WO2022126685A1 - 在体观察动物体骨中毛细血管的系统和方法 - Google Patents
在体观察动物体骨中毛细血管的系统和方法 Download PDFInfo
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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
- A61B5/48—Other medical applications
- A61B5/4887—Locating particular structures in or on the body
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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
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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
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
Definitions
- the invention belongs to the technical field of medical imaging, and in particular relates to a system for in vivo observation of capillaries in animal body bones and a corresponding method.
- Vascular endothelial cells are monolayer cells distributed in the inner wall of blood vessels, with rich heterogeneity and organ specificity. Through dense capillary branches, the vascular endothelium establishes connections with nearly all cells in various organs. Vascular endothelial cells not only serve as a pipeline system to transport nutrients and take away metabolic wastes, but also play an important role in the growth, development and homeostasis of various organs by secreting vascular secretion factors. Capillaries in different organs have different shapes, and capillaries in different locations and functions in the same organ have different shapes, which reflect the characteristics of different subtypes of endothelial cells. With the occurrence of age or disease, its morphology will also change, and its homeostasis maintenance effect on organs will also change accordingly. Therefore, observing the morphology of capillaries can provide information on the state of the corresponding organ.
- the current technology is mainly based on in vitro observation.
- the bone is cut into slices ranging from 30 microns to 300 microns, and the morphology of the capillaries is observed by light microscope or confocal microscope after specific fluorescent staining of capillaries.
- the isolated bone is made transparent after chemical treatment, and then the blood vessels are specifically fluorescently stained, and then photographed by a surface-selective illumination fluorescence microscope, and the image of capillaries is obtained by digital processing in the later stage.
- Existing in vitro observation of capillary morphology in bone limits its application range, and it is necessary to explore in vivo observation methods in order to obtain more abundant real-time observation results.
- the present invention provides a system and method for in vivo observation of capillaries in bones of animals, so as to achieve stable imaging of capillaries in bones in vivo.
- one aspect of the present invention is to provide a system for in vivo observation of capillaries in animal bones, the system includes a fixation device, a polishing device and a two-photon microscope; the fixation device is connected to the On the stage of the two-photon microscope, it is used to fix the animal body to be observed; the grinding device is used to grind the exposed bones of the animal body to be observed to thin the cortical bone; the two-photon microscope is used for grinding Image the polished bone to see the capillaries in it;
- the fixing device includes a carrier substrate, two or more supporting arms connected to the carrier substrate, and a clamping arm detachably assembled on the supporting arms, and a locking member is provided on the supporting arms , used to lock the clamping arm assembled on the support arm.
- a groove is formed on the top of the support arm, and the clamping arm is fitted into the groove.
- the number of the support arms is two or three, and each of the support arms is respectively provided with one of the clamping arms.
- the carrier substrate is provided with elongated through holes.
- one end of the elongated through hole communicates with the edge of the carrier substrate.
- the support arm is configured as a height-adjustable support arm.
- the support arm is configured as a support arm movable on the plane of the carrier substrate.
- Another aspect of the present invention is to provide a method for in vivo observation of capillaries in animal bones, using the system for in vivo observation of capillaries in animal bones to observe capillaries, the method comprising:
- the target observation area After anesthetizing the body of the animal to be observed, the target observation area is depilated, and then the skin and muscles are cut to expose the bones of the target observation area;
- An vascular imaging agent is injected into the animal to be observed, and then the two-photon microscope is activated to image the polished bone to observe the capillaries therein.
- the system and method for in vivo observation of capillaries in animal bones utilize the characteristics of deep imaging with two-photon microscopy, less damage to living cells, and longer imaging time to observe intrabony capillaries in vivo,
- the imaging position is deep and the imaging is stable;
- the animal body to be observed is fixed by designing a fixing device with a specific structure, which overcomes the problem of image displacement caused by the breathing and heartbeat of the living body, and makes the imaging more stable;
- the exposed bone to be observed is polished and thinned by using a polishing device
- the cortical bone increases the depth of the imaging position, and can image and observe the capillaries in the deeper position.
- FIG. 1 is a schematic structural diagram of a system for in vivo observation of capillaries in animal body bones according to an embodiment of the present invention
- Figures 2 and 3 are illustrations of intraosseous capillary imaging of experimental mice in an embodiment of the present invention.
- the embodiment of the present invention first provides a system for observing capillaries in animal body bone in vivo, referring to FIG.
- the two-photon microscope 1 includes a stage 11 and an imaging lens 12.
