Mouse spinal cord fixing device for two-photon microscope living imaging

Abstract

The invention discloses a mouse spinal cord fixing device for two-photon microscope in-vivo imaging, and belongs to the technical field of in-vivo imaging research, the mouse spinal cord fixing device comprises a base and two sets of supporting assemblies, the supporting assemblies are symmetrically arranged, and the two sets of supporting assemblies are provided with a first fixing part and a second fixing part respectively; the fixing teeth are arranged to be of the C-shaped tooth-shaped structure with the two tip ends, the fixing teeth can pierce into the vertebral bone surface to fix the two adjacent vertebrae during use, the fixed spinal cord is adjusted to the horizontal plane, the limiting bolt and the slide arranged on the slide bracket are utilized, the slide bracket is fixed, the slide bracket is fixed, and the slide bracket can be used for fixing the spinal cord. According to the device, the slide bracket is adjusted to the horizontal plane, so that the slide is in full contact with the spinal cord, two-photon microscope in-vivo imaging is conveniently completed, the spinal cord of a mouse can be fixed, an observation surface can be leveled, the imaging effect is good, the operation is simple, and the popularization is easy.

Description

Technical Field

[0001] The invention relates to the technical field of in vivo imaging research, and in particular to a mouse spinal cord fixing device used for in vivo imaging with a two-photon microscope. Background Art

[0002] In recent years, two-photon microscopy has been widely used in the medical field. Due to its nonlinear excitation characteristics and the use of ultrafast lasers in the infrared band as light sources, two-photon microscopy has a greater imaging penetration depth than confocal microscopy, and has low photobleaching and phototoxicity, and is widely used in living tissue imaging. In particular, the application of long-working-distance infrared high-transmittance water-immersion objectives has further improved the imaging resolution and imaging depth, providing an effective method for the study of cellular-level functions of living tissues.

[0003] Mice are one of the most commonly used experimental animals, especially widely used in genetic engineering research. Using genetically engineered mice, researchers have developed an effective way for neurobiological research through two-photon microscopy live imaging with the help of fluorescent reporter genes, or optogenetic regulation combined with calcium imaging technology. In recent years, using two-photon microscopy live imaging technology, people have achieved many results in the study of the brain and have a deeper understanding of the structure and function of neural networks. The spinal cord is an important part of the central nervous system. Live imaging of the spinal cord is of great significance for the study of touch, pain, itch, proprioception, movement, and movement rhythm. However, the mouse spine is small, has a large range of motion, and has an irregular shape of vertebrae, which makes it difficult to fix the mouse spinal cord. The traditional method of mouse spinal cord fixation is to fix the spinal cord by clamping the spinous process of the vertebra or by clamping the depression between the transverse process and the mastoid process. For the fixation method of clamping the spinous process, the spinous process is easy to be clamped off during the specific operation, and it is difficult to level the left and right. As for the fixation method of embedding the depression between the transverse process and the mastoid process, the transverse process and the mastoid process cannot be removed, resulting in inability to fully expose the spinal cord and difficulty in realizing the application of the water immersion objective of the two-photon microscope;

[0004] For example, a fixed observation device for in vivo imaging of animal spinal cord with publication number CN104287865A is composed of a base plate, a left movable adjustment device, a right movable adjustment device and an implanted observation window. The left movable adjustment device and the right movable adjustment device are fixed to the base plate by screws, and the implanted observation window is positioned by inserting and engaging the rectangular protrusions of the horizontal positioning sliders on the left movable adjustment device and the right movable adjustment device through the rectangular holes on its V-shaped frame. Although this invention fixes the spinal cord through an implanted observation window, it is beneficial to the dynamic observation of postoperative two-photon spinal cord imaging, but it cannot level the observation surface after the spinal cord is fixed, and it is difficult to carry water medium in the observation window, which limits the use of the water immersion objective of the two-photon microscope. Summary of the invention

[0005] The embodiment of the present invention provides a mouse spinal cord fixation device for two-photon microscope in vivo imaging to solve the above-mentioned technical problems.

[0006] The embodiment of the present invention adopts the following technical solution: it includes a base and a support assembly, the base is used to place a horizontal plane, the support assembly is provided in two groups and is symmetrically arranged, the two groups of support assemblies are respectively provided with a first fixing member and a second fixing member, and the fixed ends of the first fixing member and the second fixing member are arranged facing each other.

[0007] Preferably, each group of the supporting components comprises a column, a slider and a screw rod, the column is vertically arranged on the base, the column is a square column structure, a groove for placing and lifting the slider is arranged on the column, the slider is arranged in the groove of the column, and the slider and the inner wall of the groove of the column are slidably matched up and down, and a threaded groove is arranged on the slider that runs through the upper and lower ends of the slider, and the threaded groove matches the screw rod;

[0008] The screw is vertically arranged in the groove of the column, and the screw passes into the thread groove from the top end of the slider and comes out from the bottom end of the slider, so that the screw and the thread groove of the slider are threadedly matched, the length of the screw is adapted to the length of the groove, and the first scale mark is provided on the same surface of the column and the slider.

