CN217793014U

CN217793014U - Spinal cord imaging device and spinal cord imaging device with spinal cord imaging device for freely moving mouse

Info

Publication number: CN217793014U
Application number: CN202123057944.7U
Authority: CN
Inventors: 巨富荣; 蔚鹏飞; 王立平
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2022-11-15
Anticipated expiration: 2031-12-07

Abstract

The utility model provides a spinal cord image device and have its spinal cord image device of free activity mouse, include: the device comprises a pair of fixed blocks, a cover glass, a silica gel layer, a fluorescence microscope and a fixed piece; wherein, a pair of fixed blocks are oppositely arranged and used for clamping and fixing exposed vertebra from two sides; the cover glass is arranged on one side, far away from the spine, of the fixing block and used for sealing a spinal cavity of the spine and enabling the spinal cord to deform under the condition that the integrity of the spinal cord is ensured; the silica gel layer is arranged between the fixed block and the cover glass and is used for connecting the fixed block and the cover glass; the fluorescence microscope is arranged above the cover glass and is used for observing the deformed spinal cord; the fixing piece is arranged above the cover glass and is used for connecting the fluorescence microscope; the device has simple structure, convenient use, easy processing and manufacture and good imaging effect.

Description

Spinal cord imaging device and spinal cord imaging device of freely moving mouse with same

Technical Field

The utility model relates to a spinal cord imaging technology field especially relates to a spinal cord image device of free activity mouse of spinal cord image device and have it.

Background

The spinal cord is one of the important components of the central nervous system. The spinal cord, which serves as a bridge connecting the brain and the peripheral nervous system, transmits neural information with the periphery to coordinate normal activities of the living body, and plays an important role in the somatosensory and motor processes. The application of miniaturized in vivo imaging methods has made a breakthrough in the field of brain imaging related studies, but in vivo imaging studies of the spinal cord, another critical structure of the central nervous system, have been difficult to realize. This is because the irregular movement range of the spinal cord tissue caused by the respiration, heartbeat and movement of the mouse itself is large, which makes the fixation of the spinal cord tissue difficult during spinal cord imaging. In addition, the imaging speed and the optical thin slice of the two-photon microscope are limited, so that the problems of focal plane drift, image distortion or image loss and the like of the mouse during the body spinal cord imaging are caused. At present, most of the subjects reported for optical imaging of the spinal cord are anesthetized animals, and the animals are in a fresh, awake, freely-moving state. Therefore, how the spinal cord, an important neural structure, encodes brain descending commands and peripheral input signals in a real environment is not clear. Establishing a set of animal living spinal cord imaging methods in a free activity state is one of the key ways for further exploring the important functions of the spinal cord in the nervous system.

Currently, spinal cord imaging of living animals in a free-moving state is still stopped at single photon microscopy. Compared with the prior art, the two-photon microscopic imaging method has the advantages that the problems that signals are dense in the densely marked tissue and cannot be distinguished or even focused are solved, the image contrast and the penetration depth in the organism tissue are obviously improved, and high-definition imaging of the morphological structure of the subcellular cells can be realized. In 2017, the miniaturized two-photon fluorescence microscope independently developed by the Cheng and Pan team breaks the limitation of instruments on animal imaging research, and can realize rapid tissue or cell two-photon living body imaging in the free movement process of animals. At present, the application of a high-resolution miniature two-photon fluorescence microscope makes breakthrough progress on related researches in the field of in vivo imaging of free-moving animals at home and abroad, and also brings a chance for realizing spinal cord two-photon imaging in a free-moving state of a mouse.

However, the specificity of spinal cord structure presents a number of problems that have been a major impediment to the realization of free-moving animal spinal cord imaging. Among them, the spinal cord has a complex structure, a large and irregular motion amplitude of spinal cord tissue, unstable long-term imaging, and difficult stable implantation of a miniaturized microscope, which leads to a series of challenges for spinal cord in vivo imaging research.

For example, in the chinese patent "a fixed observation device for animal spinal cord living body imaging and a method for using the same" with the application publication No. CN202110686690.0, a fixed observation device for animal spinal cord living body imaging and a method for using the same "are introduced, which relate to the technical field of medical instruments. It includes: the device comprises a base, a fixing device and an observation device; the base includes: the animal support device comprises a base for placing an animal and a support fixedly arranged on the base; the fixing device includes: one end of the fixing rod is detachably fixed at the upper end of the supporting part, and the clamping part is arranged at the other end of the fixing rod and is used for fixing the spine vertebral body of the animal; the observation device includes: fix the observation frame on the holder to, set up in observation window on the observation frame. By adopting the technical scheme, the fixing device has the advantages of good fixing effect, long-term observation and avoidance of multiple operations.

