WO2023102718A1 - 基于荧光显微镜的自由活动的脊髓成像方法、装置及应用 - Google Patents

基于荧光显微镜的自由活动的脊髓成像方法、装置及应用 Download PDF

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WO2023102718A1
WO2023102718A1 PCT/CN2021/136004 CN2021136004W WO2023102718A1 WO 2023102718 A1 WO2023102718 A1 WO 2023102718A1 CN 2021136004 W CN2021136004 W CN 2021136004W WO 2023102718 A1 WO2023102718 A1 WO 2023102718A1
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spinal cord
spine
fluorescence microscope
cover glass
fixing
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PCT/CN2021/136004
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English (en)
French (fr)
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巨富荣
蔚鹏飞
王立平
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中国科学院深圳先进技术研究院
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Priority to PCT/CN2021/136004 priority Critical patent/WO2023102718A1/zh
Publication of WO2023102718A1 publication Critical patent/WO2023102718A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
    • A61D3/00—Appliances for supporting or fettering animals for operative purposes

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  • the present invention relates to the technical field of spinal cord imaging, in particular to a free-moving spinal cord imaging method, device and application based on a fluorescence microscope.
  • the spinal cord is one of the important components of the central nervous system. As a bridge connecting the brain and the peripheral nervous system, the spinal cord transmits nerve information with the periphery to coordinate the normal activities of the living body, and plays an important role in the process of body sensation and movement.
  • the application of miniaturized in vivo imaging methods has made a breakthrough in the field of brain imaging, but the in vivo imaging of another key structure of the central nervous system—the spinal cord is still difficult to achieve. This is because the range of irregular movement of the spinal cord tissue caused by the mouse's own breathing, heartbeat and exercise is large, which makes it difficult to fix the spinal cord tissue during spinal cord imaging.
  • the current mainstream spinal cord imaging technology and method is to achieve imaging through anesthesia.
  • an animal A fixed observation device for living spinal nerve imaging and a method for using the same relate to the technical field of medical instruments.
  • the base includes: a base for placing animals, and a support fixedly arranged on the base
  • the fixing device includes: one end is detachably fixed - the fixing rod at the upper end of the supporting part, and the clamping part arranged on the other end of the fixing rod for fixing the vertebral body of the animal spine
  • the observation device includes: fixed on the clamping part The observation frame on the top, and the observation window arranged on the observation frame. Adopting the above-mentioned technical scheme has the advantages of good fixation effect, long-term observation, and avoiding multiple operations.
  • the above-mentioned technologies are mainly aimed at in vivo imaging under anesthesia, and cannot achieve long-term in vivo spinal cord imaging. They can only perform acute phase imaging of the spinal cord, and will fail due to spinal cord infection in long-term imaging; moreover, the current spinal cord Imaging is suitable for anesthesia imaging, under anesthesia conditions, the tissue displacement of the spinal cord due to heartbeat and respiration is small and does not affect in vivo imaging, but in awake animals, due to the drastic spinal tissue displacement, it leads to large motion artifacts , eventually leading to imaging failure.
  • a method for long-term two-photon imaging of the spinal cord of freely moving animals in an untethered environment is urgently to be developed.
  • the present invention provides a free-moving spinal cord imaging method, device and application based on a fluorescence microscope to solve at least one of the aforementioned technical problems.
  • a fluorescence microscope-based method for imaging the freely moving spinal cord comprising:
  • the deformed spinal cord was observed through a cover glass using a fluorescence microscope.
  • the "fixing the spine to be imaged by a fixing block” includes:
  • the clamping part is fixed to complete the fixation of the spine.
  • the "using a cover glass, set on the side of the fixed block away from the spine” includes:
  • Silica gel is used to fill between the cover glass and the spine to form a silica gel layer, so as to bond the cover glass and the fixing block as a whole.