[0009] Preferably, a cylindrical groove extending to the groove area is provided at the top of the column, and a detachable first hand-tightening component is provided at the top of the screw, and the first hand-tightening component consists of a block, a hand-tightening column and a half-thread bolt. The block is fixedly arranged at the top of the screw, and the block and the screw are concentrically arranged, and a threaded hole matching the half-thread bolt is provided at the top of the block, and the lower half of the hand-tightening column can be inserted into the cylindrical groove at the top of the column and can rotate in the cylindrical groove, and a square groove matching the size of the block is provided at the bottom end of the hand-tightening column, and a placement hole matching the shape of the half-thread bolt is provided inside the hand-tightening column, and the placement hole passes through the top of the hand-tightening column and the square groove, and the half-thread bolt can be inserted into the threaded hole of the block along the placement hole from the top of the hand-tightening column.

[0010] Preferably, a clamping block is fixedly provided on the left side of the sliding block, and a through hole is provided on the clamping block, and the through hole runs through the front and rear sides of the clamping block.

[0011] Preferably, a threaded hole is provided on the left side of the card block, and the threaded hole extends from the left side of the card block to the through hole. A hand-operated hand-tightening rod is also provided on the left side of the card block, and the hand-tightening rod is provided with threads matching the threaded hole on the card block. The threaded portion of the hand-tightening rod is inserted into the threaded hole, so that the hand-tightening rod and the threaded hole on the card block form a threaded fit.

[0012] Preferably, the first fixing member and the second fixing member have the same structure. Taking the first fixing member as an example, the first fixing member includes a fixing rod and a fixing block, the fixing rod is a cylindrical structure, the fixing rod is horizontally inserted into the through hole on the corresponding card block, and the fixing rod can be laterally moved and rotated in the through hole on the corresponding card block, the fixing block is located at the front end of the fixing rod, and the fixing block is fixedly connected to the fixing rod, the front end of the fixing block is provided with an extension end, the end of the extension end is fixedly connected to the fixing block, and the front end of the extension end is inclined and extended toward the lower front part to form a fixing tooth, the front end of the fixing tooth is provided with an arc groove, so that the fixing tooth forms a C-shaped tooth structure with two sharp tips, and the two sharp tips on the fixing tooth are both serrated.

[0013] Preferably, each of the extending ends and the fixing teeth is provided with a cutting groove, so that the front end of the fixing block has two extending ends and two fixing teeth.

[0014] Preferably, a second hand-tightening component is provided on the other end of each fixing rod, that is, the end of the fixing rod. Each group of the second hand-tightening components has the same structure as the first hand-tightening component, that is, the second hand-tightening component is also composed of a square, a hand-tightening column and a half-thread bolt. The square is fixed at the end of the fixing rod in a concentric column state, and a threaded hole matching the half-thread bolt is provided at the end of the square. A square groove matching the size of the square is provided at the front end of the hand-tightening column, and an accommodation hole matching the shape of the half-thread bolt is provided inside the hand-tightening column.

[0015] Preferably, a glass slide holder and a glass slide are arranged between the first fixing member and the second fixing member, the middle section of the glass slide holder is a plate-shaped structure, a placement groove for placing the glass slide is arranged in the middle of the glass slide holder, the shapes of the two sides of the glass slide holder match the shapes of the two fixing teeth in the first fixing member and the second fixing member and the upper surface of the extension end, the two sides of the glass slide holder corresponding to each extension end are provided with a card slot, each extension end is provided with a limiting bolt corresponding to the card slot, each limiting bolt is half-toothed and is located in the lower half of the limiting bolt, a retaining ring is fixedly arranged on each limiting bolt, the distance between each retaining ring and the head of the corresponding limiting bolt matches the thickness of the two sides of the glass holder, and each extension end is provided with a threaded hole matching the thread on the corresponding limiting bolt.

[0016] Preferably, a screw is provided at the bottom of the base, and a groove matching the shape of the bottom end of the column is provided at a position corresponding to each column on the base, and each groove is provided with a nail hole passing through the upper and lower ends of the base, and the bottom end of each column is provided with a threaded hole matching the position of the nail hole in the corresponding groove, and the threaded hole matches the thread of the screw.

[0017] At least one of the above technical solutions adopted in the embodiments of the present invention can achieve the following beneficial effects:

[0018] First, in the present invention, the fixing teeth are configured as a C-shaped tooth structure with two sharp tips, and the two sharp tips on the fixing teeth are both serrated. When in use, the sharp tips of the serrated structure can penetrate into the bone surface of the vertebra, and can adapt to the irregular outer surface shape of the mouse vertebra for fixation. At the same time, the two sharp tips contact the upper and lower parts of both sides of the vertebra, so that the contact stress can be concentrated at the two sharp tips, thereby limiting the rotatable state of the vertebra, and can adapt to the irregular shapes of the outer surfaces of different mouse vertebrae for fixation, so that the mouse spine will not be displaced during the experiment, so as to cooperate with the two-photon microscope in vivo imaging.

[0019] Secondly, the front end of the fixed teeth at the extension end of the present invention is inclined and extended toward the lower front part, so that the horizontal height of each fixed tooth is lower than the fixed block. In actual application, in order to expose the L1-L2 segment spine, the tissues on both sides of the mouse spine need to be opened to both sides, and the fixed blocks located above the horizontal height of the fixed teeth are located above the tissues on both sides of the mouse spine that are opened, which can avoid squeezing injuries to the open tissues on both sides caused by the fixed blocks and displacement of the mouse spine, and reserve enough space for the lowering of the microscope objective lens.

[0020] Thirdly, the through hole on the clamp block can be used for inserting the fixing rod and supporting the fixing rod, so that when the operator rotates or translates the fixing rod, the fixing rod can maintain a fixed horizontal height. When in use, after fixing the spinal cord, the front and back height of the spinal cord can be leveled by rotating the second screws of the fixing rods on both sides.

