CN114642401A - System and method for in vivo observation of capillary blood vessels in animal bone - Google Patents
System and method for in vivo observation of capillary blood vessels in animal bone Download PDFInfo
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- CN114642401A CN114642401A CN202011507052.XA CN202011507052A CN114642401A CN 114642401 A CN114642401 A CN 114642401A CN 202011507052 A CN202011507052 A CN 202011507052A CN 114642401 A CN114642401 A CN 114642401A
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- 230000001054 cortical effect Effects 0.000 claims abstract description 7
- 238000005498 polishing Methods 0.000 claims description 8
- 210000004204 blood vessel Anatomy 0.000 claims description 5
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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
- A61B5/489—Blood vessels
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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
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- Health & Medical Sciences (AREA)
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- Heart & Thoracic Surgery (AREA)
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- Biophysics (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Surgery (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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- Microscoopes, Condenser (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention provides a system and a method for observing capillary vessels in animal bones in vivo, wherein the system comprises a fixing device, a grinding device and a two-photon microscope; the fixing device is connected to an object stage of the two-photon microscope and used for fixing an animal body to be observed; the grinding device is used for grinding the exposed bone of the animal body to be observed so as to thin the cortical bone; the two-photon microscope is used for imaging the polished bone part so as to observe capillary vessels in the bone part; the fixing device comprises a bearing substrate, more than two supporting arms connected to the bearing substrate and clamping arms detachably assembled on the supporting arms, wherein locking pieces are arranged on the supporting arms and used for locking the clamping arms assembled on the supporting arms. The invention can realize the stable imaging of the capillary vessels in the bones of the living bodies of animals, and can observe abundant forms of the capillary vessels in the bones in real time.
Description
Technical Field
The invention belongs to the technical field of medical imaging, and particularly relates to a system for observing capillary vessels in animal bones in vivo and a corresponding method.
Background
The vascular endothelial cells are monolayer cells distributed on the inner wall of the blood vessel, and have rich heterogeneity and organ specificity. Through the dense branching of capillaries, the vascular endothelium establishes a link with almost all cells within the respective organ. The 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 growth, development and steady maintenance of each organ by secreting vascular secretion factors. The capillary vessels in different organs have different shapes, and the shapes of the capillary vessels in different positions and functions in the same organ are different, which reflects the characteristics of different subtypes of endothelial cells. With age or disease, its morphology changes, and its homeostatic maintenance of organs changes accordingly. Thus, observing the morphology of the capillary vessels can provide status information of the respective organs.
The observation of capillary vessel form has two modes of in vitro and in vivo, and because the capillary vessel position in the bone is deeper, the prior art mainly takes in vitro observation as the main point at present. For example, the bone is cut into slices of 30 to 300 microns after the bone is decalcified ex vivo, and the morphology is observed by an optical microscope or a confocal microscope after the capillary vessels are specifically stained by fluorescence. For example, the blood capillary is imaged by subjecting an isolated bone to chemical treatment and then to transparentization, then subjecting the blood vessel to specific fluorescent staining and then to a fluorescence microscope with selective surface illumination, and then to digital processing at a later stage. The existing technology for observing the shape of capillary vessels in bones in vitro limits the application range of the technology, and an in-vivo observation method needs to be explored in order to obtain richer real-time observation results.
Disclosure of Invention
In view of the shortcomings of the prior art, the present invention provides a system and method for in vivo observation of capillaries in animal bones to enable stable imaging of capillaries in bones of a living body.
To achieve the above object, an aspect of the present invention provides a system for in vivo observation of capillaries in bones of an animal body, the system comprising a fixing device, a polishing device and a two-photon microscope; the fixing device is connected to an object stage of the two-photon microscope and used for fixing an animal body to be observed; the grinding device is used for grinding the exposed bone of the animal body to be observed so as to thin the cortical bone; the two-photon microscope is used for imaging the polished bone part so as to observe capillary vessels in the bone part;
the fixing device comprises a bearing substrate, more than two supporting arms connected to the bearing substrate and clamping arms detachably assembled on the supporting arms, wherein locking pieces are arranged on the supporting arms and used for locking the clamping arms assembled on the supporting arms.
Preferably, the top of the support arm is provided with a groove in which the clamp arm is fitted.
