the third recess is a rectangle;

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

[0025] In combination with the first aspect, in a possible implementation manner, second clip strips are provided on both sides of the second imaging block;

[0026] Second card slots matching the second card strip are arranged on both side surfaces of the third recess.

[0027] In combination with the first aspect, in a possible implementation manner, 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 metal.

[0028] In a second aspect, an embodiment of the present invention provides an optical in vivo imaging method applicable to an animal spleen, based on the above-mentioned optical in vivo imaging device applicable to an animal spleen, comprising:

[0029] Cut open the skin and peritoneum of the spleen area of ​​the mouse to expose the spleen, and measure the size of the spleen of the mouse;

[0030] Determine the position where the first imaging block is connected to the second imaging block according to the size of the mouse spleen, 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 use glue to install a glass slide or a PDMS film on the top surface of the closed imaging window;

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

[0032] The spleens of the mice were subjected to optical imaging studies.

[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 by the embodiment of the present invention is characterized in that the first imaging block is provided with a first notch recessed inward from the end surface, and the top projection of the first notch includes a first arc. The second imaging block is provided with a second notch recessed inward from the end surface, and the top 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 coupled and snapped to the second imaging block so that the first notch and the second notch form a closed imaging window with an adjustable imaging area. The shape of the closed imaging window is well adapted to the shape of the mouse spleen, the mouse spleen is placed in the closed imaging window, the lower part of the imaging device is sutured and fixed to the skin around the mouse wound, a glass slide or a PDMS film is installed on the top surface of the closed imaging window with glue, and then imaging is performed. The effective imaging area is large, the spleen can be imaged in full field and with high stability, and all microstructures of the spleen can be observed 100%, the marginal area and the white pulp area in a deeper position can be imaged, and real-time dynamic tracking observation across regions can be achieved. And because the imaging area of ​​the closed imaging window is adjustable, the imaging device is universal and can meet the needs of optical in vivo imaging of mice of different developmental stages and strains. In addition, the spleen is fixed to the closed imaging window, and the lower part of the imaging device is sutured and fixed to the skin around the mouse wound. The spleen can be fixed to the closed imaging window without the use of glue to prevent inflammatory reactions in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0036] Figure 1 A stereoscopic optical in vivo imaging device for animal spleen provided in an embodiment of the present application Figure 1 ;

[0037] Figure 2 A stereoscopic optical in vivo imaging device for animal spleen provided in an embodiment of the present application Figure 2 ;

[0038] Figure 3 A stereoscopic optical in vivo imaging device for animal spleen provided in an embodiment of the present application Figure 3 ;

[0039] Figure 4 A stereoscopic diagram of a first imaging block provided in an embodiment of the present application;

[0040] Figure 5 A front view of a first imaging block provided in an embodiment of the present application;

[0041] Figure 6 A right side view of a first imaging block provided in an embodiment of the present application;

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

[0043] Figure 8 A stereoscopic diagram of a second imaging block provided in an embodiment of the present application;

[0044] Fig. 9 A front view of a second imaging block provided in an embodiment of the present application;

[0045] Fig.10 A right side view of a second imaging block provided in an embodiment of the present application;

[0046] Fig.11 A top view of a second imaging block provided in an embodiment of the present application;

[0047] Fig.12 A three-dimensional diagram of a fixing member provided in an embodiment of the present application;

[0048] Fig.13 A three-dimensional diagram of a fixing plate provided in an embodiment of the present application;

[0049] Fig.14 Figure A is a fluorescent staining image of the spleen of a B cell fluorescent mouse through frozen section; Figure B is a frequency distribution histogram of the distance between the edge of the spleen white pulp and the capsule at the imaging point.

[0050] Fig.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 spleen according to an embodiment of the present application;

[0051] Fig.16 This is a cell tracking image of a B cell fluorescent mouse spleen obtained by fluorescence confocal imaging using an optical in vivo imaging device suitable for animal spleen according to an embodiment of the present application;

[0052] Fig.17 The long-term image of the spleen blood vessels of a C57 mouse was obtained by fluorescence confocal imaging using the optical in vivo imaging device suitable for the spleen of an animal according to an embodiment of the present application.