[0021] Fourthly, the lifting height of the slider can be adjusted by rotating the screw rod, and the up and down sliding cooperation between the slider and the inner wall of the groove of the column can enable the slider to maintain a vertical trajectory when it is lifted and lowered in the groove, so that a fixed orientation can be maintained when controlling the first fixing member and the second fixing member for height adjustment. In actual application, after fixing the spinal cord, the height of the spinal cord can be leveled on the left and right by rotating the first hand-tightening components on the columns on both sides.

[0022] Fifth, each component in the present invention is of detachable structural design, which is convenient for disassembly and assembly and convenient for storage. In actual application, the entire device can be disassembled, disinfected and cleaned before and after operation, so that the entire device can be thoroughly cleaned to avoid residual bacteria in the gaps between components and infection of living experimental subjects.

[0023] Sixth, the shapes of the two sides of the glass slide holder match the shapes of the two fixing teeth in the first fixing member and the second fixing member and the upper surface of the extension end, so that the glass slide holder is set in a sunken manner. In actual application, the glass slide is placed on the glass slide holder. The sunken setting of the glass slide holder allows the glass slide to fully contact the spinal cord. Dropping water on the glass slide can cooperate with the application of the water immersion objective lens of the two-photon microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0025] Figure 1 It is a schematic diagram of the three-dimensional structure of the present invention;

[0026] Figure 2 It is a schematic diagram of the local structure of the present invention;

[0027] Figure 3 It is a schematic structural diagram of the support assembly and the first fixing member in the present invention;

[0028] Figure 4 It is a structural schematic diagram of a column and a sliding block in the present invention;

[0029] Figure 5 It is a schematic diagram of the split structure of the support assembly in the present invention;

[0030] Figure 6 is a cross-sectional view of the first hand-tightening assembly in the present invention;

[0031] Figure 7 The structure of the first fixing member in the present invention is shown in FIG. Figure 1 ;

[0032] Figure 8 The structure of the first fixing member in the present invention is shown in FIG. Figure 2 ;

[0033] Fig. 9 It is a schematic diagram of the structure of the first fixing member and the second fixing member in the present invention;

[0034] Fig.10 A schematic diagram of fixing the mouse vertebrae by the first fixing member and the second fixing member in the present invention;

[0035] Fig.11 It is a schematic diagram of the installation of the slide holder in the present invention;

[0036] Fig.12 It is a structural schematic diagram of a slide holder and a slide in the present invention;

[0037] Fig.13This is a schematic diagram of the use of the water immersion objective lens of a two-photon microscope:

[0038] Fig.14 It is a schematic diagram of the installation of the neutral column of the present invention.

[0039] Reference numerals

[0040] Base 1, screw 11, embedded groove 12, support component 2, column 21, groove 211, first scale mark 212, cylindrical groove 213, slider 22, threaded groove 221, screw 23, first hand-tightening component 24, block 25, hand-tightening column 26, placement hole 261, half-thread bolt 27, block 28, through hole 281, angle mark 282, hand-tightening rod 29, silicone pad 291, first fixing part 3, fixing rod 31, straight line mark 311, second scale mark 312, fixing block 32, extension end 33, fixing teeth 34, arc groove 341, cut groove 35, second hand-tightening component 36, second fixing part 4, slide holder 5, slide 51, placement groove 52, slot 53, limit bolt 54, retaining ring 55. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention and the corresponding drawings. Obviously, the described embodiments are only 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 making creative work are within the scope of protection of the present invention.

[0042] The technical solutions provided by various embodiments of the present invention are described in detail below in conjunction with the accompanying drawings.

[0043] The embodiment of the present invention provides a mouse spinal cord fixation device for two-photon microscope in vivo imaging, comprising a base 1 and a support assembly 2;

[0044] See attached Figures 1 to 3 As shown, the base 1 is used to place a horizontal surface, and the support components 2 are provided in two groups and are symmetrically arranged. The two groups of support components 2 are respectively provided with a first fixing member 3 and a second fixing member 4, and the fixing ends of the first fixing member 3 and the second fixing member 4 are arranged facing each other;

[0045] During the operation, take the experimental mouse, anesthetize the mouse, shave the mouse hair in the surgical area on the back of the mouse, and disinfect the surgical area with 75% ethanol. Place the mouse on the base 1, cut the skin of the surgical area longitudinally along the spine, locate the first lumbar vertebra (L1) and the second lumbar vertebra (L2), separate the muscles and connective tissue near the L1-L2 vertebrae with the help of a stereo microscope, cut the transverse process during the separation process until the vertebral body is exposed, while avoiding damage to the spinal nerves and accompanying blood vessels passing through the two vertebrae. Adjust the support component 2 according to the height of the mouse's spine, so that the first fixing member 3 and the second fixing member 4 can fix the mouse spine, and use Venus scissors to cut off the vertebral lamina and part of the bone below the lamina to fully expose the spinal cord, so as to cooperate with the completion of the two-photon microscope live imaging operation of the mouse spine.

