阅读说明：本技术 捕获微通道内运动大分子形态的冷冻荧光显微成像系统及方法 (Freezing fluorescence microscopic imaging system and method for capturing moving macromolecule form in microchannel ) 是由 范亮亮 赵亮 赵宏 于 2020-05-22 设计创作，主要内容包括：一种捕获微通道内运动大分子形态的冷冻荧光显微成像系统及方法,该系统由流动快速滞止和急速冷冻系统、显微镜系统、摄像系统和图像处理系统组成；流动快速滞止和急速冷冻系统,用于准确固定原流动条件下的大分子形态,同时有效增强被染色大分子的荧光强度；显微镜系统与摄像系统相结合,实现对冷冻固化后的大分子进行三维显微成像,对于长度较长的大分子,进行分段扫描；图像处理系统通过对捕获的大分子图像进行三维重构,最终还原微通道内大分子在运动状态下的形态；本发明对于自由状态下的微纳米目标物显微成像有着极其重要的应用价值。(A freezing fluorescence microscopic imaging system and method for capturing the form of moving macromolecules in a microchannel, wherein the system comprises a flow rapid stagnation and rapid freezing system, a microscope system, a camera system and an image processing system; the system is a rapid flow stagnation and rapid freezing system, is used for accurately fixing the macromolecular form under the original flow condition and effectively enhancing the fluorescence intensity of the dyed macromolecules; the microscope system is combined with the camera system to realize three-dimensional microscopic imaging of the frozen and solidified macromolecules and sectional scanning of the macromolecules with longer length; the image processing system finally restores the form of macromolecules in the microchannel in a motion state by performing three-dimensional reconstruction on the captured macromolecule image; the invention has extremely important application value for micro-imaging of the micro-nano target object in a free state.)

1. A freezing fluorescence microscopic imaging system for capturing the moving macromolecule form in a microchannel is characterized in that: comprises a flow rapid stagnation and rapid freezing system, a microscope system (7), an image pickup system (8) and an image processing system (9); the rapid flow stagnation and rapid freezing system realizes rapid stagnation of flow in the microchannel and rapid freezing and solidification of fluid, thereby keeping the form of macromolecules in the original flow state and enhancing the fluorescence intensity of dyed macromolecules; the microscope system (7) is combined with the camera system (8) and is used for carrying out three-dimensional microscopic imaging on the frozen and solidified macromolecules, and the image processing system (9) carries out three-dimensional reconstruction on the captured macromolecule images and finally restores the forms of the macromolecules in the microchannels under the motion state.

2. The system of claim 1, wherein the system comprises: in the flow rapid stagnation and rapid freezing system, an inlet (1) is connected with an experimental section micro-channel (5) and then is connected with an outlet (3) through a three-way valve (6), and the experimental section micro-channel (5) is placed in a low-temperature liquid storage tank (4); the parallel micro-channel (2) is connected with the inlet (1) and the three-way valve (6); the rapid stagnation of the fluid flowing in the micro-channel (5) at the experimental section is realized through the combined action of the three-way valve (6) and the parallel micro-channel (2); the low-temperature liquid storage tank (4) enables the fluid in the micro-channel (5) at the experimental section to be frozen rapidly, so that the form of macromolecules is solidified, and the fluorescence intensity is enhanced.

3. The system of claim 2, wherein the system comprises: the experiment section micro-channel (5) is made of a non-deformable transparent material.

4. A method of operating a system for frozen fluorescence microscopy imaging for the capture of a mobile macromolecular species within a microchannel as claimed in any one of claims 1 to 3 wherein: dissolving macromolecules into fluid, wherein in an initial state, under the action of a three-way valve (6), an experimental section microchannel (5) is communicated with an inlet (1) and an outlet (3), and no fluid flows in parallel microchannels (2); after the required fluid condition is achieved, the three-way valve (6) acts rapidly to enable the fluid in the micro-channel (5) at the experimental section to flow rapidly; the fluid continuously flowing in from the inlet (1) flows to the outlet (3) through the parallel micro-channel (2); rapidly adding a low-temperature fluid into the low-temperature liquid storage tank (4) to rapidly freeze the fluid in the micro-channel (5) at the experimental section, thereby solidifying the form of macromolecules in the fluid and enhancing the fluorescence intensity; the microscope system (7) is combined with the camera system (8) to realize three-dimensional microscopic imaging of the frozen and solidified macromolecules and to perform segmented scanning of the macromolecules with longer length; the image processing system (9) finally restores the form of macromolecules in the micro-channel in a motion state by performing three-dimensional reconstruction on the captured macromolecule image.

5. The method of claim 4 for operating a frozen fluorescence microscopy imaging system for capturing the morphology of a moving macromolecule in a microchannel, wherein the method comprises the steps of: the macromolecules are fluorescently stained to achieve accurate capture of the morphology of macromolecules of smaller radial dimensions.