[0053] Icons: 1-first imaging block; 11-first recess; 12-first card strip; 2-second imaging block; 21-second recess; 22-first card slot; 23-outer edge platform; 3-fixing piece; 31-fixing column; 32-fixing strip; 4-screw; 5-fixing plate; 51-third recess; 52-second card slot. DETAILED DESCRIPTION

[0054] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, rather than indicating or implying that the devices or elements referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limitations on the present invention. The terms "first", "second", and "third" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance. In addition, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be a connection between the two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present invention can be understood according to the specific circumstances.

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

[0057] like Figures 4 to 7 As shown, the first imaging block 1 is provided with a first recess 11 which is recessed inward from the end surface, and the top projection of the first recess 11 includes a first arc. For example, the first arc provided in the embodiment of the present application is a semicircle. Figure 1 and Figure 2 Shown in top-down direction.

[0058] like Figures 8 to 11 As shown, the second imaging block 2 is provided with a second notch 21 inwardly recessed from the end surface, and the top 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 the embodiment of the present application is a semicircle.

[0059] The first imaging block 1 is coupled to 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. Figure 1The placement cavity formed between the first recess 11 and the second recess 21 shown in the figure has a width between the outer side walls of the first imaging block 1 that matches the width of the inner side walls of the second recess 21 of the second imaging block 2 .

[0060] The optical in vivo imaging device for animal spleen provided by the embodiment of the present invention has a first notch 11 recessed inward from the end surface, and the top projection of the first notch 11 includes a first arc. The second imaging block 2 is provided with a second notch 21 recessed inward from the end surface, and the top projection of the second notch 21 includes a first straight line, a second arc, and a second straight line connected in sequence. The first imaging block 1 is coupled and snapped to the second imaging block 2, so that the first notch 11 and the second notch 21 form a closed imaging window with adjustable imaging area. The shape of the closed imaging window is well adapted to the shape of the mouse spleen, the mouse spleen is placed in the closed imaging window, the lower part of the imaging device is sutured and fixed to the skin around the mouse wound, a glass slide or a PDMS film is installed on the top surface of the closed imaging window with glue, and then imaging is performed. The effective imaging area is large, the spleen can be imaged in full field and with high stability, and all microstructures of the spleen can be observed 100%, the marginal area and the white pulp area in a deeper position can be imaged, and real-time dynamic tracking observation across regions can be achieved. And because the imaging area of ​​the closed imaging window is adjustable, the imaging device is universal and can meet the needs of optical in vivo imaging of mice of different developmental stages and strains. In addition, the spleen is fixed to the closed imaging window, and the lower part of the imaging device is sutured and fixed to the skin around the mouse wound. The spleen can be fixed to the closed imaging window without the use of glue to prevent inflammatory reactions in mice.

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

[0062] When the optical in vivo imaging device for animal spleen of the embodiment of the present application is actually used, the first clamping strips 12 on both sides of the first imaging block 1 are slid into the first clamping slots 22 of the second imaging block 2, so that the first imaging block 1 can be clamped to the second imaging block 2. The first clamping strip 12 slides into different positions of the first clamping slot 22, and the overlapping length of the first imaging block 1 and the second imaging block 2 can be adjusted, that is, the imaging area of ​​the closed imaging window formed by the first notch 11 and the second notch 21 can be adjusted, and 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 clamped, the top surface of the closed imaging window is composed of two semicircles at the two ends and a rectangle in the middle, which is more in line with the shape of the spleen, increases the effective imaging area, and has the characteristics of full field of view, high stability, and 100% imaging of all microstructures in the spleen.

[0063] Of course, a card slot may be provided on both sides of the first imaging block 1 , and card strips matching the card strips may be provided on the surfaces where the first straight line and the second straight line of the second imaging block 2 are located.

[0064] In addition, the radius of the circle where the first arc is located may be consistent with or inconsistent with the radius of the circle where the second arc is located, but the curvature is the same, so that the first notch 11 and the second notch 21 form a closed imaging window with an adjustable imaging area. Figures 1 to 3 As shown, an embodiment of the present application provides a structural schematic diagram in which the radius of the circle where the first arc is located is inconsistent with the radius of the circle where the second arc is located.