[0046] In a further preferred embodiment, refer to the attached Figure 3 and 4 As shown, each group of the support components 2 includes a column 21, a slider 22 and a screw 23. The column 21 is vertically arranged on the base 1. The column 21 is a square column structure. A groove 211 for placing and lifting the slider 22 is arranged on the column 21. The slider 22 is arranged in the groove 211 of the column 21, and the slider 22 and the inner wall of the groove 211 of the column 21 are slidably matched up and down. The slider 22 is provided with a thread groove 221 that runs through the upper and lower ends of the slider 22, and the thread groove 221 matches the screw 23.

[0047] The screw rod 23 is vertically arranged in the groove 211 of the column 21, and the screw rod 23 passes through the thread groove 221 from the top end of the slider 22 and passes through the bottom end of the slider 22, so that the screw rod 23 and the thread groove 221 of the slider 22 are threadedly matched, and the length of the screw rod 23 is adapted to the length of the groove 211. The same surface of the column 21 and the slider 22 is provided with a first scale mark 212;

[0048] The support assembly 2 can be used to control the corresponding first fixing member 3 and the second fixing member 4 to be raised and lowered, so as to adjust the corresponding height of the mouse spine on the base 1. By rotating the screw 23, the thread on the screw 23 can be threadedly matched with the thread groove 221 on the slider 22, so that the slider 22 can be raised and lowered in the groove 211 of the column 21 as the screw 23 rotates.

[0049] Among them, setting the slider 22 and the inner wall of the groove 211 of the column 21 to slide up and down can enable the slider 22 to maintain a vertical trajectory when it is raised and lowered in the groove 211, so that the corresponding first fixing member 3 and the second fixing member 4 can maintain stable height adjustment; and the first scale mark 212 set on the same surface of the column 21 and the slider 22 can be combined with the first scale mark 212 to refer to each other and record data when adjusting the height of the sliders 22 on both sides.

[0050] In a further preferred embodiment, refer to the attached Figure 5 and 6 As shown, the top end of the column 21 is provided with a cylindrical groove 213 extending to the area of ​​the groove 211;

[0051] The top of the screw rod 23 is provided with a detachable first hand-tightening assembly 24, which is composed of a block 25, a hand-tightening plug column 26 and a half-thread bolt 27. The block 25 is fixedly arranged on the top of the screw rod 23, and the block 25 and the screw rod 23 are arranged concentrically. The top of the block 25 is provided with a threaded hole matching the half-thread bolt 27;

[0052] The lower half of the hand-tightening column 26 can be inserted into the cylindrical groove 213 at the top of the column 21 and can rotate in the cylindrical groove 213. The bottom end of the hand-tightening column 26 is provided with a square groove matching the size of the block 25. The inside of the hand-tightening column 26 is provided with a placement hole 261 matching the shape of the half-thread bolt 27. The placement hole 261 passes through the top of the hand-tightening column 26 and the square groove. The half-thread bolt 27 can be inserted into the threaded hole of the block 25 along the placement hole 261 from the top of the hand-tightening column 26.

[0053] Each support assembly 2 in the present invention is a detachable structure. When assembling, the screw rod 23 can be placed in the groove 211 of the column 21, and then the top of the hand-tightening column 26 can be inserted from the cylindrical groove 213 on the column 21, and the square groove at the bottom of the hand-tightening column 26 can be sleeved on the block 25 at the top of the screw rod 23. Then, the half-thread bolt 27 is inserted from the top of the hand-tightening column 26 along the placement hole 261 and the threaded part of the half-thread bolt 27 can be placed in the threaded hole on the block 25, so as to limit the connection state between the hand-tightening column 26, the block 25 and the screw rod 23.

[0054] When adjusting the screw rod 23, the top part of the hand-tightening plug column 26 can be manually controlled and rotated. When the hand-tightening plug column 26 rotates, the square groove part is sleeved on the block 25, so that the block 25 and the screw rod 23 can rotate synchronously with the rotation of the hand-tightening plug column 26. When in use, the lifting height of the slider 22 can be adjusted by rotating the hand-tightening plug column 26; in addition, by adjusting the hand-tightening plug column 26 on each screw rod 23, the corresponding first fixing member 3 and the second fixing member 4 can be respectively controlled to perform adaptive height adjustment, so that the first fixing member 3 and the second fixing member 4 can be adjusted to the corresponding height according to the height of the mouse spinal cord to cooperate with the spinal cord fixation operation, and after the spinal cord is fixed, the leveling operation is performed according to the height of the left and right sides of the mouse spinal cord, so that the height of the left and right sides of the spinal cord is maintained on the same horizontal plane;

[0055] Secondly, the support assembly 2 and the first hand-tightening assembly 24 consisting of the block 25, the hand-tightening plug column 26 and the half-thread bolt 27 are all detachable, so that the device is easy to maintain and clean during use.

[0056] In a further preferred embodiment, refer to the attached Figures 3 to 5 As shown, a block 28 is fixedly provided on the left side of the slider 22, and a through hole 281 is provided on the block 28. The through hole 281 passes through the front and rear sides of the block 28, and an angle scale mark 282 is provided on the front side of the block 28 and around the through hole 281. A threaded hole is provided on the left side of the block 28, and the threaded hole extends from the left side of the block 28 to the through hole 281. A hand-operated hand-tightening rod 29 is also provided on the left side of the block 28. The hand-tightening rod 29 is provided with a thread matching the threaded hole on the block 28, and the threaded part of the hand-tightening rod 29 is inserted in the threaded hole, so that the hand-tightening rod 29 and the threaded hole on the block 28 form a threaded match. When in use, the hand-tightening rod 29 can cooperate with the first fixing member 3 and the second fixing member 4 to fix and adjust the lateral movement and rotation.