  • a fluorescence microscope-based free-moving spinal cord imaging device comprising:
  • a pair of fixation blocks set opposite to each other, are used to clamp and fix the exposed spine from both sides;
  • the cover glass is arranged on the side of the fixed block away from the vertebra, and is used to close the spinal cord cavity of the vertebra and deform the spinal cord under the condition of ensuring the integrity of the spinal cord;
  • a silica gel layer arranged between the fixing block and the cover glass, for connecting the fixing block and the cover glass;
  • a fluorescence microscope is set above the cover glass for observing the deformed spinal cord.
  • the fixing block is provided with a slot facing the spine for fixing the spine.
  • the surface of the groove is provided with a drainage groove for discharging seepage.
  • the free-moving spinal cord imaging device based on a fluorescence microscope also includes:
  • a fixing part arranged above the cover glass, for connecting to a fluorescence microscope
  • An observation window is provided on the fixing member for facilitating the fluorescence microscope to observe the deformed spinal cord.
  • a fluorescence microscope-based spinal cord imaging setup for freely moving mice comprising:
  • a fixation instrument matched with the spinal cord imaging device, is used to adjust the position of the fixation block in the spinal cord imaging device to clamp the spine to be observed;
  • a blind hole is provided at the end of the fixed block away from the observation spine;
  • the fixing instrument matches the blind hole and is used to control the moving direction of the fixing block.
  • the fixation instrument includes:
  • a rod sleeve is arranged in the circumferential direction of the column and the axis of the rod sleeve is perpendicular to the axis of the column;
  • An adjustment rod arranged in the rod sleeve, is used to adjust the position of the fixed block
  • a locking bolt is arranged on the rod sleeve and is used to fix the adjusting rod;
  • the adjustment rod corresponds to the blind hole and is used for guiding the movement of the fixed block.
  • the free-moving spinal cord imaging method based on the fluorescence microscope of the present invention breaks through the limitations of anesthetized animals, fixes the spinal cord with a fixation block, and compresses the spinal cord to deformation with a cover glass, while ensuring the integrity of the spinal cord, Make it deform, compress its activity space, and realize the imaging of the spinal cord under the condition of free movement of the animal; the method has a simple process and has good imaging effect.
  • the freely movable spinal cord imaging device based on the fluorescence microscope of the present invention cooperates with the fixation block through the fixation instrument, uses the fixation instrument to fix the spine from both sides of the spine with a pair of fixation blocks, and then uses the cover glass The spinal cord is compressed until deformed, and the spinal cord is deformed while ensuring the integrity of the spinal cord. Finally, a fluorescent microscope is connected through a fixture to realize spinal cord imaging.
  • the device has the advantages of simple structure, convenient use and easy processing and manufacture.
  • Fig. 1 is the structural representation of fixed block described in the present invention
  • Fig. 2 is the schematic diagram that fixation block is implanted into spinal cord
  • FIG. 3 is a schematic diagram of the spinal cord imaging method of the present invention.
  • Fig. 4 is a figure of operation steps of the spinal cord imaging method of the present invention.
  • Fig. 5 is the structural representation of fixture among the present invention.
  • Figure 6 is a schematic diagram of a freely moving in vivo spinal cord imaging mouse
  • Fig. 7 is another embodiment of the fixed block described in Fig. 1;
  • Fig. 8 is a specific implementation of the adjustment rod.
  • modules in the devices in the implementation scenario can be distributed among the devices in the implementation scenario according to the description of the implementation scenario, or can be located in one or more devices different from the implementation scenario according to corresponding changes.
  • the modules of the above implementation scenarios can be combined into one module, or can be further split into multiple sub-modules.
  • mice used as the experimental object, and carries out the following examples to further illustrate the present invention:
  • the present invention provides an embodiment: as shown in Figures 1 to 4, a method for freely moving spinal cord imaging based on a fluorescence microscope, comprising: taking a living animal, exposing the spine at the position to be imaged through spinal cord surgery, and performing a spinal cord imaging on the surface of the spine Clean up; fix the spine; expose the anterior central artery and white matter of the spinal cord at the position of the spine, form the exposed spinal cord cavity, and seal the exposed spinal cord cavity, and deform the spinal cord while ensuring the integrity of the spinal cord and performing spinal cord imaging of said vertebral location using two-photon fluorescence microscopy.