[0065] Optionally, the first card strip 12 and the first card slot 22 are gap-matched, so that the first card strip 12 can be quickly and easily inserted into the first card slot 22. When conducting subsequent optical imaging research, a glass slide or a PDMS film will be installed on the top surface of the closed imaging window using glue, so that even if the first card strip 12 and the first card slot 22 are gap-matched, when the first card strip 12 is inserted into the second card 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 clamping strip 12 and the first clamping slot 22 may also be interference fit.

[0067] like Figure 4 , Figures 6 to 10 The side walls of the lower parts of the first notch 11 and the second notch 21 are both turned outward in a direction away from the closed imaging window, so that the inner wall of the closed imaging window formed by the first notch 11 and the second notch 21 better fits the shape of the spleen.

[0068] In addition, the outer side walls of the closed imaging window formed by the first notch 11 and the second notch 21 are both turned outward, which is convenient for the subsequent suturing and fixing of the skin around the mouse wound to the imaging device, which is convenient and quick. Specifically, during the actual imaging test, the skin and peritoneum of the mouse spleen area are first cut open, and there is an elliptical window at the cut, and a circle of sutures are added to the skin and peritoneum around the window. The lower eversion of the first imaging block 1 and the second imaging block 2 is placed in the body of the mouse and is located at the bottom of a circle of sutures, so that the skin is fixed to the concave position of the outer side wall of the eversion of the first notch 11 and the second notch 21, and then the sutures are tightened to achieve the fixation of the imaging device and the skin, that is, the purse-string suture fixation method. The imaging window is fixed to the skin around the mouse spleen area by sutures to isolate the intra-abdominal environment from the external environment and avoid infection of experimental animals.

[0069] like Figure 1 , Figure 3 and Fig.12 As shown, the optical in vivo imaging device for animal spleen further comprises at least one fixing member 3. Figure 1 As shown, an outer edge platform 23 is provided on the side of the second imaging block 2. The outer edge platform 23 is a portion of the second imaging block 2 extending outward from the side.

[0070] Each fixing member 3 includes a fixing column 31 and a fixing bar 32, and the fixing column 31 and the fixing bar 32 are integrally formed. The fixing column 31 is configured to be fixed to the outer edge platform 23. Specifically, a plurality of first internal threaded holes (such as Figure 1 and Figure 2 It is shown that three first internal thread holes are provided on the outer platform 23, and a second internal thread hole is provided on the fixing column 31. The fixing column 31 can be fixed to the outer platform 23 by the cooperation of the M1.6 screw 4, the first internal thread hole and the second internal thread hole, so that the fixing member 3 and the second imaging block 2 are fixed. The number of fixing bars 32 and the position in the horizontal direction can be flexibly determined according to the size of the spleen. In addition, by controlling the number of turns of the M1.6 screw 4, the height of the fixing bar 32 in the vertical direction can be adjusted, and the distance between the fixing bar 32 and the top surface of the imaging window can be adjusted, so as to meet the imaging requirements of the spleen of mice of different developmental stages, different strains, and different body sizes.

[0071] One end of the fixing strip 32 is fixed to the side wall of the fixing column 31 (eg Fig.12As shown, the fixing bar 32 and the fixing column 31 are integrally formed), and the fixing bar 32 gradually transitions from a convex shape to a concave shape from the fixed end to the free end, and the fixing member 3 plays a role in supporting the spleen. When the imaging measurement is actually performed, since the spleen is a long strip structure, the outer side is flat, suitable for being close to the glass slide for imaging, and the inner side is slightly convex, so that the spleen is thin on both sides and slightly thick in the middle. The fixing column 31 of the fixing member 3 is fixed to the outer edge platform 23, and the fixing bar 32 of the fixing column 31 is located below the imaging window. The concave shape of the far end of the fixing bar 32 conforms to the physiological curvature of the inner side of the spleen, which can increase the contact area between the fixing bar 32 and the spleen. The fixing bar 32 firmly supports the spleen, and further enables the imaging device to stabilize and support the spleen well without glue, and makes the outer side of the spleen stick to the closed imaging window and the upper surface stick to the glass slide or PDMS film.