However, the above-mentioned techniques mainly aim at in vivo imaging under anesthesia, and cannot achieve long-term in vivo spinal imaging, and only acute phase imaging of spinal cord is performed, in which failure due to spinal cord infection occurs; moreover, current spinal cord imaging is suitable for anesthesia imaging, under anesthesia conditions, 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 severe spinal cord tissue displacement, motion artifacts with large amplitude are caused, eventually leading to imaging failure.

A method for realizing long-term two-photon imaging of animal spinal cords which can freely move in a non-binding environment is in urgent need of research and development.

SUMMERY OF THE UTILITY MODEL

In order to overcome the defects of the prior art, the utility model provides a spinal cord imaging device and have its spinal cord imaging device of freely moving mouse for solve at least one of the aforesaid technical problems.

Specifically, the technical scheme is as follows:

a fluorescence microscope-based free-moving spinal cord imaging device, comprising:

a pair of fixing blocks, which are oppositely arranged and used for clamping and fixing exposed spines from two sides;

the cover glass is arranged on one side, far away from the spine, of the fixing block and used for sealing a spinal cavity of the spine and enabling the spinal cord to deform under the condition that the spinal cord is complete;

the silica gel layer is arranged between the fixing block and the cover glass and is used for connecting the fixing block and the cover glass;

and the fluorescence microscope is arranged above the cover glass and is used for observing the spinal cord which is deformed.

The fixing block is provided with a slot facing the spine and used for fixing the spine.

And a drainage groove for seepage to flow out is arranged on the surface of the groove.

The groove is V-shaped;

the V-shaped opening section faces the spine.

The included angle of the V shape is 25-85 degrees.

The notch is arc-shaped;

the arcuate opening section faces the spine.

The free-movement spinal cord imaging device based on the fluorescence microscope further comprises:

the fixing piece is arranged above the cover glass and is used for connecting the fluorescence microscope;

and the fixed piece is provided with an observation window for facilitating the fluorescent microscope to observe the deformed spinal cord.

A fluorescence microscope-based spinal cord imaging device for free-moving mice, comprising:

the spinal cord imaging device as described above;

the fixing instrument is matched with the spinal cord imaging device and used for adjusting the position of a fixing block in the spinal cord imaging device so as to clamp the spine to be observed;

a blind hole is formed in one end, far away from the observation vertebra, of the fixing block;

the fixing instrument is matched with the blind hole and used for controlling the moving direction of the fixing block.

The fixation instrument comprises:

a base;

the upright post is arranged on the base;

the rod sleeve is arranged on the circumferential direction of the upright column, and the axis of the rod sleeve is vertical to that of the upright column;

the adjusting rod is arranged in the rod sleeve and used for adjusting the position of the fixing block;

the locking bolt is arranged on the rod sleeve and used for fixing the adjusting rod;

the adjusting rod corresponds to the blind hole and is used for guiding the movement of the fixing block.

The adjusting lever includes:

a square section;

the middle section, one end is connected with the square section;

the cap-shaped section is connected with the other end of the middle section;

the square section corresponds to the blind hole.

The utility model discloses following beneficial effect has at least:

spinal cord imaging device and have its spinal cord imaging device of free activity mouse, cooperate with the fixed block through the fixed appearance of mouse head, utilize the fixed appearance of mouse head to fix a pair of fixed block from the both sides of backbone the backbone, then, recycle the cover glass and press the spinal cord to warping, under the complete circumstances of assurance spinal cord, make it produce deformation, at last, connect fluorescence microscope through the mounting, realize the spinal cord formation of image. The device has simple structure, convenient use, easy processing and manufacture and good imaging effect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope, and that those skilled in the art can also obtain other related drawings based on these drawings without inventive efforts.

Fig. 1 is a schematic structural view of the fixing block of the present invention;

FIG. 2 is a schematic view of a fixation block implanted in the spinal cord;

fig. 3 is a schematic view of a spinal cord imaging method according to the present invention;

fig. 4 is a diagram of the operation steps of the spinal cord imaging method according to the present invention;

fig. 5 is a schematic structural view of the mouse head fixing instrument of the present invention;

FIG. 6 is a schematic illustration of a free-moving in vivo spinal cord imaging mouse;

FIG. 7 is another embodiment of the fixing block of FIG. 1;

fig. 8 is a schematic structural view of the adjusting lever.