[0057] See attached Figure 3 and 7As shown, the first fixing member 3 and the second fixing member 4 have the same structure. Taking the first fixing member 3 as an example, the first fixing member 3 includes a fixing rod 31 and a fixing block 32. The fixing rod 31 is a cylindrical structure. The fixing rod 31 is inserted into the through hole 281 on the corresponding block 28 in a horizontal state, and the fixing rod 31 can be horizontally moved and rotated in the through hole 281 on the corresponding block 28. A straight line mark 311 is provided just above the fixing rod 31, and a second scale mark 312 is provided along the straight line mark 311. The fixing block 32 is located at the front end of the fixing rod 31, and the fixing block 32 is fixedly connected to the fixing rod 31. In operation, the first fixing member 3 and the second fixing member 4 are adjusted laterally so that the tips of the C-shaped fixing teeth of the two fixing members contact the vertebral bone By combining the front side surface of the block 28 and the second scale mark 312 on the fixing rod 31, the lateral movement distances of the first fixing member 3 and the second fixing member 4 can be referenced to each other, and the relative width of the vertebra can be recorded. The first fixing member 3 and the second fixing member 4 can be further adjusted laterally so that the tips of the C-shaped fixing teeth of the two fixing members penetrate into the vertebral bone surface, thereby achieving the fixation of the vertebra. After the spinal cord is fixed, the front and back heights of the spinal cord can be leveled by rotating the second hand-tightening component 36 so that the front and back heights of the spinal cord are maintained on the same horizontal plane. By combining the straight line mark 311 above the fixing rod 31 and the angle scale mark 282 on the periphery of the through hole 281, the rotation angles of the first fixing member 3 and the second fixing member 4 can be referenced to each other and data can be recorded.

[0058] See attached Figure 8 As shown, the front end of the fixed block 32 is provided with an extension end 33, the end of the extension end 33 is fixedly connected to the fixed block 32, and the front end of the extension end 33 is inclined and extended toward the lower part of the front side to form a fixed tooth 34. When in use, the front end of the extension end 33 is inclined and extended toward the lower part of the front side to reserve enough space for the objective lens of the two-photon microscope to descend;

[0059] The front end of the fixing tooth 34 is provided with an arc groove 341, so that the fixing tooth 34 forms a C-shaped tooth structure with two tips, and the two tips of the fixing tooth 34 are both serrated. During operation, the serrated tips can penetrate into the bone surface of the vertebral body to fix the vertebra, thereby achieving fixation of the spinal cord;

[0060] When assembling the first fixing member 3 and the second fixing member 4 with the corresponding supporting assembly 2, the fixing rod 31 can be inserted into the through hole 281 on the clamping block 28, and ensure that the extending ends 33 on the fixing rod 31 in the first fixing member 3 and the second fixing member 4 are arranged facing each other;

[0061] In practical application, each fixing rod 31 can be moved horizontally according to the position of the mouse spine on the base 1, so that the C-shaped tooth structure at the front end of the extension end 33 and the fixing tooth 34 can contact and clamp the two sides of the vertebrae;

[0062] Among them, refer to the attached Fig.10 As shown, since the outer surface of the mouse vertebra is irregular in shape, and the outer surface shape of the vertebra of each mouse is somewhat different, the fixed tooth 34 is set to a C-shaped tooth structure with two tips. When in use, only the two tips are needed to contact from the upper and lower positions on both sides of the vertebra, so that the contact stress can be concentrated on the two tip positions, thereby limiting the rotatable state of the vertebra, and the arc groove 341 area of ​​the fixed tooth 34 can be placed on the convex part of the outer surface of the vertebra. Therefore, the designed extension end 33 and the fixed tooth 34 in the present invention can adapt to the irregular shape of the outer surface of the mouse vertebra for clamping and fixing. At the same time, the two tip positions on the fixed tooth 34 are both serrated structure settings, so that the two tip positions on the fixed tooth 34 can form a tooth claw state contact at multiple points when contacting the mouse vertebra, which can fix the vertebra well and reduce the contact area between the fixed tooth 34 and the mouse vertebra, so as to reduce the damage caused by the contact pressure area on the outer surface of the vertebra;

[0063] In actual application, in order to expose the L1-L2 segment spine, the tissues on both sides of the mouse spine need to be opened to both sides, and the front end of the fixed teeth 34 at the extension end 33 is inclined and extended to the lower part of the front side, so that the horizontal height of each fixed tooth 34 is lower than the fixed block 32, and when the fixed teeth 34 fix the vertebrae, the fixed block 32 located above the horizontal height of the fixed teeth 34 is located above the tissues on both sides of the mouse spine in the open state, which can effectively avoid the fixed block 32 from causing squeezing injuries to the tissues on both sides and causing displacement of the mouse spine;

[0064] The front end of the hand-tightening rod 29 is provided with a silicone pad 291. After the displacement adjustment of each fixing rod 31 is completed, the hand-tightening rod 29 can be rotated so that the threaded portion of the hand-tightening rod 29 can be screwed into the threaded hole on the block 28, and until the silicone pad 291 at the front end of the hand-tightening rod 29 can penetrate into the through hole 281 and squeeze the fixing rod 31, and the silicone pad 291 is used to increase the friction force when the front end of the hand-tightening rod 29 contacts the fixing rod 31 to stop the fixing rod 31 in the through hole 281 of the block 28, so that the fixing rod 31 is limited in the through hole 281 of the block 28, so as to prevent the fixing rod 31 from affecting the imaging result due to displacement during operation;

[0065] Secondly, due to the physiological curvature of the mouse spinal cord, it is difficult to achieve the same horizontal position for the observed part of the spinal cord by directly fixing it. Therefore, the fixing rod 31 in the present invention can be moved laterally and rotated in the through hole 281 on the corresponding block 28. During operation, each fixing rod 31 can be rotated to drive the corresponding fixing tooth 34 to adjust the mouse spinal cord, so that the curved spinal cord to be observed in the mouse is adjusted to the same horizontal plane under the traction of the fixing teeth 34 on both sides of itself.