[0072] In addition, by setting the number of fixing parts 3 and adjusting the horizontal positions of multiple fixing strips 32, the spleen is supported at multiple points so that the spleen is close to the imaging window. The fixing strip 32 gradually transitions from a convex shape to a concave shape from the fixed end to the free end, which conforms to the physiological curvature of the inner side of the spleen. The concave arc shape at the distal end matches the shape of the spleen, which can better support the spleen, increase the stability during imaging, and avoid the inflammatory effects caused by using biological glue to fix the spleen. The fixing part 3 can be flexibly disassembled, replaced, and adjusted at a distance from the top surface of the closed imaging window to meet the full-view, high-stability imaging of the spleen of mice at different developmental stages and strains.

[0073] The imaging device provided in the embodiment of the present application adopts a first imaging block 1 and a second imaging block 2 that can be gap-fitted. According to the spleen size of the mouse in the specific experiment, the size of the closed imaging window is adjusted by changing the overlapping length of the first imaging block 1 and the second imaging block 2, so that the length of the imaging window can be flexibly adjusted within the range of 10 mm to 18 mm, and a glass slide or PDMS membrane is placed on the top surface of the imaging window using biological glue. The spleen is lifted up, and the fixing bar 32 is located between the mouse and the spleen. In addition, the height, number and horizontal position of the fixing bar 32 of the fixing member 3 can also be adjusted to support the spleen, so that the outer side of the spleen is flush with the top surface of the closed imaging window and close to the glass slide or PDMS membrane, so that spleens of different sizes can be imaged, which is suitable for mice of different strains and different developmental stages.

[0074] Furthermore, when there are multiple fixing members 3, the widths of the multiple fixing members 3 are consistent or inconsistent, so that fixing members 3 of different widths can be flexibly used according to imaging requirements.

[0075] like Figure 3 and Fig.13As shown, the optical in vivo imaging device suitable for animal spleen also includes a fixing plate 5. The fixing plate 5 can be a rectangle as shown in the figure. The fixing plate 5 is provided with a third recess 51. The top projection of the third recess 51 is a rectangle. The second imaging block 2 is engaged with the third recess 51. Second clip strips are provided on both sides of the second imaging block 2 (of course, the two side edges of the second imaging block 2 can be used as the second clip strips). Fig.13 As shown, the two side surfaces of the third notch 51 are provided with second clamping grooves 52 matched with the second clamping strip.

[0076] In actual use, the second imaging block 2 can be snapped into the third recess 51 by sliding the second clip strips on both sides of the second imaging block 2 into the second clip grooves 52 of the fixed plate 5, thereby fixing the second imaging block 2 to the fixed plate 5, and the fixed plate 5 is further fixed to the imaging stage, so as to facilitate imaging of the closed imaging window formed by the first imaging block 1 and the second imaging block 2 under different types of co-focus optical microscopes.

[0077] Furthermore, a third card slot connected to the second card slot 52 is also provided at the bottom of the third recess 51. When the second imaging block 2 is engaged with the third recess 51, the side of the second imaging block 2 facing away from the first imaging block 1 can be engaged with the third card slot, thereby making the second imaging block 2 and the fixing plate 5 more firmly fixed.

[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 materials with good biocompatibility. 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 parts are made of titanium, the weight of the imaging device is heavier than the weight of the mouse itself, and it is inconvenient for the mouse to carry the imaging device, which has an impact on its subsequent life. Polyamide is light in weight, and the first imaging block 1 and the second imaging block 2 are made of polyamide, which can reduce the total weight of the imaging device. The structure of the fixing part 3 is relatively small. The fixing part 3 is made of titanium and can be made using 3D printing technology to achieve the required accuracy of the fixing part 3. The fixing plate 5 is made of titanium to improve the bearing capacity and strength of the fixing plate 5. The M1.6 screw 4 will contact the mouse abdominal cavity when in use. The M1.6 screw 4 is also made of titanium alloy with good biocompatibility, which can reduce damage to the mouse, and the M1.6 screw 4 of titanium alloy is easy to purchase.

[0080] The dimensions of the optical in vivo imaging device suitable for animal spleen according to the embodiment of the present application are illustrated by way of example.

[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 portion of the first notch 11 is a semicircle with a radius of 3 mm, and the first clip 12 is 0.5 mm high and 0.8 mm wide. The side wall of the first imaging block 1 is 1.5 mm high, and the outer diameter of the bottom surface is 9.3 mm wide.