Wherein, 1, a fixed block; 2. the spine; 3. covering glass slides; 4. a silica gel layer; 5. a fluorescence microscope; 6. a fixing member; 7. a mouse head fixing instrument; 1B, blind holes; 2A, slotting; 2B, a drainage groove; 2C, a gap; 6A, an observation window; 701. an adjusting lever; 702. locking the bolt; 703. a base; 704. a column; 705. a rod sleeve; 701A, a square section; 701B, a middle section; 701C, a cap-shaped section;

alpha in fig. 1 is the included angle of the V-shape.

Detailed Description

Those skilled in the art will appreciate that the modules in the devices in the implementation scenario may be distributed in the devices in the implementation scenario according to the description of the implementation scenario, or may be located in one or more devices different from the present implementation scenario with corresponding changes. The modules of the implementation scenario may be combined into one module, or may be further split into multiple sub-modules.

In order to solve the problems of the prior art, the utility model discloses regard as the experimental objective with the mouse, carry out following embodiment, right the utility model discloses further explanation:

the principle of the utility model is that: as shown in fig. 1-4, a living animal is taken, the spine of a position to be imaged is exposed through spinal surgery, and the surface of the spine is cleaned; fixing the spine; exposing central anterior artery blood vessels and spinal cord white matter at the vertebral position to form an exposed spinal cavity, sealing the exposed spinal cavity, and deforming the spinal cord under the condition of ensuring the integrity of the spinal cord; spinal cord imaging of the spinal location is performed using a two-photon fluorescence microscope.

The method comprises the following specific steps:

the method comprises the following steps: after exposing 3-6 sections of spines of mouse lumbar segments through spinal surgery, cleaning tissues attached to the surfaces of spines, and stably embedding the tissues below the spinous processes of the spines by means of the fixing block 1; in order to reduce motion artifacts as much as possible, a complete transverse process is placed above the fixed blocks on the two sides, and the fixed blocks 1 are adjusted until the shaking of the vertebrae is basically eliminated;

step two: adopting a skull drill to polish a spinal plate, exposing a central anterior artery blood vessel and peripheral spinal cord white matter, adopting sterile normal saline to clean the surface of the spinal cord, and then adopting silica gel and a cover glass 3 to seal an exposed spinal cavity;

step three: because the spinal cord shakes greatly during movement, the spinal cord is pressed by the cover glass 3, and the spinal cord is deformed under the condition of ensuring the integrity of the spinal cord, so that movement artifacts are further reduced;

step four: the miniature two-photon microscope, such as a fluorescent microscope 5, is stably arranged on the fixing piece 6, and the components of the whole device are small enough and light enough to be conveniently carried by mice;

preferably, a cover glass 3 with 3-7mm of glass is adopted to press the spinal cord, so that the spinal cord is deformed under the condition of ensuring the integrity of the spinal cord.

The utility model also provides an embodiment: a fluorescence microscope based ambulatory spinal cord imaging device, comprising: a pair of fixing blocks 1, a cover glass 3, a silica gel layer 4, a fluorescence microscope 5 and a fixing block 6; wherein, a pair of fixed blocks 1 are oppositely arranged and used for clamping and fixing exposed vertebra 2 from two sides; the cover glass 3 is arranged on one side of the fixed block 1, which is far away from the vertebra 2, and is used for sealing a spinal cavity of the vertebra and enabling the spinal cord to deform under the condition of ensuring the integrity of the spinal cord; the silica gel layer 4 is arranged between the fixed block 1 and the cover glass 3 and is used for connecting the fixed block 1 and the cover glass 3; the fluorescence microscope 5 is arranged above the cover glass 3 and is used for observing the spinal cord which is deformed; the fixing piece 6 is arranged above the cover glass 3 and is used for connecting a fluorescence microscope; the fixing member 6 is provided with an observation window 5A for facilitating observation of the deformed spinal cord by the fluorescence microscope 5.

In order to facilitate clamping, a slot 2A facing the spine 2 is formed in the fixing block 1 and used for fixing the spine 2; the slot 2A can be in various structures, preferably, like a V-shape shown in fig. 1, and the included angle of the V-shape is 25-85 degrees, preferably, 35 degrees; the spine 2 can be stably held by the two paired slits 2A.

