Sleeve for multiphoton fluorescence microscope in-vivo intracerebral imaging

Abstract

The utility model belongs to the technical field of biological instruments, and relates to an in-vivo multi-photon microscopic imaging sleeve for long-term recording. The sleeve is a transparent cylinder made of quartz glass, and a hollow aluminum foil sheet for fixing the skull is adhered to the sleeve; the inner diameter of the sleeve can be selected by a user as required; the hollow aluminum foil sheet can be pressed according to the diameter of the quartz glass column, the inner diameter of the hollow aluminum foil sheet is consistent with the diameter of the quartz glass column, the hollow aluminum foil sheet is embedded in the periphery of the sleeve, and the sleeve is embedded in the hollow inner diameter of the hollow aluminum foil sheet through UV shadowless glue. The use result shows that the sleeve for in-vivo multi-photon microscopic imaging is simple in structure and convenient to use, not only can be flexibly adapted to in-vivo multi-photon microscopic imaging of nuclei in different brain regions, but also can greatly improve the image resolution of cells in a specified region, so that the in-vivo multi-photon microscopic imaging can be realized. The method is a powerful means for improving information data acquisition and obtaining more accurate and effective information.

Description

Technical Field

[0001] The utility model belongs to the technical field of biological instruments and relates to a medical instrument, specifically to a sleeve for multiphoton fluorescence microscope live brain imaging, which can be used to improve imaging resolution when multiphoton fluorescence microscope detects deep tissue imaging in the brain of living animals and can perform long-term observation of animals. Background Art

[0002] Currently, in brain science research, exploring the cognitive activities of awake animals and the activity patterns of neurons in different brain regions is a key research direction in neurobiology. Multiphoton microscopy has the advantage of excitation light being able to penetrate thick specimens, allowing scientists to conduct research on living animals, thereby more accurately exploring the workings of the brain at the subcellular level. However, although two-photon excitation light can penetrate thick tissue, the area currently probed by two-photon microscopy in most domestic laboratories is limited to the cerebral cortex. If the real-time dynamic changes in deep brain tissue of living animals can be observed over a long period of time, it will be possible to more accurately explore the pathogenesis of diseases and promote the development of brain science research.

[0003] Some imported cannulas currently offered by some companies are not only expensive but also have fixed height and diameter specifications, making them difficult to customize based on the desired area of ​​observation. These cannulas are constructed with a thin glass sheet as the base, embedded in the center of a hollow aluminum tube. This can lead to some loss of laser light as it passes through the glass sheet, limiting the distance it can reach deep into tissue and significantly reducing imaging resolution.

[0004] Currently, most domestic laboratories can only use two-photon microscopy to observe changes in superficial brain tissue (such as blood vessels and the cortex), while imaging of deeper structures is often blurry or impossible. Due to the high fixation of the cannulas sold by some companies, after implantation into the animal's brain, the limited penetration of the two-photon fluorescence microscope laser and the limitations of scattered light prevent the laser from reaching the bottom of the cannula. This results in low-resolution and unclear imaging, significantly limiting the exploration and research of deep brain structures. Summary of the Invention

[0005] The purpose of this utility model is to solve existing technical problems: due to the limited penetration distance of the excitation light wavelength of a two-photon fluorescence microscope and the divergence of scattered light, how to make the laser penetrate and reach the target tissue with low loss is one of the main problems that need to be solved. In addition, how to achieve long-term observation of animals is also a problem that needs to be solved by the technology of this utility model. Therefore, in order to make the specifications of the cannula meet the needs of observing different deep brain regions, while also improving the imaging resolution of deep tissue in the brain of in vivo animals under a two-photon fluorescence microscope, so as to facilitate real-time observation of dynamic changes in deep brain tissue, this application provides a cannula for multiphoton fluorescence microscopy of living brain imaging.