- the fixing device 1 is connected to the stage 11 of the two-photon microscope 1, and is used to fix the object to be observed.
- the imaging lens 12 of the microscope 1 is used to image the polished bone to observe the capillaries therein.
- the grinding device is used for grinding the exposed bones of the animal body to be observed, so as to thin the cortex of the bone, so as to increase the depth of the imaging position, and the intraosseous capillaries in the deeper position can be imaged and observed.
- the fixing device 2 includes a carrier substrate 21 , two or more support arms 22 connected to the carrier substrate 21 , and a clamping arm detachably assembled on the support arms 22 .
- the support arm 22 is provided with a locking member 24 for locking the clamping arm 23 assembled on the support arm 22 .
- the number of the support arms 22 is two or three, and each of the support arms 22 is configured with one of the clamping arms 23 respectively.
- the fixation device 2 shown in FIG. 1 comprises three support arms 22, and the area between the three support arms 22 is formed as a space for placing the animal body to be observed.
- the support arm 22 is configured as a height-adjustable support arm, so that the height of the space for placing the animal object to be observed is adjustable, so as to be suitable for animal objects of different sizes.
- the support arm 22 is configured as a movable support arm on the plane of the carrier substrate 21, so that the lateral width for placing the animal to be observed is adjustable to suit different body sizes. animal body.
- a groove 25 is formed on the top of the support arm 22 , the clamping arm 23 is assembled in the groove 25 , and the locking member 24 is set as a thread locking piece.
- an elongated through hole 26 is formed on the carrier substrate 21 , and the fixing device 2 can be fixedly connected through the elongated through hole 26 and a threaded fastener.
- the fixing device 2 can be fixedly connected through the elongated through hole 26 and a threaded fastener.
- one end of the elongated through hole 26 communicates with the edge of the carrier substrate 21 .
- Another aspect of the present invention is to provide a method for in vivo observation of capillaries in animal bones, using the system for in vivo observation of capillaries in animal bones to observe capillaries.
- the animal body to be observed is an experimental mouse as an example for description.
- the method includes the following steps:
- the fixing device 2 is fixedly connected to the stage 11 of the two-photon microscope 1 .
- a micro-drill is used to gently remove part of the cortical bone, with the limit of breaking the vascular structure inside the bone without grinding bleeding.
- TRITC-labeled dextran TRITC-Dextran, average molecular weight 65000-85000, final injection concentration 0.1 mg/kg
- mice After the imaging, the muscle and skin tissues of the experimental mice were sutured, so that the mice could survive after recovery from anesthesia, and the same mice could be used to repeat the related experiments afterwards.
- FIG. 2 and FIG. 3 are illustrations of intraosseous capillary imaging of the thigh region of experimental mice in an embodiment of the present invention.
- Figure 2 shows the capillaries of the arched column structure near the growth plate (gp) of the upper epiphysis (mp)
- Figure 3 shows the capillaries of the diaphyseal end (dp) network structure. It can be known from FIG. 2 and FIG. 3 that the system and method provided by the embodiments of the present invention can realize in vivo observation of capillaries of different shapes in animal body bones.
- vascular endothelial cells in the arched columnar structure near the epiphyseal (mp) growth plate (gp) mainly promote bone formation
- the diaphyseal (dp) reticular Structural blood vessels have a regulatory effect on hematopoiesis of bone marrow cells. Therefore, by distinguishing different shapes of intraosseous capillaries, the heterogeneity of capillary endothelial cells can be judged and their physiological state can be judged.
- the system and method for in vivo observation of capillaries in animal bones utilize the characteristics of deep imaging with two-photon microscopy, less damage to living cells, and longer imaging capability to observe living cells.
- Intraosseous capillaries the imaging position is deeply incised, and the imaging is stable; by designing a fixed device with a specific structure to fix the animal body to be observed, the problem of image displacement caused by living breathing and heartbeat is overcome, and the imaging is more stable; the exposure to be observed is exposed by using a polishing device
- the bone is polished to thin the cortical bone, which increases the depth of the imaging position and enables imaging and observation of the capillaries in the bone at a deeper position.