Preferably, the number of the supporting arms is two or three, and each supporting arm is provided with one clamping arm.
Preferably, the bearing substrate is provided with a strip-shaped through hole.
Preferably, one end of the elongated through hole is connected to the edge of the carrier substrate.
Preferably, the support arm is provided as a height-adjustable support arm.
Preferably, the support arm is provided as a support arm movable in the plane of the carrier substrate.
Another aspect of the present invention provides a method for in vivo observation of capillaries in animal bone, the method comprising:
fixedly connecting the fixing device to the objective table of the two-photon microscope;
placing an animal body to be observed between more than two supporting arms of the fixing device, controlling the clamping arm to clamp the animal body to be observed on the bearing substrate and locking the clamping arm;
anaesthetizing an animal body to be observed, performing unhairing treatment on a target observation area, and then cutting skin and muscle to expose bones of the target observation area;
polishing the exposed bone using the polishing device to thin cortical bone;
injecting a blood vessel imaging agent into the animal body to be observed, starting the two-photon microscope to image the ground bone part, and observing capillary vessels in the bone part.
According to the system and the method for observing the capillary vessels in the animal bone in vivo, provided by the embodiment of the invention, the capillary vessels in the living bone are observed by utilizing the characteristics that the two-photon microscope has deeper imaging and less damage to living cells and can image for a longer time, and the imaging position is deeply cut and stably imaged; the fixing device with a specific structure is designed to fix the animal body to be observed, so that the problem of image displacement caused by living body respiration and heartbeat is solved, and imaging is more stable; utilize grinding device to treat the exposed bone of observing and polish attenuate cortex lycii, promoted the degree of depth of formation of image position, can carry out the formation of image to the intraosseous capillary vessel of more dark position and observe.
Drawings
FIG. 1 is a schematic structural diagram of a system for in vivo observation of capillaries in bones of an animal body according to an embodiment of the present invention;
fig. 2 and 3 are illustrations of imaging of intraosseous capillaries of experimental mice in an example of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention are described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the invention shown in the drawings and described in accordance with the drawings are exemplary only, and the invention is not limited to these embodiments.
It should be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and/or processing steps closely related to the scheme according to the present invention are shown in the drawings, and other details not so relevant to the present invention are omitted.
The embodiment of the present invention first provides a system for in vivo observation of capillary vessels in bones of an animal body, which comprises a two-photon microscope 1, a polishing device (not shown in the figure) and a fixing device 2, referring to fig. 1. The two-photon microscope 1 comprises an object stage 11 and an imaging lens 12, the fixing device 1 is connected to the object stage 11 of the two-photon microscope 1 and used for fixing an object to be observed, and the imaging lens 12 of the two-photon microscope 1 is used for imaging a bone part after polishing to observe capillary vessels therein. Grinding device is used for treating the bone that the animal body that observes exposes and polishes to the bone of attenuate cortex lycii, with the degree of depth that increases the formation of image position, can carry out the formation of image to the capillary vessel in the bone of more dark position and observe.
As shown in fig. 1, the fixing device 2 includes a carrying substrate 21, two or more supporting arms 22 connected to the carrying substrate 21, and a clamping arm 23 detachably assembled on the supporting arms 22, wherein a locking member 24 is disposed on the supporting arms 22 for locking the clamping arm 23 assembled on the supporting arms 22.
In a preferred embodiment, the number of the supporting arms 22 is two or three, and each supporting arm 22 is configured with one of the clamping arms 23. The fixture 2 shown in fig. 1 comprises three support arms 22, the area between the three support arms 22 forming a space for placing the animal body to be observed.
In a preferred embodiment, the supporting arm 22 is a height-adjustable supporting arm, so that the height of the space for placing the animal body to be observed is adjustable to adapt to animal bodies with different sizes.
In a further preferred scheme, the supporting arm 22 is provided as a supporting arm which is movable on the plane of the bearing substrate 21, so that the transverse width for placing the animal body to be observed is adjustable to adapt to animal bodies with different body sizes.
In a preferred embodiment, as shown in fig. 1, the top of the support arm 22 is provided with a recess 25, the holding arm 23 is fitted into the recess 25, and the locker 24 is provided as a screw locker.