[0066] In a further preferred embodiment, refer to the attached Figure 8 and 9 As shown, each of the extension ends 33 and the fixed teeth 34 is provided with a slot 35, so that the front end of the fixed block 32 has two extension ends 33 and two fixed teeth 34;

[0067] In the prior art, in order to realize two-photon microscopy in vivo imaging of the mouse spinal cord, when fixing the spinal cord across two or more vertebrae, it is easy to cause cutting damage to the spinal nerves and accompanying blood vessels. In the present invention, a groove 35 is provided between the two fixing teeth 34 at the front end of each fixing block 32. When fixing is performed from both sides of the vertebrae, the two fixing teeth 34 at the front end of each fixing block 32 can fix two adjacent vertebrae respectively, and the spinal nerves and accompanying blood vessels between the adjacent vertebrae can pass through the groove 35, which can effectively avoid cutting damage to the spinal nerves and accompanying blood vessels.

[0068] Among them, since the vertebrae of the mouse thoracic spine and lumbar spine are of different sizes, multiple groups of fixing teeth 34 with different widths and gap sizes can be customized to be used for adaptive fixation of different segments of vertebrae.

[0069] In a further preferred embodiment, refer to the attached Figure 3 and 7 As shown, a second hand-tightening assembly 36 is provided at the other end of each fixing rod 31 located at the fixing teeth 34, that is, at the end of the fixing rod 31;

[0070] Each group of the second hand-tightening components 36 has the same structure as the first hand-tightening components 24, that is, the second hand-tightening components 36 are also composed of a block 25, a hand-tightening plug column 26 and a half-thread bolt 27. The block 25 is fixedly arranged at the end of the fixing rod 31 in a concentric column state, and a threaded hole matching the half-thread bolt 27 is arranged at the end of the block 25, and a square groove matching the size of the block 25 is arranged at the front end of the hand-tightening plug column 26. The inside of the hand-tightening plug column 26 is provided with a placement hole 261 matching the shape of the half-thread bolt 27;

[0071] After the fixing rod 31 is inserted into the through hole 281 on the corresponding block 28, the square groove at the bottom of the hand-tightening plug column 26 can be sleeved on the block 25, and then the half-thread bolt 27 is inserted from the placement hole 261 at the top of the hand-tightening plug column 26 and the threaded part of the half-thread bolt 27 is screwed into the threaded hole on the block 25, so that the hand-tightening plug column 26 and the fixing rod 31 are connected as a whole;

[0072] When in use, the hand-tightening plug column 26 can be held to perform lateral translation and rotation adjustment on the entire fixing rod 31, so that the fixing teeth 34 at the front end of the fixing rod 31 can be adjusted to an adaptive angle according to the actual shape of the mouse vertebrae, and the hand-tightening plug column 26 can cooperate with the fixing block 32 at the front end of the fixing rod 31 to limit the movable distance of the fixing rod 31 in the through hole 281 on the corresponding card block 28, so as to prevent the fixing rod 31 from falling off from the through hole 281 due to overload, so as to complete the fixation and leveling operation of the spinal cord;

[0073] Secondly, the second hand-tightening assembly 36 is a detachable structure, which can facilitate cleaning and maintenance of the device before and after use;

[0074] In the operation of fixing the spinal cord, the first hand-tightening component 24, the second hand-tightening component 36 and the hand-tightening insert rod 29 are used in conjunction with each other to adjust the first hand-tightening component 24 of the two side columns 21 so that the C-shaped fixing teeth 34 of the first fixing member 3 and the second fixing member 4 are located on both sides of the L1 and L2 vertebrae and reach the height of the vertebral body. The second hand-tightening component 36 is used in conjunction with the hand-tightening insert rod 29 to move the first fixing member 3 and the second fixing member 4 laterally inward so that the serrated structure at the tip of the C-shaped fixing teeth 34 contacts the vertebral body. Combined with the length of the fixing rod 31, the first fixing member 3 and the second fixing member 4 are moved laterally inward. The scale mark and the through hole 281 of the block 28 can obtain and record the relative width of the vertebral body. The first fixing member 3 and the second fixing member 4 are further adjusted to move laterally inward so that the serrated structure at the tip of the C-shaped fixing tooth 34 penetrates into the vertebral bone surface of the vertebra to fix the vertebra. The second hand-tightening assembly 36 of the first fixing member 3 and the second fixing member 4 and the hand-tightening insertion rod 29 are used together to adjust the front and back heights of the spinal cord to the same horizontal plane, and the first hand-tightening assembly 24 of the two side columns 21 is adjusted to adjust the left and right heights of the spinal cord to the same horizontal plane.