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Abstract
一种在体观察动物体骨中毛细血管的系统和方法，所述系统包括固定装置(2)、打磨装置和双光子显微镜(1)；所述固定装置(2)连接于所述双光子显微镜(1)的载物台(11)上，用于对待观察的动物体进行固定；所述打磨装置用于对待观察的动物体暴露出的骨头进行打磨，以减薄骨皮质；所述双光子显微镜(1)用于对打磨后的骨头部位进行成像，以观察其中的毛细血管；其中，所述固定装置(2)包括承载基板(21)、连接在所述承载基板(21)上的两个以上的支撑臂(22)以及可拆卸地装配于所述支撑臂(22)上的夹持臂(23)，所述支撑臂(22)上设置有锁紧件(24)，用于锁紧装配在所述支撑臂(22)上的夹持臂(23)。本系统和方法能够实现动物活体的骨内毛细血管的稳定成像，在体实时观察到丰富的骨内毛细血管形态。
Description
本发明属于医学成像技术领域，具体涉及一种在体观察动物体骨中毛细血管的系统以及相应的方法。
血管内皮细胞是分布在血管内壁的单层细胞，具有丰富的异质性与器官特异性。通过密集的毛细血管分支，血管内皮在各个器官内与几乎所有的细胞建立联系。血管内皮细胞不单单作为管道系统运输营养物质和带走代谢废物，更通过分泌血管分泌因子对各个器官的生长发育和稳态维持有着重要作用。不同器官内的毛细血管有着不同的形态，同一器官内不同位置和功能的毛细血管形态也有所不同，它反映了不同亚型的内皮细胞的特性。随着年龄或者疾病的发生，其形态也会发生变化，其对器官产生的稳态维持作用也会相应出现变化。因此，观察毛细血管的形态可以提供相应器官的状态信息。
毛细血管形态的观察有离体和在体两种方式，由于骨内毛细血管位置较深，目前现有的技术主要以离体观察为主。例如将离体的骨脱钙后将骨切成30微米至300微米的薄片，通过对毛细血管进行特异性荧光染色后通过光学显微镜或者共聚焦显微镜观察其形态。又例如，将离体的骨进行化学处理后透明化，再对血管进行特异性荧光染色后通过择面照明萤光显微镜进行拍摄，后期数字化处理得到毛细血管的成像。现有的离体观察骨内毛细血管形态的技术局限了它的应用范围，需要探索在体的观察方法，以期得到更为丰富的实时观察结果。
发明内容
鉴于现有技术存在的不足，本发明提供一种在体观察动物体骨中毛细血管的系统和方法，以能够做到活体的骨内毛细血管的稳定成像。
为实现上述发明目的，本发明的一方面是提供了一种在体观察动物体骨中毛细血管的系统，所述系统包括固定装置、打磨装置和双光子显微镜；所述固定装置连接于所述双光子显微镜的载物台上，用于对待观察的动物体进行固定； 所述打磨装置用于对待观察的动物体暴露出的骨头进行打磨，以减薄骨皮质；所述双光子显微镜用于对打磨后的骨头部位进行成像，以观察其中的毛细血管；
其中，所述固定装置包括承载基板、连接在所述承载基板上的两个以上的支撑臂以及可拆卸地装配于所述支撑臂上的夹持臂，所述支撑臂上设置有锁紧件，用于锁紧装配在所述支撑臂上的夹持臂。
优选地，所述支撑臂的顶部设置形成有凹槽，所述夹持臂装配于所述凹槽中。
优选地，所述支撑臂的数量为两个或三个，每一所述支撑臂分别配置有一个所述夹持臂。
优选地，所述承载基板上开设有长条形通孔。
优选地，所述长条形通孔的一端连通至所述承载基板的边缘。
优选地，所述支撑臂设置为高度可调的支撑臂。
优选地，所述支撑臂设置为在所述承载基板的平面上可移动的支撑臂。
本发明的另一方面是提供一种在体观察动物体骨中毛细血管的方法，采用如上所述的在体观察动物体骨中毛细血管的系统对毛细血管进行观察，所述方法包括：
将所述固定装置固定连接于所述双光子显微镜的载物台上；
将待观察的动物体放置于所述固定装置的两个以上的支撑臂之间，控制所述夹持臂将所述待观察的动物体夹持于所述承载基板上并锁紧所述夹持臂；
将待观察的动物体麻醉后，对目标观察区域进行脱毛处理，再将皮肤和肌肉划开以暴露出目标观察区域的骨头；
使用所述打磨装置对暴露出的骨头进行打磨，以减薄骨皮质；
向所述待观察的动物体注射血管成像剂，然后启动所述双光子显微镜对打磨后的骨头部位进行成像，观察其中的毛细血管。