In the process of implanting the fixing block 1 and putting silica gel, seepage is easy to occur, and in order to facilitate the outflow of the seepage, the side wall of the open groove 2A is provided with a drainage groove 2B for the outflow of the seepage; one end of the drainage groove 2B is communicated with the gap end of the open groove 2A of the fixed block 1; the other end is arranged at the bottom of the open slot 2A; the fixed block 1 is also provided with a notch 2C for matching with the cover glass 3.

The utility model provides an embodiment: a fluorescence microscope-based spinal cord imaging device for free-moving mice, comprising: spinal cord imaging device, fixation device 7 as described in embodiment I; the spinal cord imaging device is matched with the fixing instrument 7 and used for adjusting the position of the fixing block 1 in the spinal cord imaging device so as to clamp the spine to be observed; a blind hole 1B is formed in one end, far away from the observation vertebra, of the fixing block 1; the end of the adjusting rod 701 in the fixing instrument corresponds to the blind hole 1B and is used for controlling the moving direction of the fixing block 1.

Specifically, the fixing instrument 1 includes: an adjusting rod 701, a locking bolt 702, a base 703, two upright posts 704 which are oppositely arranged, and a rod sleeve 705; wherein, the upright post 704 is vertically arranged on the base 703; the rod sleeve 705 is arranged on the circumference of the upright post 704, and the axis of the rod sleeve 705 is perpendicular to the axis of the upright post 704; the adjusting rod 701 is arranged in the rod sleeve 705, and an operator can push and pull the adjusting rod 701 to adjust the position of the fixing block; a locking bolt 702 is arranged on the rod sleeve 705 and used 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.

Fig. 7, the utility model discloses another alternative of fixed block 1: compared with the specific embodiment I, the fixing block 1 is provided with the slot 2A facing the spine 2, the slot 2A is arc-shaped, and the spine 2 can be stably clamped by the two paired slots 2A.

The adjusting rod 701 may be cylindrical as shown in fig. 5, and the end of the adjusting rod 701 may have a tip, which may be conical as shown in fig. 5 and corresponds to the blind hole 1B; at this time, the blind hole 1B may be a square hole in fig. 2 or may be a circular hole as required;

alternatively, the tip may be a quadrangular pyramid corresponding to the blind hole 1B in fig. 2.

Preferably, the adjusting rod 701 is stepped as shown in FIG. 8; the square section 701A at the small-sized end of the step corresponds to the blind hole 1B in fig. 2; the middle section 701B is circular and matched with the rod sleeve 705; the size of the cap section 701C is larger than the inner diameter of the rod sleeve 705 to prevent the adjusting rod 701 from coming out of the rod sleeve 705.

The above disclosure is only a few specific implementation scenarios of the present invention, however, the present invention is not limited thereto, and any changes that can be considered by those skilled in the art shall fall within the protection scope of the present invention. The utility model discloses the serial number is only for the description, does not represent the goodness of implementing the scene.

Claims

1. A fluorescence microscope-based ambulatory spinal cord imaging device, comprising:

the cover glass is arranged on one side, far away from the spine, of the fixing block and used for sealing a spinal cavity of the spine and enabling the spinal cord to deform under the condition that the integrity of the spinal cord is ensured;

the silica gel layer is arranged between the fixed block and the cover glass and is used for connecting the fixed block and the cover glass;

and the fluorescence microscope is arranged above the cover glass and is used for observing the deformed spinal cord.

2. The fluorescence microscope-based ambulatory spinal cord imaging device according to claim 1, wherein:

3. The fluorescence microscope-based ambulatory spinal cord imaging device according to claim 2, wherein:

4. A fluorescence microscope based free-moving spinal cord imaging device as claimed in claim 2, wherein:

the groove is V-shaped;

the V-shaped opening section faces the spine.

5. The fluorescence microscope-based ambulatory spinal cord imaging device according to claim 4, wherein:

the included angle of the V shape is 25-85 degrees.

6. A fluorescence microscope based free-moving spinal cord imaging device as claimed in claim 2, wherein:

the notch is arc-shaped;

the arcuate opening section faces the spine.

7. The fluorescence microscope-based ambulatory spinal cord imaging device according to claim 1, further comprising:

8. A fluorescence microscope-based spinal cord imaging apparatus for free-moving mice, comprising:

a spinal cord imaging device as claimed in any one of claims 1-7;

9. The fluorescence microscope-based spinal cord imaging apparatus of free-moving mice according to claim 8, wherein the fixation instrument comprises: