X-ray light sheet confocal three-dimensional fluorescence microscopic imaging system and method

Abstract

The application discloses an X-ray light sheet confocal three-dimensional fluorescence microscopic imaging system and method, and belongs to the technical field of X-ray fluorescence marked microscopic imaging. The system comprises a fan-shaped X-ray source, an X-ray slit, an electric translation stage, a three-dimensional electric object table, a microscopic scanning module, a confocal module, a lens and a photoelectric detector. A sample is placed on the three-dimensional electric object table, and the fan-shaped X-ray source is located on one side of the three-dimensional electric object table. The X-ray slit is located between the three-dimensional electric object table and the fan-shaped X-ray source and is controlled to be lifted and lowered by the electric translation stage. The microscopic scanning module is located above the three-dimensional electric object table, and the confocal module, the lens and the photoelectric detector are sequentially located on an emergent light path of the microscopic scanning module. The application combines tomography technology based on the fan-shaped X-ray source and a fluorescence confocal microscopic system based on laser, has high spatial resolution and fast scanning speed.

Description

Technical Field

[0001] The present invention relates to the technical field of X-ray fluorescence labeling microscopic imaging, and in particular to an X-ray light sheet confocal three-dimensional fluorescence microscopic imaging system and method. Technical Background

[0002] X-rays are a common detection method. When X-rays interact with materials, they will be absorbed by the material and excite the inner electrons in the atoms to a high-energy state. Subsequently, the electrons in the high-energy state will release energy back to a low-energy state through collisions, radiation, etc. For different materials, their absorption of X-rays and the characteristic spectral lines emitted will also vary. Therefore, X-rays can be used for material detection. In addition, compared with visible light, X-ray photons have higher energy and stronger penetrating power. This characteristic enables X-ray detection to analyze the internal structure and element distribution of samples.

[0003] In order to obtain three-dimensional information inside the sample, there are currently two main methods: one is X-ray computed tomography technology (CN02283444.3; CN02155046.8), which is based on the principle of slicing and uses a fan-beam X-ray source to irradiate the sample with X-rays along the axial plane for analysis at each section, and reconstructs a three-dimensional image of the sample in combination with a three-dimensional algorithm. However, the maximum resolution of this method can only reach 0.5mm, which cannot achieve microscopic imaging; the other is to use a micro-focus X-ray source to scan each area of ​​the sample point by point (based on a microbeam single capillary ellipsoid mirror). X-ray fluorescence imaging [J]. Tao Fen, Feng Binggang, Deng Biao, et al. Spectroscopy and Spectral Analysis, 2020, 40(7):5; Using confocal X-ray method to study the three-dimensional distribution of elements and chemical states in living plants [C]. Wei Xiangjun, Jiang Zheng, Zou Yang, et al. National X-ray Fluorescence Spectroscopy Academic Conference. Geological Society of China, 2010.), and then reconstruct the three-dimensional information of the sample. This method has higher spatial resolution, but it has high requirements for the X-ray source and its supporting system. In addition, the scanning process takes a long time and cannot be applied to the detection of dynamic processes, such as metabolism in biological tissues. Summary of the Invention

[0004] The present invention combines tomography technology based on a sector X-ray source and a laser-based fluorescence confocal microscopy system. By using the fluorescence confocal system to detect fluorescence signals, the spatial resolution can be greatly improved. In combination with placing a micron-level X-ray slit on the side of the sample to shape the X-rays, the fluorescence signal layer excited in the sample is made thinner, which can further improve the spatial resolution in the depth direction. In addition, in combination with a two-dimensional scanning galvanometer system, the scanning speed can be significantly improved, greatly shortening the time required for the scanning process.

[0005] This invention provides an X-ray sheet confocal three-dimensional fluorescence microscopy system and method. Through a unique system design, it enables rapid, high-resolution three-dimensional fluorescence microscopy of samples labeled with X-ray fluorescence scintillators. This system is suitable for studying microscopic dynamic processes such as metabolism in transparent biological tissues, as well as for studying the microstructure and elemental composition within other transparent X-ray fluorescence scintillator materials.