本发明实施例提供的在体观察动物体骨中毛细血管的系统和方法，利用双光子显微镜成像较深且对活体细胞伤害较小而能较长时间成像的特点，观察活体骨内毛细血管，成像位置深切成像稳定；通过设计特定结构的固定装置来固 定待观察的动物体，克服活体呼吸和心跳造成的图像位移的问题，使得成像更加稳定；利用打磨装置对待观察的暴露骨头进行打磨减薄骨皮质，提升了成像位置的深度，可以对更深位置的骨内毛细血管进行成像观察。
图1是本发明实施例的在体观察动物体骨中毛细血管的系统的结构示意图；
图2和图3是本发明实施例中对实验小鼠的骨内毛细血管成像的图示。
为使本发明的目的、技术方案和优点更加清楚，下面结合附图对本发明的具体实施方式进行详细说明。这些优选实施方式的示例在附图中进行了例示。附图中所示和根据附图描述的本发明的实施方式仅仅是示例性的，并且本发明并不限于这些实施方式。
在此，还需要说明的是，为了避免因不必要的细节而模糊了本发明，在附图中仅仅示出了与根据本发明的方案密切相关的结构和/或处理步骤，而省略了与本发明关系不大的其他细节。
本发明实施例首先提供了一种在体观察动物体骨中毛细血管的系统，参阅图1，所述系统包括双光子显微镜1、打磨装置(图中未示出)和固定装置2。所述双光子显微镜1包括载物台11和成像镜头12，所述固定装置1连接于所述双光子显微镜1的载物台11上，用于对待观察的动物体进行固定，所述双光子显微镜1的成像镜头12用于对打磨后的骨头部位进行成像，以观察其中的毛细血管。所述打磨装置用于对待观察的动物体暴露出的骨头进行打磨，以减薄骨皮质，以增加成像位置的深度，可以对更深位置的骨内毛细血管进行成像观察。
其中，如图1所示，所述固定装置2包括承载基板21、连接在所述承载基板21上的两个以上的支撑臂22以及可拆卸地装配于所述支撑臂22上的夹持臂23，所述支撑臂22上设置有锁紧件24，用于锁紧装配在所述支撑臂22上的夹持臂23。
在优选的方案中，所述支撑臂22的数量为两个或三个，每一所述支撑臂22分别配置有一个所述夹持臂23。图1中示出的固定装置2包含有三个支撑臂22，三个支撑臂22之间的区域形成为用于放置待观察的动物体的空间。
在优选的方案中，所述支撑臂22设置为高度可调的支撑臂，以使得用于放置待观察的动物体的空间的高度可调，以适应于不同体型大小的动物体。
进一步优选的方案中，所述支撑臂22设置为在所述承载基板21的平面上可移动的支撑臂，以使得用于放置待观察的动物体的横向宽度可调，以适应于不同体型大小的动物体。
在优选的方案中，如图1所示，所述支撑臂22的顶部设置形成有凹槽25，所述夹持臂23装配于所述凹槽25中，所述锁紧件24设置为螺纹锁紧件。
在优选的方案中，如图1所示，所述承载基板21上开设有长条形通孔26，通过所述长条形通孔26配合螺纹紧固件可将所述固定装置2固定连接在所述双光子显微镜1的载物台11上，并且设置为长条形通孔使得连接位置易于调整。在进一步优选的方案中，如图1所示，所述长条形通孔26的一端连通至所述承载基板21的边缘。
本发明的另一方面是提供一种在体观察动物体骨中毛细血管的方法，采用如上所述的在体观察动物体骨中毛细血管的系统对毛细血管进行观察。本发明实施例中以待观察的动物体为实验小鼠为例进行说明。结合图1的结构示意图，所述方法包括以下步骤：
(1)、将所述固定装置2固定连接于所述双光子显微镜1的载物台11上。
(2)、将实验小鼠(图1中未示出)放置于所述固定装置2的支撑臂22之间，控制所述夹持臂23将所述待观察的动物体夹持于所述承载基板21上并锁紧所述夹持臂23。
(3)、将实验小鼠麻醉后，对目标观察区域大腿骨头使用脱毛膏进行脱毛处理，再将皮肤和肌肉划开以暴露出目标观察区域的骨头。
(4)、使用所述打磨装置对暴露出的骨头进行打磨，以减薄骨皮质。例如，使用微型钻头轻轻磨去部分皮质骨，以不磨出血破坏骨内部血管结构为界限。
(5)、向所述实验小鼠注射血管成像剂。例如，从鼠眼眶注射TRITC标记葡聚糖(TRITC-Dextran，平均分子量65000～85000，注射终浓度0.1mg/kg)。