[0006] Based on the above background, the present invention solves its technical problems by employing the following technical solutions: Optical quartz glass is a material that can change the direction of optical propagation and possesses excellent spectral properties, including birefringence, which allows it to transmit both visible light and a continuous spectrum from the far ultraviolet to the near infrared. Quartz glass has a visible light transmittance exceeding 93% and over 80% in the ultraviolet spectral region, while scattering is negligible. Based on these advantages, cylindrical quartz glass is used to replace the existing cannula's tube body, wall, and base glass. This allows laser light to reach the target tissue with minimal loss and scattering. Furthermore, the quartz glass can be customized to specific specifications (thickness: 0.5mm-5mm; diameter: 0.2mm-4mm) by adjusting the cutting laser parameters, depending on the depth of the target tissue within the brain and the observation range.

[0007] The present invention discloses a cannula for in vivo multiphoton microscopy with long-term recording, which consists of a cannula body and a hollow aluminum foil. The cannula body is a transparent cylinder made of quartz glass with an aluminum foil adhered thereto.

[0008] In the present invention, the thickness and diameter of the quartz glass column of the cannula body can be customized according to the requirements of the brain area (thickness: 0.5mm-5mm; diameter: 0.2mm-4mm). In one embodiment of the present invention, the thickness of the cannula body is 1.5mm and the diameter is 2mm.

[0009] In the present invention, the hollow aluminum foil is arranged on the outer periphery of the sleeve body, and the sleeve body is embedded into the hollow inner diameter of the hollow aluminum foil by UV shadowless glue;

[0010] In the present invention, the inner diameter of the hollow aluminum foil can be changed according to the size of the sleeve body (inner diameter: 0.2mm-4mm, outer diameter: 1mm-8mm). In one embodiment of the present invention, the inner diameter of the hollow aluminum foil is 2mm and the outer diameter is 6mm.

[0011] During use, the quartz glass column is fixedly connected to a hollow aluminum foil with the same inner diameter as the glass column. The purpose of this is to attach the aluminum foil to the skull and serve as an anchor to fix the quartz glass column implanted in the mouse brain. This not only ensures that the glass column is in contact with the target tissue and is parallel to the skull at a height, but also facilitates subsequent fixation with dental cement after the aluminum foil serves as a base to the skull, thereby achieving the purpose of long-term observation. This cannula for in vivo multiphoton microscopy with long-term recording consists of a cannula and a hollow aluminum foil. The cannula is a transparent cylinder made of quartz glass with a hollow aluminum foil adhered to its periphery.

[0012] The beneficial effects of this invention are: after the cannula of this invention is implanted into the animal brain, both imaging resolution and observation depth are significantly improved to a level unattainable using previous cannulas. Furthermore, when the cannula is implanted at the appropriate angle, it is even possible to observe morphological changes in dendritic spines on dendrites, a submicroscopic structure that was not visible using previous cannulas. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a three-dimensional structural diagram of a cannula used for multiphoton fluorescence microscopy in vivo brain imaging in this embodiment;

[0014] Figure 2 Schematic diagram of the implementation of the cannula used for multiphoton fluorescence microscopy in vivo brain imaging in this embodiment;

[0015] Figure 3 It is two-photon fluorescence imaging obtained using the cannula of the existing technology;

[0016] Figure 4 is the two-photon fluorescence imaging obtained using the cannula of this embodiment;

[0017] Figure numerals: 1. cannula body; 2. aluminum foil; 3. multiphoton microscopy system; 4. skull; 5. cortical tissue; 6. hippocampal tissue. DETAILED DESCRIPTION

[0018] The structure and effects of the present application are further described in detail below with reference to the embodiments. It is understood that the specific embodiments described herein are merely for explaining the present invention, and are not intended to limit the present invention. It should also be noted that, for ease of description, the accompanying drawings only show portions, not all, of the structure relevant to the present invention.

[0019] Because the hippocampus plays a role in memory and spatial orientation, it is closely linked to various central nervous system diseases such as Alzheimer's disease, epilepsy, and depression. Here, a multiphoton fluorescence microscopy intracerebral imaging cannula implanted into the hippocampus of C57BL / 6 mice is used as an example of a specific implementation method.

