A clamping device of a single-photon fluorescence microscope and a clamping method thereof

Abstract

The present application relates to the technical field of neurobiology, and particularly relates to a single-photon fluorescence microscope clamping device and a clamping groove mounting method thereof; the present application comprises a clamping groove body, the lower end of the clamping groove body is shaped like a microscope lens, the upper end of the clamping groove body is provided with a mounting hole for being connected to a stereotaxic instrument, and the lower end of the clamping groove body is used for inserting a clamping groove; the present application inserts a clamping groove in advance through the clamping groove body, and then inserts a single-photon fluorescence microscope into the clamping groove, so that the process of indirectly using a microscope lens as a holder to clamp the clamping groove for embedding in the prior art is omitted, the clamping groove embedding process is simplified, and the efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of neurobiology, and in particular to a slot clamping device for a single-photon fluorescence microscope and a method for installing the slot. Background Technology

[0002] Miniature single-photon fluorescence imaging is a novel neuroimaging method based on calcium imaging. Its basic principle is as follows: a virus carrying a calcium ion fluorescent indicator is injected into a target brain region. The virus then infects neurons, expressing the calcium ion indicator within the neurons. When the neurons are excited, an influx of calcium ions causes a burst of transient peaks in intracellular calcium ions. Since the calcium ion indicator binds to calcium ions and emits fluorescence, at the moment of neuronal excitation, along with the calcium ion peak, the burst of fluorescence peak can be captured using a fluorescence microscope. The activity and state of neurons can be analyzed based on the fluorescence information.

[0003] The use of a miniature single-photon fluorescence microscope mainly consists of two parts. First, a microlens is implanted at the viral injection site. This lens, acting as an extension of the microscope lens, captures fluorescence signals from deep brain tissue. One end of the lens is inserted into the brain tissue, while the other end protrudes from the skull to work with the microscope. Second, a microscope lens holder is installed on the surface of the mouse skull. Because recording fluorescence signals with a miniature microscope is a long-term, regularly intermittent process, and only one microscope is available, the microscope must be mounted on the mouse skull during recording, allowing the mouse to move freely to study its neural activity under specific behaviors. After recording, the microscope is removed. Therefore, this process requires a fixed device on the mouse skull surface for inserting the microscope; this device is the holder.

[0004] In the current mainstream implementation method, when installing the slot, the slot needs to be fixed to the microscope lens, becoming an integral part of the microscope. A microscope clamping device is used to hold the microscope, and then the clamping device is installed on a stereo positioning device. The positioning device accurately adjusts the microscope and the slot as a whole to the appropriate position on the skull surface. Then, light-cured resin is applied to the outside of the slot and the skull surface. After the light-cured resin solidifies, the slot and the skull are tightly bonded. At this point, the microscope is removed, thus achieving the installation of the slot on the mouse skull. This process essentially uses the microscope lens as a clamp to hold the slot, because there are only microscope clamps on the market and no dedicated slot clamps. Therefore, this method can only be adopted, that is, using a microscope clamp to hold the microscope and using the microscope to hold the slot for installation. In subsequent formal experiments, the microscope is inserted into the slot and the screw is tightened to achieve neural imaging of freely moving mouse brain tissue. Because the miniature microscope is small and easily damaged, and because the development stage of miniature single-photon fluorescence microscopy technology is limited, the current focus in this field is on the miniature single-photon fluorescence microscope itself, and there is a lack of matching accessories. This leads to significant obstacles in the later use of the microscope after purchase, especially since there is currently no device specifically designed to hold the slot separately. The microscope can only be used indirectly as a holder for the slot, which undoubtedly increases the risk of damage to the miniature microscope and makes the operation complicated. Summary of the Invention

[0005] The main technical problem solved by this invention is to provide a slot clamping device for a single-photon fluorescence microscope. The device pre-installs a slot by using a slot clamping body, and then inserts the single-photon fluorescence microscope into the slot. This eliminates the need for the microscope lens to be used as an indirect clamping device for embedding the slot in the existing solution, thus simplifying the slot embedding process and improving efficiency. The invention also provides a slot installation method for a single-photon fluorescence microscope.

[0006] To solve the above-mentioned technical problems, one technical solution adopted by the present invention is: to provide a slot mounting clamping device for a single-photon fluorescence microscope, which is connected to a stereo positioning instrument, wherein the device includes a slot clamping body, the lower end of the slot clamping body is shaped like a microscope lens, the upper end of the slot clamping body is provided with a mounting hole for connecting to the stereo positioning instrument, and the lower end of the slot clamping body is used for inserting into the slot.

[0007] As an improvement of the present invention, the slot clamping body is cylindrical.

[0008] As a further improvement of the present invention, the stereo positioning device is provided with a coronal axial arm, and the coronal axial arm is provided with a screw hole. The mounting hole of the slot clamping body is connected to the screw hole of the coronal axial arm by a screw, thereby fixing the slot clamping body to the coronal axial arm.