Technical Field

The invention relates to a microscopic imaging method. In particular to a freezing fluorescence microscopic imaging system and a method for capturing the form of moving macromolecules in a microchannel.

Background

The morphological measurement of moving macromolecules in the microchannel has important value in the fields of biomedicine, chemical engineering and the like. For example, a large number of microscale channels exist in the human body, which constitute microchannels for blood flow. Blood exhibits viscoelastic fluid properties, mainly due to the presence of protein macromolecules in the plasma. The characteristics of the protein macromolecules such as the shape and the distribution in the microvasculature directly influence the hemodynamic characteristics and are directly related to the occurrence of certain diseases (such as thrombus). Therefore, by capturing microscopic detail characteristics such as the form and distribution of the moving macromolecules in the microchannel, the mechanism of disease occurrence is disclosed, and the method has important significance for preventing and treating diseases. At present, no relevant measurement means is reported, and the difficulty is that the form of a moving macromolecule can be changed in a microchannel, and the form of the moving macromolecule cannot be accurately captured by means of a conventional laser confocal microscope and the like. Secondly, the length of the macromolecule chain is long and can reach dozens of micrometers, and great difficulty is brought to three-dimensional capture of the full chain length of the moving macromolecule. Finally, the radial size of the macromolecule is small, nanometer or even smaller, and cannot be captured by a conventional fluorescence microscope. Therefore, capturing the moving macromolecule form in the microchannel is a difficult problem in the field of microscopic imaging, and a new means is urgently needed to be developed for solving the problem.

Disclosure of Invention

In order to solve the above problems, the present invention aims to provide a frozen fluorescence microscopy imaging system and method for capturing the form of a moving macromolecule in a microchannel, which has the advantages of high precision, capability of realizing three-dimensional capture of the form of the moving macromolecule in the microchannel, and the like, and has great application potential in the fields of biomedicine, chemical engineering, and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

a freezing fluorescence microscopic imaging system for capturing the movement macromolecule form in a microchannel comprises a flow rapid stagnation and rapid freezing system, a microscope system 7, a camera system 8 and an image processing system 9; the rapid flow stagnation and rapid freezing system realizes rapid stagnation of flow in the microchannel and rapid freezing and solidification of fluid, thereby keeping the form of macromolecules in the original flow state and enhancing the fluorescence intensity of dyed macromolecules; the microscope system 7 is combined with the camera system 8 to perform three-dimensional microscopic imaging on the frozen and solidified macromolecules, and the image processing system 9 performs three-dimensional reconstruction on the captured macromolecule images to finally restore the form of the macromolecules in the microchannel in a motion state.

In the flow rapid stagnation and rapid freezing system, an inlet 1 is connected with an experimental section micro-channel 5 and then is connected with an outlet 3 through a three-way valve 6, and the experimental section micro-channel 5 is placed in a low-temperature liquid storage tank 4; the parallel micro-channel 2 is connected with the inlet 1 and the three-way valve 6; the rapid stagnation of the fluid flowing in the micro-channel 5 at the experimental section is realized through the combined action of the three-way valve 6 and the parallel micro-channel 2; the low-temperature liquid storage tank 4 enables the fluid in the micro-channel 5 at the experimental section to be frozen rapidly, so that the form of macromolecules is solidified, and the fluorescence intensity is enhanced.

The working method of the freezing fluorescence microscopic imaging system for capturing the movement macromolecule form in the microchannel is characterized in that the macromolecule is dissolved in fluid, and in an initial state, under the action of a three-way valve 6, an experimental section microchannel 5 is communicated with an inlet 1 and an outlet 3, and no fluid flows in a parallel microchannel 2; after the required fluid condition is achieved, the three-way valve 6 acts rapidly, so that the fluid in the micro-channel 5 at the experimental section flows rapidly; the fluid continuously flowing into the inlet 1 flows to the outlet 3 through the parallel micro-channel 2; rapidly adding a low-temperature fluid into the low-temperature liquid storage tank 4 to rapidly freeze the fluid in the micro-channel 5 at the experimental section, thereby solidifying the form of macromolecules in the fluid and enhancing the fluorescence intensity; the microscope system 7 is combined with the camera system 8 to realize three-dimensional microscopic imaging of the frozen and solidified macromolecules and sectional scanning of the macromolecules with longer length; the image processing system 9 performs three-dimensional reconstruction on the captured macromolecule image to finally restore the form of the macromolecule in the microchannel in a motion state.

The invention solves the key problem of capturing the form of the moving macromolecules in the microchannel, and has the following advantages:

1) the form of macromolecules in the original flowing state in the microchannel is effectively kept through a flowing rapid stagnation and rapid freezing system, and the problem that the macromolecules are continuously subjected to form change is solved;

2) through freezing and solidification, the fluorescence intensity of the dyed macromolecules can be effectively enhanced, so that the capturing of the morphology of the macromolecules by using an optical microscope becomes possible;

3) by combining a rapid flow stagnation and rapid freezing system, a microscope system, a camera system and an image processing system, the full-chain-length three-dimensional capture of moving macromolecules can be realized, and the method has great application value in the fields of biomedicine, chemical engineering and the like.