[0006] The technical solutions of the present invention are as follows:

[0007] An X-ray sheet confocal three-dimensional fluorescence microscopy imaging system includes a sector X-ray source, an X-ray slit, an electric translation stage, a three-dimensional electric stage, a microscopy scanning module, a confocal module, a lens, and a photodetector;

[0008] The sample to be microscopically imaged is placed on the three-dimensional electric stage, and the sector X-ray source is located on one side of the three-dimensional electric stage; the X-ray slit is located between the three-dimensional electric stage and the sector X-ray source, and is raised and lowered by the electric translation stage;

[0009] The microscopic scanning module is located above the three-dimensional electric stage and is used to collect the fluorescence signal emitted by the sample and scan it point by point. The confocal module, lens and photodetector are located in sequence on the output light path of the microscopic scanning module. The confocal module filters the fluorescence signal emitted by the microscopic scanning module, and then focuses it through the lens and receives it from the photodetector.

[0010] As a preferred embodiment of the present invention, the X-ray slit is parallel to the loading surface of the three-dimensional electric loading platform.

[0011] As a preferred embodiment of the present invention, the microscopic scanning module includes a microscope objective lens, a tube mirror, a scanning mirror, a two-dimensional scanning galvanometer, an electric push-pull beam splitter / reflector, and a camera;

[0012] The microscope objective lens is used to collect the fluorescence signal in the sample and realize the microscopic function, and the tube lens transmits the signal to the electric push-pull beam splitter / reflector;

[0013] The cylindrical mirror and scanning mirror are used to form a two-dimensional scanning system with a two-dimensional scanning galvanometer, and two-dimensional scanning of the sample in the horizontal direction is achieved through the deflection of the two-dimensional scanning galvanometer; the electric push-pull beam splitter / reflector can realize the switching of beam splitting and reflection functions, and the camera is located on the beam splitting light path of the electric push-pull beam splitter / reflector and is used to image the sample; the scanning mirror and the two-dimensional scanning galvanometer are located in turn on the reflection light path of the electric push-pull beam splitter / reflector.

[0014] As a preferred embodiment of the present invention, the confocal module includes a group of coaxially placed confocal lenses and a pinhole diaphragm. The pinhole diaphragm is located on the confocal plane of the confocal lens group and is conjugate with the focus of the microscopic scanning module. It is used to filter out fluorescence outside the focus of the microscopic scanning module and improve the spatial resolution of three-dimensional imaging.

[0015] The working process of the above system is as follows: the X-rays emitted by the fan-shaped X-ray source are shaped by the X-ray slit and then incident on the sample, exciting the scintillator to emit fluorescence in a very thin horizontal cross-section inside the sample. The fluorescence signal of the sample is then collected by the microscope objective lens, passed through the microscope scanning module, the confocal module, and the lens, and received by the photodetector, thereby obtaining the fluorescence signal of a certain point in the sample. Combined with the two-dimensional scanning galvanometer in the microscope scanning module to scan the entire cross-section, a fluorescence microscopic image of the entire cross-section can be obtained. After that, the three-dimensional electric stage is controlled to move up and down to perform fluorescence imaging on each cross-section of the sample, and finally a three-dimensional fluorescence microscopic image of the sample can be reconstructed.

[0016] Compared with the prior art, the advantages of the present invention are:

[0017] (1) Compared with traditional computed tomography technology, the present invention combines a confocal microscopy system, which can greatly improve the imaging resolution of the system and obtain microscopic three-dimensional information of the sample.

[0018] (2) Compared with the imaging technology based on micro-focus X-ray source scanning, the present invention uses a galvanometer scanning system as a scanning module. By controlling the two-dimensional scanning galvanometer to scan the entire cross section, the scanning speed can be greatly improved, the detection time can be shortened, and it is more suitable for three-dimensional scanning of dynamic processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the structure of the X-ray light sheet confocal three-dimensional fluorescence microscopy imaging system. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to the accompanying drawings.