[0009] As a further improvement of the present invention, the stereo positioning device is also provided with a vertical axis arm and a sagittal axis arm.

[0010] As a further improvement of the present invention, the coronal axial arm is connected to the vertical axial arm.

[0011] As a further improvement of the present invention, a microlens is carried in the cylindrical cavity below the bottom surface of the card slot.

[0012] A method for mounting a slot in a single-photon fluorescence microscope, comprising the following steps:

[0013] Step S1: Fix the mouse head on a stereotaxic instrument, cut open its scalp to expose its skull, and establish a coordinate system on the skull through the sutures on the skull surface according to neurobiological methods to find the target site. Use a skull drill to drill a hole at the target site to penetrate the skull.

[0014] Step S2: Insert the card slot into the lower end of the card slot holder, and then connect the upper end of the card slot holder to the stereo positioning device with screws;

[0015] Step S3: Operate the stereotactic instrument and move the slot holder above the mouse's head through its coronal axial arm so that the slot connected to the lower end of the slot holder is at the target position on the mouse's head.

[0016] Step S4: Operate the stereotactic instrument, and use its vertical axis arm and coronal axis arm to move the slot holder downward, so that the microlens in the slot will be inserted into the brain tissue.

[0017] Step S5: When the microlens in the slot is inserted to the target depth, dental cement is used to bond the outer wall of the slot to the surface of the skull. After the dental cement solidifies, the slot holder is pulled out, and the slot is firmly attached to the surface of the mouse skull. At this point, the installation process of the slot is completed.

[0018] The beneficial effects of this invention are as follows: Compared with the prior art, this invention pre-installs the card slot with the card slot holder and then inserts the single-photon fluorescence microscope into the card slot, thereby eliminating the process of using the microscope lens indirectly as a holder to clamp the card slot for embedding in the prior art, simplifying the card slot embedding process and improving efficiency. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the application of the present invention;

[0020] Figure 2 This is a schematic diagram of the structure of the card slot clamping body of the present invention;

[0021] Figure 3 This is a schematic diagram of the card slot structure of the present invention;

[0022] Reference numerals: 1-Screw hole, 2-Vertical axial arm, 3-Sagittal axial arm, 4-Coronal axial arm, 5-Fixing screw, 6-Card slot holder, 7-Mouse. Detailed Implementation

[0023] Please refer to Figures 1 to 3 The present invention discloses a slot mounting and clamping device for a single-photon fluorescence microscope, which is connected to a stereo positioning instrument. The device includes a slot clamping body 6, the lower end of which is shaped like a microscope lens, the upper end of which is provided with a mounting hole for connecting to the stereo positioning instrument, and the lower end of which is used to insert into a slot.

[0024] In this invention, the card slot is pre-installed by the card slot holder 6, and then the single-photon fluorescence microscope is inserted into the card slot. This eliminates the need for the microscope lens to be used as a holder to hold the card slot for embedding in the existing solution, thus simplifying the card slot embedding process and improving efficiency.

[0025] In this invention, the slot holder 6 is cylindrical.

[0026] In this invention, the stereo positioning device is provided with a coronal axial arm 4, and the coronal axial arm 4 is provided with a screw hole 1. The mounting hole of the slot clamping body 6 is connected to the screw hole 1 of the coronal axial arm 4 by screws, thereby fixing the slot clamping body 6 to the coronal axial arm 4. The stereo positioning device is also provided with a vertical axial arm 2 for vertical lifting and lowering and a sagittal axial arm 3 for forward and backward movement. The coronal axial arm 4 is connected to the vertical axial arm 2.

[0027] In this invention, a microlens is carried in a cylindrical cavity below the bottom surface of the card slot.

[0028] Specifically, based on the size of the miniature single-photon microscope lens, a slot holder 6 was designed using 3D printing technology to stably hold the miniature microscope lens slot. The overall shape of the slot holder 6 is cylindrical, and its upper end can be connected to the screw hole 1 on the coronal axis arm 4 of the stereo positioning instrument with screws. Its lower end is designed in the shape of a microscope lens.

[0029] The present invention also provides a slot mounting method for a single-photon fluorescence microscope, comprising the following steps:

[0030] Step S1: Fix the mouse head on a stereotaxic instrument, cut open its scalp to expose its skull, and establish a coordinate system on the skull through the sutures on the skull surface according to neurobiological methods to find the target site. Use a skull drill to drill a hole at the target site to penetrate the skull.

[0031] Step S2: Insert the card slot into the lower end of the card slot holder 6, and then connect the upper end of the card slot holder 6 to the stereo positioning device with screws.

[0032] Step S3: Operate the stereotactic instrument and move the slot holder to above the mouse's head through its coronal axial arm 4, so that the slot connected to the lower end of the slot holder 6 is at the target position on the mouse's head.

[0033] Step S4: Operate the stereotactic instrument, and use its vertical axis arm 2 and coronal axis arm 4 to move the slot clamping body 6 downward, so that the microlens in the slot will be inserted into the brain tissue;

[0034] Step S5: When the microlens in the slot is inserted to the target depth, dental cement is used to bond the outer wall of the slot to the surface of the skull. After the dental cement solidifies, the slot holder is pulled out, and the slot is firmly attached to the surface of the mouse skull. At this point, the installation process of the slot is completed.

[0035] Specifically, the mouse's head is first fixed to the stereotactic instrument, the scalp is cut open to expose the skull, and a coordinate system is established on the skull using neurobiological methods through the sutures on the skull surface to locate the target site. A skull drill is then used to drill a hole at the target site to penetrate the skull. The lower end of the slot holder 6 is inserted into the slot, and its upper end is connected to the stereotactic instrument with a screw. The entire slot holder 6 is then fixed to the stereotactic instrument. At this point, the stereotactic instrument is manipulated to adjust the slot to be above the drill hole. A microlens is carried in the cylindrical cavity below the bottom surface of the slot. Then, the slot holder 6 is manipulated downward using the vertical axis arm 2 of the stereotactic instrument. The microlens carried in the lower part of the slot will be inserted into the brain tissue. When it is inserted to the target depth, dental cement is used to bond the outer wall of the slot to the skull surface. After the dental cement solidifies, the screw on the slot is loosened, and the slot holder 6 is pulled out. The slot is then firmly attached to the surface of the mouse skull, and the slot installation process is complete. In formal experiments, simply insert the lens of the miniature microscope into the slot to begin recording calcium ion fluorescence signals.

[0036] This invention simulates the manufacturing of a microscope lens slot holder, reducing the previous two-step process of first holding the microscope with a microscope holder, then installing the slot on the microscope, and finally embedding it using this whole unit, to a single process of directly using the holder to hold the slot and embed it.

[0037] This invention solves the problem of difficult clamping of the microscope slot when mounting a miniature single-photon fluorescence microscope on the surface of a mouse skull. It changes the previous method where only microscope holders were available on the market, but no dedicated slot holder was available. This method required the use of a miniature microscope, first installing the slot on the microscope, then using the microscope holder to install the whole assembly, and finally removing the microscope after installation. This invention eliminates the involvement of the miniature microscope in this process, reduces the risk of microscope damage, and greatly simplifies the slot installation procedure.

[0038] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A slot-mounted clamping device for a single-photon fluorescence microscope, which is connected to a stereoscopic locator, characterized in that, The device includes a card slot holder, the lower end of which is shaped like a microscope lens and is used to directly insert into the card slot to replace the microscope lens during the card slot installation process; the upper end of the card slot holder is provided with a mounting hole for connecting to the stereo positioning instrument, which is also provided with a vertical axial arm.

2. The slot mounting and clamping device for a single-photon fluorescence microscope according to claim 1, characterized in that, The slot holder is cylindrical.

3. The slot mounting and clamping device for a single-photon fluorescence microscope according to claim 2, characterized in that, The stereo positioning device is provided with a coronal axial arm, which has screw holes. The mounting holes of the slot clamping body are connected to the screw holes of the coronal axial arm by screws, thereby fixing the slot clamping body to the coronal axial arm.

4. The slot mounting and clamping device for a single-photon fluorescence microscope according to claim 3, characterized in that, The coronal axial arm is connected to the vertical axial arm.

5. The slot mounting and clamping device for a single-photon fluorescence microscope according to claim 4, characterized in that, A microlens is carried in the cylindrical cavity below the bottom surface of the card slot.

6. A method for mounting a single-photon fluorescence microscope using a slot, characterized in that, Includes the following steps: Step S1: Fix the mouse head on the stereotaxic instrument, cut open its scalp to expose its skull, and establish a coordinate system on the skull through the sutures on the skull surface according to neurobiological methods to find the target site. Use a skull drill to drill a hole at the target site to penetrate the skull. Step S2: Insert the card slot into the lower end of the card slot holder, and then connect the upper end of the card slot holder to the stereo positioning device with screws; Step S3: Operate the stereotactic instrument and move the slot holder above the mouse's head through its coronal axial arm so that the slot connected to the lower end of the slot holder is at the target position on the mouse's head. Step S4: Operate the stereotactic instrument, and use its vertical axis arm and coronal axis arm to move the slot holder downward, so that the microlens in the slot will be inserted into the brain tissue. Step S5: When the microlens in the slot is inserted to the target depth, dental cement is used to bond the outer wall of the slot to the surface of the skull. After the dental cement solidifies, the slot holder is pulled out, and the slot is firmly attached to the surface of the mouse skull. At this point, the installation process of the slot is completed.

Images