Drawings

FIG. 1 is a schematic diagram of the system of the present invention.

Detailed Description

The invention is described in further detail below with reference to the following figures and detailed description:

a freezing fluorescence microscopic imaging system for capturing the movement macromolecule form in a microchannel comprises a flow rapid stagnation and rapid freezing system, a microscope system 7, a camera system 8 and an image processing system 9; the rapid flow stagnation and rapid freezing system realizes rapid stagnation of flow in the microchannel and rapid freezing and solidification of fluid, thereby keeping the form of macromolecules in the original flow state and enhancing the fluorescence intensity of dyed macromolecules; the microscope system 7 is combined with the camera system 8 to perform three-dimensional microscopic imaging on the frozen and solidified macromolecules, and the image processing system 9 performs three-dimensional reconstruction on the captured macromolecule images to finally restore the form of the macromolecules in the microchannel in a motion state.

In the flow rapid stagnation and rapid freezing system, an inlet 1 is connected with an experimental section micro-channel 5 and then is connected with an outlet 3 through a three-way valve 6, and the experimental section micro-channel 5 is placed in a low-temperature liquid storage tank 4; the parallel microchannel 2 is connected with an inlet 1 and a three-way valve 6. The rapid stagnation of the fluid flowing in the experiment section micro-channel 5 is realized through the combined action of the three-way valve 6 and the parallel micro-channel 2. The low-temperature liquid storage tank 4 enables the fluid in the micro-channel 5 at the experimental section to be frozen rapidly, so that the macromolecular form is solidified, and the fluorescence intensity is enhanced.

As a preferred embodiment of the present invention, the cryogenic fluid in the cryogenic fluid reservoir 4 is liquid nitrogen.

As a preferred embodiment of the present invention, the experimental microchannel 5 in which the macromolecules are located can be made of a non-deformable transparent material such as glass.

As a preferred embodiment of the present invention, the macromolecules of the present invention are fluorescently stained to achieve accurate capture of the morphology of macromolecules of smaller radial dimensions.

The working method of the freezing fluorescence microscopic imaging system for capturing the movement macromolecule form in the microchannel is characterized in that the macromolecule is dissolved in fluid, and in an initial state, under the action of a three-way valve 6, an experimental section microchannel 5 is communicated with an inlet 1 and an outlet 3, and no fluid flows in a parallel microchannel 2; after the required fluid condition is achieved, the three-way valve 6 acts rapidly, so that the fluid in the micro-channel 5 at the experimental section flows rapidly; the fluid continuously flowing into the inlet 1 flows to the outlet 3 through the parallel micro-channel 2; rapidly adding a low-temperature fluid into the low-temperature liquid storage tank 4 to rapidly freeze the fluid in the micro-channel 5 at the experimental section, thereby solidifying the form of macromolecules in the fluid and enhancing the fluorescence intensity; the microscope system 7 is combined with the camera system 8 to realize three-dimensional microscopic imaging of the frozen and solidified macromolecules and sectional scanning of the macromolecules with longer length; the image processing system 9 performs three-dimensional reconstruction on the captured macromolecule image to finally restore the form of the macromolecule in the microchannel in a motion state.

The following describes the implementation of the present invention in one embodiment:

taking the three-dimensional form capture of the long-chain protein macromolecules in the micro-channel as an example, the dyed long-chain protein macromolecules flow in the micro-channel 5 of the experimental section. In the initial state, under the action of the three-way valve 6, the experiment section micro-channel 5 is communicated with the inlet 1 and the outlet 3, and no fluid flows in the parallel micro-channel 2. After the required fluid conditions are reached, the three-way valve 6 acts rapidly to rapidly arrest the fluid flow in the experimental section microchannel 5. The fluid which flows in continuously from the inlet 1 flows to the outlet 3 through the parallel micro-channels 2. And (3) rapidly adding liquid nitrogen and other low-temperature fluids into the low-temperature liquid storage tank 4 to rapidly freeze the fluid in the micro-channel 5 at the experimental section, so that the form of the large molecules of the long-chain protein is solidified, and the fluorescence intensity of the gas is enhanced. The microscope system 7 is combined with the camera system 8, so that three-dimensional microscopic imaging of the frozen and solidified long-chain protein macromolecules is realized, and segmented scanning is carried out on the long-chain protein macromolecules. And the image processing system 9 is utilized to carry out three-dimensional reconstruction on the captured protein macromolecule image, and finally, the form of the protein macromolecule in the microchannel under the motion state is reduced. In conclusion, the invention solves the problem of capturing the form of the moving macromolecules in the microchannel, provides a novel microscopic imaging system and a novel microscopic imaging method, and has huge application potential in the fields of biomedicine, chemical engineering and the like.