  • Step 1 After exposing the 3-6 segments of the lumbar spine of the mouse through spinal cord surgery, after cleaning up the tissue attached to the surface of the spinal bone, use the fixation block 1 to stably embed it in the position below the condyle of the spine; in order to reduce motion artifacts as much as possible , place the complete transverse process above the fixed blocks on both sides, and adjust the fixed block 1 until the shaking of the vertebrae is basically eliminated;
  • Step 2 Use a skull drill to polish the spinal plate to expose the anterior central artery and surrounding spinal cord white matter, clean the surface of the spinal cord with sterile saline, and then seal the exposed spinal cord cavity with silica gel and cover glass 3;
  • Step 3 Since the spinal cord shakes greatly during movement, compress the spinal cord through the cover glass 3, and deform it while ensuring the integrity of the spinal cord, further reducing motion artifacts;
  • Step 4 Install the miniature two-photon microscope, such as the fluorescence microscope 5, stably on the fixture 6.
  • the components of the whole device are small enough and light enough to be carried by the mouse;
  • a 3-7 mm glass cover glass 3 is used to compress the spinal cord, and deform the spinal cord while ensuring the integrity of the spinal cord.
  • the present invention also provides an embodiment: a freely movable spinal cord imaging device based on a fluorescent microscope, comprising: a pair of fixed blocks 1, a cover glass 3, a silica gel layer 4, a fluorescent microscope 5 and a fixing member 6; wherein, one The fixing block 1 is arranged oppositely, and is used for clamping and fixing the exposed vertebra 2 from both sides; the cover glass 3 is arranged on the side of the fixing block 1 away from the vertebra 2, and is used for closing the spinal cord cavity of the vertebra and Under the condition of ensuring the integrity of the spinal cord, the spinal cord is deformed; the silica gel layer 4 is arranged between the fixed block 1 and the cover glass 3 for connecting the fixed block 1 and the cover glass 3 The fluorescence microscope 5 is arranged above the cover glass 3 for observing the deformed spinal cord; the fixture 6 is arranged above the cover glass 3 for connecting the fluorescence microscope; the fixture 6 is set There is an observation window 5A for facilitating the fluorescence microscope 5 to observe the deformed spinal cord.
  • the fixed block 1 is provided with a slot 2A facing the spine 2 for fixing the spine 2; various structures can be selected for the slot 2A, preferably, a V-shape as shown in Figure 1 , the spine 2 can be stably clamped through two pairs of slots 2A.
  • the side wall of the slot 2A is provided with a drainage groove 2B for flowing out the leakage; one end of the drainage groove 2B is connected to the The notch end of the slot 2A of the fixing block 1 is connected;
  • the present invention provides an embodiment: a spinal cord imaging device based on a fluorescent microscope for free-moving mice, including: the spinal cord imaging device as described in Embodiment 1, and a fixation instrument 7; wherein, the fixation instrument 7, the spinal cord
  • the imaging device is matched, and is used to adjust the position of the fixed block 1 in the spinal cord imaging device to clamp the spine to be observed; the end of the fixed block 1 away from the observed spine is provided with a blind hole 1B; the adjustment in the fixation instrument
  • the end of the rod 701 corresponds to the blind hole 1B and is used to control the moving direction of the fixed block 1 .
  • the fixed instrument 1 includes: an adjustment rod 701, a locking bolt 702, a base 703, two opposite columns 704, and a rod cover 705; wherein, the column 704 is vertically arranged on the base 703; the rod cover 705 is set in the circumferential direction of the column 704 and the axis of the rod sleeve 705 is perpendicular to the axis of the column 704; the adjustment rod 701 is arranged in the rod sleeve 705, and the operator can push and pull the adjustment rod 701 to adjust the position of the fixed block. Position; the locking bolt 702 is arranged on the rod sleeve 705 for fixing the adjusting rod 701; the adjusting rod 701 corresponds to the blind hole 1B and is used for guiding the movement of the fixing block.
  • the present invention discloses other alternatives of the fixed block 1: compared with the specific embodiment 1, the fixed block 1 is provided with a slot 2A towards the vertebra 2, and the slot 2A is arc-shaped, The spine 2 can be stably clamped through the two pairs of slots 2A.
  • the adjusting rod 701 in specific embodiment III can be cylindrical in Fig. 5, and the end of this adjusting rod 701 has a tip, and this tip can be conical in Fig. 5, and with blind hole 1B corresponds; at this time, the blind hole 1B can be a square hole in Figure 2 or a circular hole as required;
  • the above-mentioned tip can also be a quadrangular pyramid corresponding to the blind hole 1B in FIG. 2 .
  • the adjustment rod 701 is stepped as shown in Figure 8; the square section 701A at the small end of the ladder corresponds to the blind hole 1B in Figure 2; the middle section 701B is round and matches the rod sleeve 705; the hat-shaped section The size of 701C is larger than the inner diameter of the rod sleeve 705 to prevent the adjustment rod 701 from coming out of the rod sleeve 705 .

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Abstract

一种基于荧光显微镜（5）的自由活动的脊髓成像方法、装置及应用，包括：通过固定块（1）固定待成像的脊椎（2）；将盖玻片（3）设在固定块（1）中间位置，压紧脊髓；利用荧光显微镜（5），在自由移动的情况下，通过盖玻片（3）观测稳定固定的脊髓。通过脊髓手术暴露出腰段3～6节段脊柱（2）后，借助固定块（1）嵌入脊椎（2）横突下方位置，并将完整的横突置于两侧凹型槽上方，消除椎骨的抖动；采用颅骨钻打磨脊椎板，暴露出中央前动脉血管和周围脊髓，利用硅胶和盖玻片（3）封闭暴露的脊髓腔；通过玻璃片压迫脊髓，在保证脊髓完整的情况下，使其产生形变；利用牙科水泥封闭暴露的脊椎骨及手术窗口，最后通过荧光显微镜（5）对脊髓成像；流程简单，具有良好的成像效果。

Description

基于荧光显微镜的自由活动的脊髓成像方法、装置及应用 技术领域
本发明涉及一种脊髓成像技术领域，尤其涉及一种基于荧光显微镜的自由活动的脊髓成像方法、装置及应用。
背景技术
脊髓是中枢神经系统的重要组成结构之一。脊髓作为连接大脑与周围神经系统的桥梁，传送与外周之间的神经信息以协调生命体正常活动，在躯体感觉和运动过程中发挥重要作用。微型化活体成像方法的应用使脑成像领域的相关研究取得了突破性进展，但中枢神经系统的另一关键结构—脊髓的活体成像研究仍难以实现。这是因为小鼠自身呼吸、心跳以及运动引起的脊髓组织无规则运动范围较大，导致脊髓成像时对脊髓组织固定困难。加之双光子显微镜成像速度和光学薄片有限，导致小鼠在体脊髓成像时出现焦面漂移、图像扭曲或丢失等问题。目前，已报道的关于脊髓光学成像的研究对象多为麻醉动物，鲜有清醒的自由活动状态的动物。所以脊髓这一重要的神经结构在真实环境下如何编码大脑下行指令和外周输入信号尚不清楚。建立一套在自由活动状态下的动物活体脊髓成像方法，是进一步探索脊髓在神经系统中重要功能的关键途径之一。
目前，自由活动状态下的动物活体脊髓成像仍止步于单光子显微成像。相较而言，双光子显微成像不仅解决了标记密集的组织中信号密集导致无法分辨甚至无法聚焦等问题，显著提高了图像比度和在生物体组织内的穿透深度，而且能够实现亚细胞的形态结构高清晰成像。2017年，程和平团队自主研发的微 型化双光子荧光显微镜打破了仪器对动物成像研究的限制，能够在动物自由活动过程中实现快速的组织或细胞双光子活体成像。目前，高分辨率微型化双光子荧光显微镜的应用，使国内外自由活动动物在体成像领域的相关研究取得了突破性进展，也为实现在小鼠自由活动状态下的脊髓双光子成像带来了契机。
但是，脊髓结构的特异性带来的诸多问题，是实现自由活动的动物脊髓成像的一大阻碍。其中，脊髓结构复杂、脊髓组织运动幅度较大且不规则、长期成像不稳定以及微型化显微镜稳定植入困难等，导致脊髓活体成像研究面临一系列挑战。
目前主流的脊髓成像技术和方法是通过麻醉来实现成像，例如，申请公布号为CN202110686690.0的中国专利《一种动物脊髓活体成像的固定观察装置及其使用方法》中，介绍了一种动物脊神经活体成像的固定观察装置及其使用方法，它涉及医疗器械技术领域。它包括：基座、固定装置和观察装置；所述基座包括：用于供动物放置的底座，以及，固定设置于所述底座上的支撑件；所述固定装置包括：一端可拆卸地固定-在所述在支撑件上端的固定杆，以及，设置于所述固定杆另一端上的、用于固定动物脊柱椎体的夹持件；所述观察装置包括：固定在所述夹持件上的观察框，以及，设置于所述观察框上的观察窗。采用上述技术方案，具有固定效果好，可长时程观察，且避免多次手术的优势。
可是，上述提到的技术主要针对于麻醉状态下的活体成像，无法实现长期的脊髓活体成像，只能进行脊髓的急性期成像，在长期成像中会由于脊髓感染而失败；而且，目前的脊髓成像适合麻醉成像，在麻醉条件下，由于心跳和呼吸导致的脊髓的组织位移很小，不影响活体成像，但在清醒的动物中，由于剧烈的脊髓组织位移，导致幅度较大的运动伪影，最终导致成像失败。
一种能够实现无束缚环境中对自由活动的动物脊髓长期的双光子成像方法急待研发。
发明内容
为了克服现有技术的不足，本发明提供了一种基于荧光显微镜的自由活动的脊髓成像方法、装置及应用，用于解决前述技术问题中的至少一个。
具体地，其技术方案如下：
一种基于荧光显微镜的自由活动的脊髓成像方法，包括：
通过固定块固定待成像的脊椎；
利用盖玻片，设置在所述固定块远离所述脊椎的一侧，压紧所述脊椎；
利用荧光显微镜，通过盖玻片观测产生形变的所述脊髓。
所述“通过固定块固定待成像的脊椎”，包括：
通过夹持部件左右两侧相对夹持所述脊椎；
固定所述夹持部件，完成所述脊椎的固定。
所述“利用盖玻片，设置在所述固定块远离所述脊椎的一侧”，包括：
对固定后的所述脊椎进行消毒；
采用硅胶填充在盖玻片与所述脊椎之间，形成硅胶层，以将所述盖玻片与所述固定块粘结为一体。
一种基于荧光显微镜的自由活动的脊髓成像装置，包括：
一对固定块，相对设置，用于从两侧夹持固定暴露的脊椎；
盖玻片，设置在所述固定块远离所述脊椎的一侧，用于封闭所述脊椎的脊髓腔并在保证脊髓完整的情况下，使所述脊髓产生形变；
硅胶层，设置在所述固定块与所述盖玻片之间，用于连接所述固定块与所述盖玻片；
荧光显微镜，设置在所述盖玻片上方，用于观测产生形变的所述脊髓。
所述固定块上设置有朝向所述脊椎的开槽，用于固定所述脊椎。
所述开槽的表面上设置有用于流出渗液的引流槽。
所述的一种基于荧光显微镜的自由活动的脊髓成像装置，还包括：
固定件，设置在所述盖玻片上方，用于连接荧光显微镜；
所述固定件上设置有观察窗，用于便于所述荧光显微镜观察变形的所述脊髓。
一种基于荧光显微镜的自由活动小鼠的脊髓成像装置，包括：
如上所述的脊髓成像装置；
固定仪，与所述脊髓成像装置匹配，用于调整所述脊髓成像的装置中的固定块的位置以夹持待观测脊椎；
所述固定块远离所述观测脊椎的一端设置有盲孔；
所述固定仪与所述盲孔匹配，用于控制所述固定块的移动方向。
所述固定仪，包括：
底座；
两个立柱，相对的设置在所述底座上；
杆套，设置在所述立柱的周向上且所述杆套的轴线与所述立柱的轴线垂直；
调整杆，设置在所述杆套内，用于调整固定块的位置；
锁紧螺栓，设置在所述杆套上，用于固定所述调整杆；
所述调整杆与所述盲孔对应，用于对所述固定块的移动导向。
一种如上所述的基于荧光显微镜的自由活动的脊髓成像方法在小鼠脊髓成像方向上的应用。
本发明至少具有以下有益效果：
本发明所述的基于荧光显微镜的自由活动的脊髓成像方法，突破了麻醉动物的限制，利用固定块对脊髓进行固定，并使用盖玻片将脊髓压至变形，在保证脊髓完整的情况下，使其产生形变，压缩其活动空间，实现在动物自由活动的情况下，对脊髓进行成像；本方法流程简单，具有良好的成像效果。
本发明所述的基于荧光显微镜的自由活动的脊髓成像装置，通过固定仪与固定块进行配合，利用固定仪将一对固定块从脊柱的两侧固定所述脊柱，然后，再利用盖玻片将脊髓压至变形，在保证脊髓完整的情况下，使其产生形变，最后，通过固定件连接荧光显微镜，实现脊髓成像。本装置结构简单，使用方便，易于加工制作。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明所述固定块的结构示意图；
图2为固定块植入脊髓的示意图；
图3为本发明所述脊髓成像方法的示意图；
图4为本发明所述脊髓成像方法的手术操作步骤图；
图5为本发明中固定仪的结构示意图；
图6为自由移动的活体脊髓成像小鼠示意图；
图7为图1所述固定块的另一种实施方式；
图8为调整杆的一种具体实施方式。
其中，1.固定块；2.脊椎；3.盖玻片；4.硅胶层；5.荧光显微镜；6.固 定件；7.固定仪；1B.盲孔；2A.开槽；2B.引流槽；2C.缺口；6A.观察窗；701.调整杆；702.锁紧螺栓；703.底座；704.立柱；705.杆套；701A.方形段；701B.中间段；701C.帽形段。
具体实施方式
本领域技术人员可以理解实施场景中的装置中的模块可以按照实施场景描述进行分布于实施场景的装置中，也可以进行相应变化位于不同于本实施场景的一个或多个装置中。上述实施场景的模块可以合并为一个模块，也可以进一步拆分成多个子模块。
为了解决现有技术的问题，本发明以小鼠作为实验目标，进行以下实施例，对本发明进行进一步的说明：
具体实施例I：
本发明提供一种实施例：如图1～4，一种基于荧光显微镜的自由活动的脊髓成像方法，包括：取活体动物，通过脊髓手术暴露待成像位置的脊椎，并对所述脊椎表面进行清理；固定所述脊椎；暴露所述脊椎位置的中央前动脉血管和脊髓白质，形成暴露的脊髓腔，并封闭所述暴露的脊髓腔，在保证脊髓完整的情况下，使所述脊髓产生形变；利用双光子荧光显微镜对所述脊椎位置进行脊髓成像。