[0082] The second imaging block 2 is 17mm wide, 15mm long and 2.9mm high. The top surface is 1.4mm thick, and the upper part of the second recess 21 is composed of a rectangle with a length of 15mm and a width of 8mm and a semicircle with a radius of 4mm. The two side walls of the rectangle are provided with a first card slot 22 with a length of 8mm, a width of 0.8mm and a height of 0.7mm, which is used to cooperate with the first card strip 12 of the first imaging block 1. One side wall of the second imaging block 2 is 4mm wide, and is distributed with three first internal threaded holes that cooperate with the M1.6 screws 4. The distance between two adjacent first internal threaded holes is 4mm, and the width of the other side wall of the second imaging block 2 is 2mm. The bottom surface width of the inner side wall of the second imaging block 2 is 9.5mm, which just fits the clearance of the first imaging block 1. After the clearance of the first imaging block 1 and the second imaging block 2 is matched, 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 threaded hole matches the M1.6 screw 4, the fixing strip 32 is 6mm long, 1.5mm or 2.5mm wide, 1mm thick, and the distance between the distal concave shape and the fixing column 31 is 4mm.

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

[0085] The fixing plate 5 has an overall length of 55 mm, a width of 48 mm, and a height of 2.6 mm. The second slots 52 arranged on both sides of the third notch 51 have a height of 1.6 mm, and are used to fix the second imaging block 2 for imaging under a confocal microscope.

[0086] Another embodiment of the present invention provides an optical in vivo imaging method applicable to an animal spleen, based on the above-mentioned optical in vivo imaging device applicable to an animal spleen, comprising the following steps.

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

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

[0089] When the fixing device 3 is used to support the spleen, the steps also include: determining the width, quantity and horizontal position of the fixing bars 32 of the fixing device 3 used according to the size of the mouse spleen, and connecting the two through the second internal threaded hole of the fixing column 31 of the fixing device 3 and the first internal threaded hole of the second imaging block 2 by means of an M1.6 screw 4.

[0090] The spleen of the mouse is lifted up and placed on the fixing bar 32 , and the height of the fixing bar 32 is adjusted so that the outer side of the spleen is in close contact with the glass slide or the PDMS membrane.

[0091] The skin around the wound of the mouse is fixed to the lower part of the closed imaging window. Specifically, the skin around the wound is fixed to the outer side wall of the lower part of the closed imaging window by purse-string suture to form a closed environment.

[0092] Conduct optical imaging studies on the spleen of mice. Specifically, conduct optical imaging studies on the spleen of mice through confocal scanning imaging, two-photon imaging, etc. The research content includes but is not limited to: dynamically analyzing the heterogeneous transport process of polymer carriers with different characteristics in the spleen and the dynamic interaction of various cells in the spleen in the normal or infected state of the body.

[0093] Fig.14 Figure A is a frozen section fluorescence staining image of the spleen of a B cell fluorescent mouse, and the white arrow is the outer side of the spleen, i.e., the imaging surface; Figure B is a frequency distribution histogram of the distance between the edge of the white pulp of a single spleen and the capsule at the imaging location. As can be seen from the figure, the white pulp of the spleen is mostly distributed at a depth of 100μm-500μm from the capsule. In a single spleen, there is very little white pulp below 100μm that is suitable for fluorescence confocal microscopy imaging. It is difficult to observe the white pulp area using the existing spleen optical imaging device. The optical in vivo imaging device suitable for animal spleens provided in an embodiment of the present invention increases the effective imaging area, and can image the entire spleen, ensuring that white pulp suitable for imaging can be found, so as to minimize the number of mice used in the experiment and fulfill the "3R principle" of animal experiments.

[0094] Fig.15 This is an optical section image of a C57 mouse spleen obtained by performing fluorescence confocal imaging using the optical in vivo imaging device suitable for animal spleen according to an embodiment of the present application. Fig.15The left side is a panoramic view of the spleen, wherein the white pulp area is contained in the white box, and the right side is an enlarged image of a certain area of ​​the panoramic view, wherein the yellow dotted line is the white pulp area. As can be seen from the figure, by using the imaging device of the embodiment of the present application, a good and complete spleen fluorescence image can be obtained, and all microstructures of the spleen as well as immune cells and white pulp areas in the spleen can be clearly observed.