In a preferred embodiment, as shown in fig. 1, the supporting substrate 21 is provided with an elongated through hole 26, the fixing device 2 can be fixedly connected to the stage 11 of the two-photon microscope 1 through the elongated through hole 26 in cooperation with a threaded fastener, and the elongated through hole is configured to facilitate adjustment of the connection position. In a further preferred scheme, as shown in fig. 1, one end of the elongated through hole 26 is connected to the edge of the carrier substrate 21.
Another aspect of the present invention is to provide a method for observing capillaries in animal bones in vivo, wherein the capillaries are observed by using the system for observing capillaries in animal bones in vivo as described above. In the examples of the present invention, an animal to be observed is exemplified as an experimental mouse. In conjunction with the schematic structure of fig. 1, the method comprises the following steps:
(1) and fixedly connecting the fixing device 2 to the objective table 11 of the two-photon microscope 1.
(2) Placing a test mouse (not shown in fig. 1) between the supporting arms 22 of the fixing device 2, controlling the clamping arm 23 to clamp the animal body to be observed on the bearing substrate 21 and locking the clamping arm 23.
(3) After the experimental mouse is anesthetized, the thigh bone of the target observation area is depilated by using depilatory cream, and then the skin and the muscle are cut to expose the bone of the target observation area.
(4) And using the grinding device to grind the exposed bone to thin the cortical bone. For example, a micro-drill is used to gently abrade away a portion of the cortical bone, as defined by the destruction of the vascular structure within the bone without abrading blood.
(5) And injecting a vascular imaging agent into the experimental mouse. For example, TRITC-labeled Dextran (TRITC-Dextran, average molecular weight 65000-85000, final concentration of injection 0.1mg/kg) is injected from the orbital region of a mouse.
(6) And starting the two-photon microscope to image the polished bone part and observe capillary vessels in the bone part.
Further, after imaging is completed, the muscle skin tissue of the experimental mouse is sutured, so that the mouse can survive after recovery from anesthesia, and then the relevant experiment can be repeated by using the same mouse.
Fig. 2 and 3 are diagrams for imaging of intraosseous capillaries in the femoral region of an experimental mouse in the example of the present invention. Fig. 2 shows capillaries of a vaulted structure near the epiphyseal (mp) growth plate (gp) above, and fig. 3 shows capillaries of a reticular structure at the metaphysis (dp). As can be understood from fig. 2 and 3, the system and method provided by the embodiment of the present invention can realize in vivo observation of capillary vessels of different shapes in animal bones. Two different forms of capillary endothelial cells have different physiological effects on bone tissues: the endothelial cells of the arched column structure near the epiphyseal (mp) growth plate (gp) mainly promote the generation of bone, and the reticular structure blood vessels of the diaphyseal (dp) have the regulation and control function on the hematopoiesis of bone marrow cells. Therefore, by distinguishing the capillary vessels in the bones of different forms, the heterogeneity of the capillary endothelial cells can be judged, and the physiological state can be judged.
In summary, the system and the method for observing capillary vessels in animal bones in vivo provided by the embodiment of the invention utilize the characteristics that the two-photon microscope has deeper imaging and less damage to living cells, so that the living bones can be imaged for a longer time, and the capillary vessels in the living bones are observed, and the imaging position is deeply cut and stably imaged; the fixing device with a specific structure is designed to fix the animal body to be observed, so that the problem of image displacement caused by living body respiration and heartbeat is solved, and imaging is more stable; utilize grinding device to treat the exposed bone of observing and polish attenuate cortex lycii, promoted the degree of depth of formation of image position, can carry out the formation of image to the intraosseous capillary vessel of more dark position and observe.
The foregoing is directed to embodiments of the present application and it is noted that numerous modifications and adaptations may be made by those skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.
Claims (8)
1. A system for in vivo observation of capillaries in bones of an animal, said system comprising a fixation device, a polishing device and a two-photon microscope; the fixing device is connected to an object stage of the two-photon microscope and used for fixing an animal body to be observed; the grinding device is used for grinding the exposed bone of the animal body to be observed so as to thin the cortical bone; the two-photon microscope is used for imaging the polished bone part so as to observe capillary vessels in the bone part;
the fixing device comprises a bearing substrate, more than two supporting arms connected to the bearing substrate and clamping arms detachably assembled on the supporting arms, wherein locking pieces are arranged on the supporting arms and used for locking the clamping arms assembled on the supporting arms.