[0021] like Figure 1 As shown in the figure, the X-ray sheet confocal three-dimensional fluorescence microscopy imaging system mainly consists of a fan-shaped X-ray source, an X-ray slit, an electric translation stage, a three-dimensional electric stage, a microscopy scanning module, a confocal module, a lens, and a photodetector. The basic system structure is shown in the attached Figure 1 :

[0022] Wherein, the sector-shaped X-ray source is used to excite the X-ray fluorescence scintillator marker in the sample to generate a fluorescence signal;

[0023] The X-ray slit is used to reduce the thickness of the fan-shaped X-ray, thereby reducing the thickness of the excited fluorescent signal layer in the sample and improving the accuracy of sample sectioning;

[0024] The electric translation stage is used to control the up and down position of the X-ray slit so that the fluorescence signal layer excited by the X-rays is exactly located on the focal plane of the microscopy scanning module;

[0025] The three-dimensional electric stage is used to place the sample to be tested and can adjust the position of the sample;

[0026] In this embodiment, the microscopic scanning module includes a microscope objective lens, a tube lens, a scanning mirror, a two-dimensional scanning galvanometer, an electric push-pull beam splitter / reflector, and a camera. The microscope objective lens is used to collect the fluorescence signal in the sample and realize the microscopic function. The tube lens and the scanning mirror are used to form a conjugate system with the two-dimensional scanning galvanometer. The two-dimensional scanning galvanometer is deflected to realize two-dimensional scanning of the sample in the horizontal direction. The electric push-pull beam splitter / reflector is used to cooperate with the camera to image the sample, thereby facilitating the adjustment of the position of the sample and the X-ray fluorescence layer.

[0027] In this embodiment, the confocal module includes a group of coaxially placed confocal lenses and a pinhole diaphragm. The pinhole diaphragm is located on the confocal plane of the confocal lens group and forms an object-image conjugate with the focus of the microscopic scanning module. It can filter out noise signals outside the focus of the microscopic scanning module, thereby improving the spatial resolution of three-dimensional imaging.

[0028] The lens is used to focus the fluorescent signal so that it can be completely received by the photodetector;

[0029] The photoelectric detector is used to receive the collected fluorescence signal. The present invention can select an ordinary photoelectric detection device, or a detection device suitable for weak signals such as a photomultiplier tube, an avalanche diode, etc., or even use a spectrometer to realize spectral detection of the fluorescence signal.

[0030] The following will be combined with the Figure 1 Explain the working process of the whole system:

[0031] Turn on the power of all equipment, open the X-ray protection door, place the sample labeled with X-ray fluorescent scintillator on the three-dimensional electric stage, adjust the three-dimensional electric stage so that the sample is in the appropriate position, and then close the X-ray protection door;

[0032] Turn on the fan-shaped X-ray source and switch the push-pull beam splitter / reflector to the beam splitter. At this time, the sample can be imaged on the camera. While observing the image on the camera, the height of the X-ray slit is controlled by the electric translation stage so that the fluorescence signal layer excited in the sample after the X-ray passes through the slit is exactly located on the focal plane of the microscopy scanning system. Then, switch the push-pull beam splitter / reflector to the reflector and perform a three-dimensional scan of the sample by controlling the two-dimensional scanning galvanometer and the three-dimensional electric stage.

[0033] During the three-dimensional scanning process, the fluorescent signal emitted by the fluorescent signal layer of the sample is collected by the microscope objective lens in the microscopic scanning module, and is emitted after passing through the cylindrical lens, the reflector, the scanning mirror, and the two-dimensional scanning galvanometer in sequence. The emitted fluorescent signal is then incident on the confocal module, filtered by the pinhole diaphragm, and emitted from the confocal module, and finally received by the photodetector, thereby obtaining the fluorescent signal of a certain point in the sample. In other words, only the fluorescent signal at the focus on the sample enters the detector at a time. As the two-dimensional galvanometer deflects, the focus on the sample will move on the focal plane, scanning the entire focal plane. Since the focal plane and the fluorescent plane have been overlapped in the above, by controlling the two-dimensional scanning galvanometer, it is possible to scan the cross section of the sample where the entire fluorescent signal layer is located, thereby obtaining the fluorescent information of the sample in the fluorescent signal layer.

[0034] The sample's height is then adjusted using a three-dimensional motorized stage, and fluorescence imaging is performed on each cross-section of the sample to obtain fluorescence information for the entire sample. By analyzing and processing the fluorescence data from the entire sample, a three-dimensional fluorescence microscopic image of the sample can be reconstructed.

[0035] In a specific embodiment of the present invention, other optical elements such as filters and attenuators can be added to the optical path to filter the fluorescence signal, reduce background noise, and prevent detector saturation. In addition, the photodetector can be replaced by a spectrometer, combined with multiple X-ray fluorescence scintillator markers with different fluorescence peaks, to achieve three-dimensional microscopic fluorescence imaging of multiple analytes.