[0095] Fig.16 This is a cell tracking image of the spleen of a B cell fluorescent mouse obtained by performing fluorescence confocal imaging using the optical in vivo imaging device suitable for the spleen of an animal according to an embodiment of the present application. Fig.16 Time-lapse imaging of splenic B cells after intravenous injection of nanoparticles can clearly track the migration trajectory of B cells in the spleen, and the window maintains a high degree of stability during the entire imaging process. White arrows indicate B cells in motion.

[0096] Fig.17 The long-term image of the spleen blood vessels of a C57 mouse was obtained by fluorescence confocal imaging using the optical in vivo imaging device suitable for the spleen of an animal according to an embodiment of the present application. Fig.17 Imaging of blood vessels within the spleen revealed that the window remained highly stable after intravenous injection of the nanoparticles and during subsequent imaging.

[0097] The imaging devices and surgical instruments in all the above steps were sterilized with alcohol or sterilized with ultraviolet light, and the experimental mice were subjected to intraperitoneal anesthesia or gas anesthesia during the surgery and imaging process.

[0098] In summary, the imaging device of the embodiment of the present application, by designing the matching first imaging block 1 and 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-view, high-stability imaging of the spleen, and can 100% observe all microstructures of the spleen, and the imaging device is universal and meets the imaging requirements of mice of different strains and different developmental stages. The imaging device avoids the use of biological glue to fix the spleen through the multi-point support of multiple fixing bars 32, greatly reducing the inflammation phenomenon during the imaging process.

[0099] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other. Each embodiment focuses on the differences from other embodiments.

[0100] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application.

Claims

1. An optical in vivo imaging device suitable for animal spleen, characterized in that: including a first imaging block and a second imaging block; The first imaging block is provided with a first recess which is recessed inwardly from the end surface, and the top projection of the first recess includes a first arc line; The second imaging block is provided with a second notch which is indented inwardly from the end surface, and the top projection of the second notch includes a first straight line, a second arc line and a second straight line which are connected in sequence; The first imaging block is engaged with the second imaging block so that the first recess and the second recess form a closed imaging window with an adjustable imaging area.

2. The optical in vivo imaging device for animal spleen according to claim 1, characterized in that: A first clamping strip is respectively arranged on both sides of the first imaging block; The first straight line of the second imaging block and the surface where the second straight line is located are respectively provided with a first clamping groove matched with the first clamping 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 loosely matched.

4. The optical in vivo imaging device for animal spleen according to claim 1, characterized in that: The side walls of the lower portions of the first recess and the second recess are both turned outward 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: Also includes at least one fixing member; The side surface of the second imaging block is provided with an outer edge platform; Each of the fixing members comprises a fixing column and a fixing bar; The fixing column 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 in a direction from the fixing end to the free end.

6. The optical in vivo imaging device for animal spleen according to claim 5, characterized in that: When there are multiple fixing members, the widths of the multiple fixing members are consistent or inconsistent.

7. The optical in vivo imaging device for animal spleen according to claim 5, characterized in that: Also included is a fixing plate; The fixing plate is provided with a third notch; The top projection of the third recess is a rectangle; The second imaging block is engaged with the third recess.

8. The optical in vivo imaging device for animal spleen according to claim 7, characterized in that: Second clamping strips are provided on both sides of the second imaging block; Second card slots matching the second card strip are arranged on both side surfaces of the third recess.

9. The optical in vivo imaging device for animal spleen according to claim 7, 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 metal.

10. An optical in vivo imaging method for animal spleen, characterized in that: The optical in vivo imaging device for animal spleen according to any one of claims 1 to 9 comprises: Cut open the skin and peritoneum of the spleen area of ​​the mouse to expose the spleen, and measure the size of the spleen of the mouse; Determine the position where the first imaging block is connected to the second imaging block according to the size of the mouse spleen, 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 use glue to install a glass slide or a PDMS film on the top surface of the closed imaging window; Fixing the skin around the wound of the mouse to the lower part of the closed imaging window; The spleens of the mice were subjected to optical imaging studies.

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