[0075] In a further preferred embodiment, refer to the attached Fig.11 and 12 As shown, a glass slide holder 5 and a glass slide 51 are arranged between the first fixing member 3 and the second fixing member 4;

[0076] The middle section of the glass slide holder 5 is a plate-like structure, and a placement groove 52 for placing a glass slide 51 is provided in the middle of the glass slide holder 5. The shapes of the two sides of the glass slide holder 5 match the shapes of the two fixing teeth 34 and the upper surface of the extension end 33 in the first fixing member 3 and the second fixing member 4, and are arranged in a sunken manner;

[0077] A slot 53 is provided at the position corresponding to each extension end 33 on both sides of the glass slide holder 5. The slot 53 is a long slot-shaped opening, which can adapt to the lateral movement of the first fixing member 3 and the second fixing member 4 in practical applications. A limit bolt 54 is provided at the position corresponding to the slot 53 on each extension end 33. Each limit bolt 54 is half-threaded and located at the lower half of the limit bolt 54. A retaining ring 55 is fixedly provided on each limit bolt 54. The distance between each retaining ring 55 and the head of the corresponding limit bolt 54 matches the thickness of the two sides of the glass holder. A threaded hole matching the thread on the corresponding limit bolt 54 is provided on each extension end 33.

[0078] After the first fixing member 3 and the second fixing member 4 have completed the fixing and leveling operation on the mouse vertebrae, the glass slide 51 can be placed in the placement groove 52, and the portion between the head and the retaining ring 55 on each limiting bolt 54 is inserted into the slot 53 through the opening of the slot 53, so that the retaining ring 55 and the head on each limiting bolt 54 can support both sides of the glass slide holder 5. After each limiting bolt 54 is inserted into the corresponding threaded hole on the extension end 33, the glass slide 51 can be placed in the placement groove 52 on the glass slide holder 5, and then the height of the mouse spinal cord can be adjusted. Each limiting bolt 54 is rotated and adjusted to adjust the height of the glass slide holder 5. The limiting bolt 54 can be adjusted to match the height of the mouse spinal cord so that the glass slide 51 on the glass slide holder 5 can fully contact the spinal cord and remain horizontal. The experimental mouse with the spinal cord fixed is then placed on the stage of the two-photon microscope together with the fixing device. Water drops are then dripped on the glass slide 51, and the water immersion objective lens of the two-photon microscope is immersed in the water drops on the glass slide 51 to observe the spinal cord, thereby obtaining imaging results.

[0079] In a further preferred embodiment, refer to the attached Fig.13 As shown, a screw 11 is provided at the bottom of the base 1, and a groove 12 matching the shape of the bottom end of the column 21 is provided at a position corresponding to each column 21 on the base 1, and each groove 12 is provided with a nail hole penetrating the upper and lower ends of the base 1, and the bottom end of each column 21 is provided with a threaded hole matching the position of the nail hole in the corresponding groove 12, and the threaded hole matches the thread of the screw 11;

[0080] The embedding groove 12 on the base 1 can be used for the bottom end of the column 21 to be inserted, so as to limit the translatable state of each column 21. After the screw 11 is inserted from the bottom end of the base 1 and screwed into the threaded hole at the bottom end of the column 21, the column 21 can be connected to the base 1, thereby providing a limiting effect for the column 21 in subsequent use. At the same time, the detachable structure between the column 21 and the base 1 can also facilitate comprehensive disinfection and cleaning of the column 21.

[0081] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A mouse spinal cord fixation device for two-photon microscopy in vivo imaging, characterized in that: include: A base (1), the base (1) being used to place on a horizontal surface; A support assembly (2), wherein the support assembly (2) is provided in two groups and is symmetrically arranged; The two groups of support components (2) are respectively provided with a first fixing member (3) and a second fixing member (4), and the fixing ends of the first fixing member (3) and the second fixing member (4) are arranged facing each other.

2. A mouse spinal cord fixation device for two-photon microscopy in vivo imaging according to claim 1, characterized in that: Each group of the support components (2) comprises a column (21), a slider (22) and a screw rod (23); The column (21) is vertically arranged on the base (1), and the column (21) is a square column structure. A groove (211) is arranged on the column (21) for placing and lifting the slider (22); The slider (22) is arranged in the groove (211) of the column (21), and the slider (22) and the inner wall of the groove (211) of the column (21) are slidably matched up and down, and the slider (22) is provided with a thread groove (221) penetrating the upper and lower ends of the slider (22), and the thread groove (221) matches the screw rod (23); The screw rod (23) is vertically arranged in the groove (211) of the column (21), and the screw rod (23) passes through the thread groove (221) from the top end of the slider (22) and passes out from the bottom end of the slider (22), so that the screw rod (23) and the thread groove (221) of the slider (22) are threadedly matched; The same surface of the column (21) and the sliding block (22) is provided with a first scale mark (212).

3. A mouse spinal cord fixation device for two-photon microscopy in vivo imaging according to claim 2, characterized in that: The top end of the column (21) is provided with a cylindrical groove (213) extending to the area of ​​the groove (211); A detachable first hand-tightening assembly (24) is provided at the top end of the screw rod (23); The first hand-tightening assembly (24) is composed of a block (25), a hand-tightening plug (26) and a half-thread bolt (27); The block (25) is fixedly arranged on the top of the screw rod (23), and the block (25) and the screw rod (23) are arranged concentrically, and the top of the block (25) is provided with a threaded hole matching the half-thread bolt (27); The lower half of the hand-tightening plug post (26) can be inserted into the cylindrical groove (213) at the top of the column (21) and can rotate in the cylindrical groove (213), and the bottom end of the hand-tightening plug post (26) is provided with a square groove matching the size of the block (25); The hand-tightening plug post (26) is provided with a placement hole (261) matching the shape of the half-thread bolt (27) inside, and the placement hole (261) passes through the top end of the hand-tightening plug post (26) and the square groove; The half-thread bolt (27) can be inserted into the threaded hole of the block (25) along the placement hole (261) from the top end placement hole (261) of the hand-tightened insertion column (26).