(6)、启动所述双光子显微镜对打磨后的骨头部位进行成像，观察其中的毛细血管。
进一步地，成像结束后，缝合实验小鼠的肌肉皮肤组织，使得麻醉苏醒后小鼠能够存活，其后仍能够使用同一只小鼠重复进行相关实验。
图2和图3是本发明实施例中对实验小鼠的大腿部位的骨内毛细血管成像的图示。其中，图2为上方骨骺端(mp)生长板(gp)附近拱柱状结构的毛细血管，图3则为骨干端(dp)网状结构的毛细血管。从图2和图3可以获知，本发明实施例提供的系统和方法，可以实现在体观察动物体骨中不同形态的毛细血管。两种不同形态的毛细血管内皮细胞，对骨组织有着不同的生理作用：骨骺端(mp)生长板(gp)附近拱柱状结构血管内皮细胞主要促进骨质生成，而骨干端(dp)网状结构血管对骨髓细胞造血有调控作用。因此，通过分辨不同形态的骨内毛细血管，能够判断毛细血管内皮细胞的异质性，对其生理状态做出判断。
综上所述，本发明实施例提供的在体观察动物体骨中毛细血管的系统和方法，利用双光子显微镜成像较深且对活体细胞伤害较小而能较长时间成像的特点，观察活体骨内毛细血管，成像位置深切成像稳定；通过设计特定结构的固定装置来固定待观察的动物体，克服活体呼吸和心跳造成的图像位移的问题，使得成像更加稳定；利用打磨装置对待观察的暴露骨头进行打磨减薄骨皮质，提升了成像位置的深度，可以对更深位置的骨内毛细血管进行成像观察。
以上所述仅是本申请的具体实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本申请的保护范围。
Claims (10)
- 一种在体观察动物体骨中毛细血管的系统，其中，所述系统包括固定装置、打磨装置和双光子显微镜；所述固定装置连接于所述双光子显微镜的载物台上，用于对待观察的动物体进行固定；所述打磨装置用于对待观察的动物体暴露出的骨头进行打磨，以减薄骨皮质；所述双光子显微镜用于对打磨后的骨头部位进行成像，以观察其中的毛细血管；其中，所述固定装置包括承载基板、连接在所述承载基板上的两个以上的支撑臂以及可拆卸地装配于所述支撑臂上的夹持臂，所述支撑臂上设置有锁紧件，用于锁紧装配在所述支撑臂上的夹持臂。
- 根据权利要求1所述的在体观察动物体骨中毛细血管的系统，其中，所述支撑臂的顶部设置形成有凹槽，所述夹持臂装配于所述凹槽中。
- 根据权利要求1所述的在体观察动物体骨中毛细血管的系统，其中，所述支撑臂的数量为两个或三个，每一所述支撑臂分别配置有一个所述夹持臂。
- 根据权利要求2所述的在体观察动物体骨中毛细血管的系统，其中，所述支撑臂的数量为两个或三个，每一所述支撑臂分别配置有一个所述夹持臂。
- 根据权利要求1所述的在体观察动物体骨中毛细血管的系统，其中，所述承载基板上开设有长条形通孔。
- 根据权利要求5所述的在体观察动物体骨中毛细血管的系统，其特征在于，所述长条形通孔的一端连通至所述承载基板的边缘。
- 根据权利要求1所述的在体观察动物体骨中毛细血管的系统，其中，所述支撑臂设置为高度可调的支撑臂。
- 根据权利要求7所述的在体观察动物体骨中毛细血管的系统，其中，所述支撑臂设置为在所述承载基板的平面上可移动的支撑臂。
- 根据权利要求2所述的在体观察动物体骨中毛细血管的系统，其中，所述支撑臂设置为高度可调的支撑臂，所述支撑臂还设置为在所述承载基板的平面上可移动的支撑臂。
- 一种在体观察动物体骨中毛细血管的方法，其中，采用如权利要求1-9 任一所述的在体观察动物体骨中毛细血管的系统对毛细血管进行观察，所述方法包括：将所述固定装置固定连接于所述双光子显微镜的载物台上；将待观察的动物体放置于所述固定装置的两个以上的支撑臂之间，控制所述夹持臂将所述待观察的动物体夹持于所述承载基板上并锁紧所述夹持臂；将待观察的动物体麻醉后，对目标观察区域进行脱毛处理，再将皮肤和肌肉划开以暴露出目标观察区域的骨头；使用所述打磨装置对暴露出的骨头进行打磨，以减薄骨皮质；向所述待观察的动物体注射血管成像剂，然后启动所述双光子显微镜对打磨后的骨头部位进行成像，观察其中的毛细血管。
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