2. A system for in vivo observation of capillaries in animal bone according to claim 1 wherein the top of the support arm is provided with a groove formed therein into which the retaining arm fits.
3. The system for in vivo observation of capillaries in animal bone according to claim 1, wherein the number of said supporting arms is two or three, and each of said supporting arms is provided with one of said holding arms.
4. The system of claim 1, wherein the carrier substrate has a through hole with a long strip shape.
5. The system for in vivo observation of capillaries in animal bones as claimed in claim 4, wherein one end of said elongated through hole is connected to the edge of said carrier substrate.
6. A system for in vivo observation of capillaries in animal bones as in any one of claims 1-5, wherein the support arm is configured as a height-adjustable support arm.
7. A system for in vivo observation of capillaries in animal bone according to claim 6, wherein the support arm is provided as a support arm movable in the plane of the carrier substrate.
8. A method for in vivo observation of capillaries in animal bone, wherein the capillaries are observed using the system for in vivo observation of capillaries in animal bone according to any one of claims 1 to 7, the method comprising:
fixedly connecting the fixing device to the objective table of the two-photon microscope;
placing an animal body to be observed between more than two supporting arms of the fixing device, controlling the clamping arm to clamp the animal body to be observed on the bearing substrate and locking the clamping arm;
anaesthetizing an animal body to be observed, performing unhairing treatment on a target observation area, and then cutting skin and muscle to expose bones of the target observation area;
polishing the exposed bone using the polishing device to thin cortical bone;
injecting a blood vessel imaging agent into the animal body to be observed, starting the two-photon microscope to image the ground bone part, and observing capillary vessels in the bone part.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011507052.XA CN114642401A (en) | 2020-12-18 | 2020-12-18 | System and method for in vivo observation of capillary blood vessels in animal bone |
| PCT/CN2020/138080 WO2022126685A1 (en) | 2020-12-18 | 2020-12-21 | System and method for in vivo observation of intraosseous capillary in animal body |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011507052.XA CN114642401A (en) | 2020-12-18 | 2020-12-18 | System and method for in vivo observation of capillary blood vessels in animal bone |
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| CN114642401A true CN114642401A (en) | 2022-06-21 |
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| CN202011507052.XA Pending CN114642401A (en) | 2020-12-18 | 2020-12-18 | System and method for in vivo observation of capillary blood vessels in animal bone |
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| CN (1) | CN114642401A (en) |
| WO (1) | WO2022126685A1 (en) |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4842578A (en) * | 1986-03-12 | 1989-06-27 | Dyonics, Inc. | Surgical abrading instrument |
| WO2005017115A2 (en) * | 2003-08-11 | 2005-02-24 | Mount Sinai School Of Medicine Of New York University | Cord blood-derived hematopoietic progenitor cells |
| CN101416995A (en) * | 2007-10-24 | 2009-04-29 | 南京宇道科技开发有限公司 | Epimedium extract and preparation method, preparation and use thereof |
| CN105962893A (en) * | 2016-04-25 | 2016-09-28 | 上海交通大学 | Endogenous photo-imaging system used for moving animal |
| CN110151726A (en) * | 2018-02-11 | 2019-08-23 | 上海市第六人民医院 | Load purposes of the human pluripotent stem cells excretion body of resveratrol on preparation treatment bone and joint degenerative disease drug |
| TW201944990A (en) * | 2018-04-04 | 2019-12-01 | 大陸商天士力醫藥集團股份有限公司 | Traditional chinese medicine composition for preventing and/or treating ischemic reperfusion injury |
| CN208705563U (en) * | 2018-08-21 | 2019-04-05 | 河北医科大学第二医院 | A kind of Two Photon Fluorescence adapter for the observation of mouse head |
| CN109481077A (en) * | 2018-12-06 | 2019-03-19 | 沈阳建筑大学 | A kind of adjustable mouse experiment fixator |
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- 2020-12-18 CN CN202011507052.XA patent/CN114642401A/en active Pending
- 2020-12-21 WO PCT/CN2020/138080 patent/WO2022126685A1/en active Application Filing
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| WO2022126685A1 (en) | 2022-06-23 |
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