4. The mouse spinal cord fixation device for two-photon microscopy in vivo imaging according to claim 2, characterized in that: A clamping block (28) is fixedly arranged on the left side of the slider (22), and a through hole (281) is arranged on the clamping block (28). The through hole (281) passes through the front and rear sides of the clamping block (28), and an angle scale mark (282) is arranged on the front side surface of the through hole (281).

5. The mouse spinal cord fixation device for two-photon microscopy in vivo imaging according to claim 4, characterized in that: A threaded hole is provided on the left side of the clamping block (28), and the threaded hole extends from the left side of the clamping block (28) to the through hole (281). A hand-operated hand-tightening rod (29) is also provided on the left side of the clamping block (28), and a thread matching the threaded hole on the clamping block (28) is provided on the hand-tightening rod (29). The threaded portion of the hand-tightening rod (29) is inserted into the threaded hole, so that the hand-tightening rod (29) and the threaded hole on the clamping block (28) form a threaded fit.

6. The mouse spinal cord fixation device for two-photon microscopy in vivo imaging according to claim 4, characterized in that: The first fixing member (3) and the second fixing member (4) have the same structure. Taking the first fixing member (3) as an example, the first fixing member (3) comprises a fixing rod (31) and a fixing block (32); The fixing rod (31) is a cylindrical structure, and the fixing rod (31) is inserted into the through hole (281) on the corresponding card block (28) in a horizontal state, and the fixing rod (31) can move horizontally and rotate in the through hole (281) on the corresponding card block (28); The fixing block (32) is located at the front end of the fixing rod (31), and the fixing block (32) is fixedly connected to the fixing rod (31); The front end of the fixed block (32) is provided with an extension end (33), the end of the extension end (33) is fixedly connected to the fixed block (32), and the front end of the extension end (33) is extended obliquely toward the lower front portion to form a fixed tooth (34), and the front end of the fixed tooth (34) is provided with an arc groove (341), so that the fixed tooth (34) forms a C-shaped tooth structure with two tips, and the two tip portions of the fixed tooth (34) are both serrated.

7. The mouse spinal cord fixation device for two-photon microscopy in vivo imaging according to claim 6, characterized in that: A cutting groove (35) is provided on each of the extending ends (33) and the fixing teeth (34), so that the front end of the fixing block (32) has two extending ends (33) and two fixing teeth (34).

8. The mouse spinal cord fixation device for two-photon microscopy in vivo imaging according to claim 6, characterized in that: A second hand-tightening assembly (36) is provided at the other end of each fixing rod (31) located at the fixing tooth (34), that is, at the end of the fixing rod (31); Each group of the second hand-tightening components (36) has the same structure as the first hand-tightening components (24), that is, the second hand-tightening components (36) are also composed of a block (25), a hand-tightening column (26) and a half-thread bolt (27). The block (25) is fixedly arranged at the end of the fixing rod (31) in a concentric column state, and a threaded hole matching the half-thread bolt (27) is arranged at the end of the block (25). A square groove matching the size of the block (25) is arranged at the front end of the hand-tightening column (26), and a placement hole (261) matching the shape of the half-thread bolt (27) is arranged inside the hand-tightening column (26).

9. The mouse spinal cord fixation device for two-photon microscopy in vivo imaging according to claim 6, characterized in that: A glass slide holder (5) and a glass slide (51) are arranged between the first fixing member (3) and the second fixing member (4); The middle section of the glass slide holder (5) is a plate-shaped structure, a placement groove (52) for placing a glass slide (51) is provided in the middle of the glass slide holder (5), and the shapes of the two sides of the glass slide holder (5) match the shapes of the two fixing teeth (34) in the first fixing member (3) and the second fixing member (4) and the upper surface of the extension end (33); A slot (53) is provided at a position corresponding to each extension end (33) on both sides of the glass slide holder (5); a limit bolt (54) is provided at a position corresponding to the slot (53) on each extension end (33); each limit bolt (54) is half-threaded and located at the lower half of the limit bolt (54); a retaining ring (55) is fixedly provided on each limit bolt (54); and the distance between each retaining ring (55) and the head of the corresponding limit bolt (54) matches the thickness of both sides of the glass holder; Each extension end (33) is provided with a threaded hole matching the thread on the corresponding limiting bolt (54).

10. The mouse spinal cord fixation device for two-photon microscopy in vivo imaging according to claim 2, characterized in that: The bottom of the base (1) is provided with a screw (11), and a portion of the base (1) corresponding to each column (21) is provided with an embedding groove (12) matching the shape of the bottom end of the column (21), and each embedding groove (12) is provided with a nail hole penetrating the upper and lower ends of the base (1), and the bottom end of each column (21) is provided with a threaded hole matching the position of the nail hole in the corresponding embedding groove (12), and the threaded hole matches the thread of the screw (11).

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