[0036] The above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples, and many variations are possible. All variations that can be directly derived or imagined by a person skilled in the art from the disclosure of the present invention should be considered to be within the scope of protection of the present invention.

Claims

1. An X-ray sheet confocal three-dimensional fluorescence microscopy system, characterized in that: It includes a fan-shaped X-ray source, an X-ray slit, an electric translation stage, a three-dimensional electric stage, a microscopy scanning module, a confocal module, a lens and a photodetector; The sample to be microscopically imaged is placed on the three-dimensional electric stage, and the fan-shaped X-ray source is located on one side of the three-dimensional electric stage; The X-ray slit is located between the three-dimensional electric stage and the fan-shaped X-ray source, parallel to the loading surface of the three-dimensional electric stage, and is raised and lowered by the electric translation stage; The microscopic scanning module is located above the three-dimensional motorized stage and is used to collect the fluorescent signal emitted by the sample and scan it point by point. The confocal module, lens, and photodetector are sequentially located on the output light path of the microscopic scanning module. The confocal module filters the fluorescent signal emitted by the microscopic scanning module, which is then focused by the lens and received by the photodetector. The microscopic scanning module includes a microscope objective lens, a tube lens, a scanning mirror, a two-dimensional scanning galvanometer, an electric push-pull beam splitter / reflector, and a camera; the microscope objective lens is used to collect fluorescence signals in the sample and realize the microscopic function, and the tube lens transmits the signal to the electric push-pull beam splitter / reflector; the tube lens and the scanning mirror are used to form a two-dimensional scanning system with the two-dimensional scanning galvanometer, and two-dimensional scanning of the sample in the horizontal direction is realized by deflecting the two-dimensional scanning galvanometer; the electric push-pull beam splitter / reflector can realize the switching of beam splitting and reflection functions, and the camera is located on the beam splitting optical path of the electric push-pull beam splitter / reflector for imaging the sample; the scanning mirror and the two-dimensional scanning galvanometer are located in turn on the reflection optical path of the electric push-pull beam splitter / reflector; The confocal module includes a group of coaxially placed confocal lenses and a pinhole diaphragm. The pinhole diaphragm is located on the confocal plane of the confocal lens group and is conjugate with the focus of the microscopic scanning module. It is used to filter out fluorescence outside the focus of the microscopic scanning module and improve the spatial resolution of three-dimensional imaging.

2. An imaging method based on the X-ray sheet confocal three-dimensional fluorescence microscopy imaging system according to claim 1, characterized in that: include: Place the sample labeled with X-ray fluorescence scintillator on the three-dimensional electric stage, and adjust the position of the three-dimensional electric stage so that the sample is located within the working area; Start the sector X-ray source and switch the push-pull beam splitter / reflector to the beam splitter function. The light emitted by the sector X-ray source passes through the X-ray slit and excites a fluorescent signal layer inside the sample. Observe the sample through a camera and adjust the height of the X-ray slit using the motorized translation stage so that the fluorescent signal layer is located on the focal plane of the microscope objective. Switch the push-pull beam splitter / reflector to the reflector function. The fluorescence signal emitted by the sample is imaged by the microscope objective. The imaging signal is output through the cylindrical mirror, reflector, scanning mirror, and two-dimensional scanning galvanometer mirror in sequence. The confocal module filters the fluorescence out of focus. By controlling the deflection of the two-dimensional scanning galvanometer mirror, the sample is scanned in the horizontal direction of the fluorescence signal layer. Finally, the photodetector receives the fluorescence signal on the focal plane of the microscope objective. The three-dimensional motorized stage is controlled to move the sample up and down, and fluorescence imaging is performed on each cross section of the sample. The fluorescence information of the entire sample is obtained by the photodetector, and finally a three-dimensional fluorescence microscopic image of the sample is reconstructed.

3. The imaging method of the X-ray sheet confocal three-dimensional fluorescence microscopy imaging system according to claim 2, characterized in that: The height of the sample is adjusted by a three-dimensional electric stage, so that a fluorescent signal layer from low to high or from high to low is excited inside the sample, and the sample is sequentially sliced ​​along the height direction.

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