[0020] Example

[0021] This embodiment discloses a cannula for multiphoton fluorescence microscopy in vivo brain imaging, referring to Figure 1 The cannula comprises a cannula body 1 and an aluminum foil sheet 2. The cannula body 1 is a transparent cylindrical body made of quartz glass. The aluminum foil sheet 2 has a hollow structure in the middle, which is adapted to the dimensions of the cannula body 1. In this embodiment, commercially available quartz glass was cut into the required thickness (0.5mm-5mm) and diameter (0.2mm-4mm) as needed for the experiment. Aluminum foil sheets with the same inner diameter (0.2mm-4mm) and outer diameter (1mm-8mm) as the quartz glass column were ordered from a hardware factory. Based on the location and size of the hippocampus in the brain of C57BL / 6 mice, the parameters of a laser cutting instrument were set to produce a quartz glass column with a thickness of 1.5mm and a diameter of 2mm, which is the cannula body 1 of this embodiment. The aluminum foil sheet was then pressed into a metal ring with an inner diameter of 2mm and an outer diameter of 6mm, which is the aluminum foil sheet 2 of this embodiment. Both were rinsed in 100% acetone and allowed to dry. In a clean work environment, use tweezers to dip a corner of the cannula (1) into the UV-curing optical adhesive. Carefully place the cannula (1) in the center of the aluminum foil (2) and evenly spread the curing agent with a needle. Now, illuminate the cannula with a UV-curing LED light for one minute. If any glue solidifies on the glass surface, gently peel it off with a sharp needle. Leave the semi-fixed cannula (1) for 24 hours until the adhesive is fully bonded. Then, soak the cannula in 75% alcohol until ready for use.

[0022] The operating principle of the cannula used for multiphoton fluorescence microscopy in vivo brain imaging in this embodiment is as follows:

[0023] Reference Figure 2In the process of burying the cannula body 1, we first expose the mouse skull 4 and aspirate the cortical tissue 5 above the hippocampus 6 until the hippocampus 6 is exposed. The cannula body 1 is slowly pressed vertically onto the hippocampus 6 (the cannula body 1 is facing down and the aluminum foil 2 is facing up). At this time, it can be seen that the hippocampus 6 close to the cannula body 1 is slightly squeezed and deformed. After filling the aspirated cortical tissue 5, the hollow aluminum foil 2 is adhered to the skull, and fast-bonding glue is used to adhere the hollow aluminum foil 2 to the skull 4. At the same time, fast-bonding dental cement is prepared to further anchor the connection between the aluminum foil 2 and the skull 4 to increase the firmness. Imaging preparation can be carried out after the animal recovers. The animal is placed under the two-photon microscopy system 3, the lens is aligned with the cannula 1, and the height is adjusted in time until the image is clear.

[0024] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A cannula for multiphoton fluorescence microscopy intravital brain imaging, characterized in that: The invention is composed of a sleeve body (1) and an aluminum foil sheet (2), wherein the sleeve body (1) is a transparent cylinder made of quartz glass, and a hollow structure is provided in the middle of the aluminum foil sheet (2), and the hollow structure is adapted to the outer diameter of the sleeve body (1), and the sleeve body (1) is fixedly arranged in the hollow structure of the aluminum foil sheet (2).

2. The cannula for multiphoton fluorescence microscopy intravital brain imaging according to claim 1, characterized in that: The wall thickness of the sleeve body (1) is 0.5 mm to 5 mm, and the diameter of the sleeve body (1) is 0.2 mm to 4 mm.

3. The cannula for multiphoton fluorescence microscopy intravital brain imaging according to claim 2, characterized in that: The wall thickness of the sleeve body (1) is 1.5 mm, and the diameter of the sleeve body (1) is 2 mm.

4. The cannula for multiphoton fluorescence microscopy intravital brain imaging according to claim 1, characterized in that: The aluminum foil (2) is arranged on the outer periphery of the sleeve body (1), and the sleeve body (1) is embedded in the inner diameter of the hollow structure of the aluminum foil (2) through UV shadowless adhesive.

5. The cannula for multiphoton fluorescence microscopy intravital brain imaging according to claim 1, characterized in that: The hollow structure of the aluminum foil (2) has an inner diameter of 0.2 mm to 4 mm and an outer diameter of 1 mm to 8 mm.

6. The cannula for multiphoton fluorescence microscopy intravital brain imaging according to claim 5, characterized in that: The hollow structure of the aluminum foil (2) has an inner diameter of 2 mm and an outer diameter of 6 mm.

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