具体步骤如下：
步骤一：通过脊髓手术暴露出小鼠腰段3～6节段脊柱后，清理干净脊柱骨头表面附着的组织后，借助固定块1，稳定嵌入脊椎骨突下方位置；为了尽可能的减少运动伪影，将完整的横突置于两侧的固定块的上方，调整固定块1直至基本消除椎骨的抖动；
步骤二：采用颅骨钻打磨脊椎板，暴露出中央前动脉血管和周围脊髓白质， 采用无菌生理盐水清理干净脊髓表面后，之后采用硅胶和盖玻片3封闭暴露的脊髓腔；
步骤三：由于脊髓在运动时存在大幅度的抖动，通过盖玻片3压迫脊髓，在保证脊髓完整的情况下，使其产生形变，进一步减少运动伪影；
步骤四：将微型双光子显微镜，如荧光显微镜5，稳定的安装在固定件6上，整个装置的部件足够小，足够轻，方便老鼠携带；
优选的，采用3-7mm的玻璃的盖玻片3压迫所述脊髓，在保证所述脊髓完整的情况下，使所述脊髓产生形变。
具体实施例II：
本发明还提供一种实施例：一种基于荧光显微镜的自由活动的脊髓成像装置，包括：一对固定块1、盖玻片3、硅胶层4、荧光显微镜5以及固定件6；其中，一对固定块1相对设置，用于从两侧夹持固定暴露的脊椎2；盖玻片3设置在所述固定块1远离所述脊椎2的一侧，用于封闭所述脊椎的脊髓腔并在保证脊髓完整的情况下，使所述脊髓产生形变；硅胶层4设置在所述固定块1与所述盖玻片3之间，用于连接所述固定块1与所述盖玻片3；荧光显微镜5设置在所述盖玻片3上方，用于观测产生形变的所述脊髓；固定件6设置在所述盖玻片3上方，用于连接荧光显微镜；所述固定件6上设置有观察窗5A，用于便于所述荧光显微镜5观察变形的所述脊髓。
为了方便夹持，所述固定块1上设置有朝向所述脊椎2的开槽2A，用于固定所述脊椎2；开槽2A可选择多种结构，优选的，如附图1的V字形，通过两个成对的开槽2A能够稳定的夹持脊椎2。
在植入固定块1和放入硅胶的过程中，容易渗液，为了便于渗液流出，所述开槽2A的侧壁上设置有用于流出渗液的引流槽2B；引流槽2B的一端与固定 块1的开槽2A的缺口端连通；另一端设置在开槽2A的底部；固定块1上还设置有用于与盖玻片3匹配的缺口2C。
具体实施例III：
本发明提供一种实施例：一种基于荧光显微镜的自由活动小鼠的脊髓成像装置，包括：如具体实施例I所述的脊髓成像装置、固定仪7；其中，固定仪7，所述脊髓成像装置匹配，用于调整所述脊髓成像的装置中的固定块1的位置以夹持待观测脊椎；所述固定块1远离所述观测脊椎的一端设置有盲孔1B；固定仪中的调整杆701的端部与所述盲孔1B对应，用于控制所述固定块1的移动方向。
具体的，所述固定仪1，包括：调整杆701、锁紧螺栓702、底座703、相对设置的两个立柱704、杆套705；其中，立柱704垂直设置在所述底座703上；杆套705设置在所述立柱704的周向上且所述杆套705的轴线与所述立柱704的轴线垂直；调整杆701设置在所述杆套705内，操作人员可以推拉调整杆701调整固定块的位置；锁紧螺栓702设置在所述杆套705上，用于固定所述调整杆701；所述调整杆701与所述盲孔1B对应，用于对所述固定块的移动导向。
具体实施例IV：
如图7，本发明公开固定块1的其他的替代方案：与具体实施例I相比，所述固定块1上设置有朝向所述脊椎2的开槽2A，开槽2A为圆弧形，通过两个成对的开槽2A能够稳定的夹持脊椎2。
具体实施例V：在具体实施例III中的调整杆701可以是图5中的圆柱形，该调整杆701的端部带有尖端，该尖端可以为图5中的圆锥形，并与盲孔1B对应；此时盲孔1B可以是图2中的方形孔也可以根据需要设置为圆形孔；
作为替代方案，上述的尖端也可以是与图2中的盲孔1B对应的四棱锥形。
优选的，调整杆701为如图8所示的阶梯形；位于阶梯小尺寸一端的方形段701A与图2中的盲孔1B对应；中间段701B为圆形与杆套705匹配；帽形段701C的尺寸大于杆套705的内径，防止调整杆701从杆套705中脱出。
以上公开的仅为本发明的几个具体实施场景，但是，本发明并非局限于此，任何本领域的技术人员能思之的变化都应落入本发明的保护范围。上述本发明序号仅仅为了描述，不代表实施场景的优劣。

Claims (10)

  1. 一种基于荧光显微镜的自由活动的脊髓成像方法，其特征在于，包括：
    通过固定块固定待成像的脊椎；
    利用盖玻片，设置在所述固定块远离所述脊椎的一侧，压紧所述脊椎；
    利用荧光显微镜，通过盖玻片观测产生形变的所述脊髓。
  2. 根据权利要求1所述的一种基于荧光显微镜的自由活动的脊髓成像方法，其特征在于，所述“通过固定块固定待成像的脊椎”，包括：
    通过夹持部件左右两侧相对夹持所述脊椎；
    固定所述夹持部件，完成所述脊椎的固定。
  3. 根据权利要求1所述的一种基于荧光显微镜的自由活动的脊髓成像方法，其特征在于，所述“利用盖玻片，设置在所述固定块远离所述脊椎的一侧”，包括：
    对固定后的所述脊椎进行消毒；
    采用硅胶填充在盖玻片与所述脊椎之间，形成硅胶层，以将所述盖玻片与所述固定块粘结为一体。
  4. 一种基于荧光显微镜的自由活动的脊髓成像装置，其特征在于，包括：
    一对固定块，相对设置，用于从两侧夹持固定暴露的脊椎；
    盖玻片，设置在所述固定块远离所述脊椎的一侧，用于封闭所述脊椎的脊髓腔并在保证脊髓完整的情况下，使所述脊髓产生形变；
    硅胶层，设置在所述固定块与所述盖玻片之间，用于连接所述固定块与所述盖玻片；
    荧光显微镜，设置在所述盖玻片上方，用于观测产生形变的所述脊髓。
  5. 根据权利要求4所述的一种基于荧光显微镜的自由活动的脊髓成像装置，其特征在于：
    所述固定块上设置有朝向所述脊椎的开槽，用于固定所述脊椎。
  6. 根据权利要求5所述的一种基于荧光显微镜的自由活动的脊髓成像装置，其特征在于：
    所述开槽的表面上设置有用于流出渗液的引流槽。
  7. 根据权利要求4所述的一种基于荧光显微镜的自由活动的脊髓成像装置，其特征在于，还包括：
    固定件，设置在所述盖玻片上方，用于连接荧光显微镜；
    所述固定件上设置有观察窗，用于便于所述荧光显微镜观察变形的所述脊髓。
  8. 一种基于荧光显微镜的自由活动小鼠的脊髓成像装置，其特征在于，包括：
    如权利要求4-7任一权利要求所述的脊髓成像装置；
    固定仪，与所述脊髓成像装置匹配，用于调整所述脊髓成像的装置中的固定块的位置以夹持待观测脊椎；
    所述固定块远离所述观测脊椎的一端设置有盲孔；
    所述固定仪与所述盲孔匹配，用于控制所述固定块的移动方向。
  9. 根据权利要求8所述的基于荧光显微镜的自由活动小鼠的脊髓成像装置，其特征在于，所述固定仪，包括：
    底座；
    立柱，设置在所述底座上；
    杆套，设置在所述立柱的周向上且所述杆套的轴线与所述立柱的轴线垂直；
    调整杆，设置在所述杆套内，用于调整固定块的位置；
    锁紧螺栓，设置在所述杆套上，用于固定所述调整杆；
    所述调整杆与所述盲孔对应，用于对所述固定块的移动导向。
  10. 一种如权利要求1～3任一权利要求所述的基于荧光显微镜的自由活动的脊髓成像方法在小鼠脊髓成像方向上的应用。
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