High vacuum environment light mirror electron microscope correlation imaging system

Abstract

The present application relates to a kind of high-vacuum environment light mirror electron microscope correlation imaging system, including microscope main body, high-vacuum chamber, sample transmission rod and structured light illumination system. Wherein, microscope main body is provided with imaging system, high-vacuum chamber is located on microscope main body, upper light hole is equipped on the top cover of high-vacuum chamber, bottom is equipped with lower light hole, upper light hole and lower light hole are vertically aligned, and pass through each other. The objective lens of imaging system is located in high-vacuum chamber and is located between upper light hole and lower light hole. Frozen sample is arranged on the storage end of sample transmission rod, and the storage end is arranged in high-vacuum chamber, so that frozen sample is arranged above the objective lens. Structured light illumination system is arranged at the rear side of microscope main body, for emitting structured light to high-vacuum chamber, structured light is irradiated on frozen sample by passing through lower light hole, realizes frozen structured light illumination imaging, solves the problem of low resolution in prior art optical imaging and low positioning accuracy of light mirror electron microscope imaging correlation.

Description

Technical Field

[0001] This invention relates to the technical field of frozen sample microscopic imaging, and more particularly to a high-vacuum environment light and electron microscopy co-imaging system. Background Technology

[0002] Cryo-electron microscopy (cryo-electron microscopy) has become one of the most important experimental techniques in high-resolution structural biology research. Rapid cryo-preparation of biological samples allows for their fixation in near-physiological conditions, avoiding the adverse effects of chemical fixation methods such as sample deformation and damage to ultrastructure, thus providing more realistic information about the biological sample structure. Furthermore, by combining fluorescence labeling, cryo-fluorescence microscopy, and cryo-electron tomography, researchers can utilize fluorescence localization and electron microscopy to specifically identify and perform high-resolution ultrastructural analysis of molecular machines at the same location within the same cell. This technique is known as cryo-correlative light and electron microscopy (Cryo-CLEM).

[0003] Cryo-optical-electron microscopy (cryo-EM) co-imaging technology can integrate the localization and structural information of target molecules, thereby resolving the high-resolution three-dimensional structure of target molecules in situ within cells. This allows for accurate statistical analysis of the in-situ dynamic changes, biological functions, and mechanisms of action of target molecules. However, existing cryo-fluorescence imaging systems are limited by the cryo-imaging environment and the numerical aperture of the optical objectives, severely restricting the resolution achievable by cryo-optical imaging systems. Furthermore, they are inconvenient to use and operate, and samples are easily contaminated or damaged. This further limits the correlation localization accuracy, experimental success rate, and widespread application of cryo-optical-electron microscopy co-imaging technology. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a high-vacuum environment optical-electron microscopy correlation imaging system, which solves the problems of low resolution and low experimental success rate caused by poor cryogenic imaging environment in the prior art.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides a high-vacuum environment optical-electron microscopy correlation imaging system, the specific technical solution of which is as follows:

[0008] A high-vacuum environment optical-electron microscopy correlation imaging system includes:

[0009] The microscope body is equipped with an imaging system;

[0010] The high-vacuum chamber is fixedly mounted on the microscope body. The top cover plate has an upper light-transmitting hole, and the bottom has a lower light-transmitting hole. The upper and lower light-transmitting holes are interconnected.

[0011] The objective lens of the imaging system is located in a high vacuum chamber, between the upper and lower light-transmitting apertures;

[0012] The sample transfer rod includes a placement end and a pull-out end, and extends between the placement end and the pull-out end;

[0013] The frozen sample is placed at the placement end, which passes through the high vacuum chamber so that the frozen sample is positioned above the objective lens.

[0014] The structured light illumination system, located at the rear of the microscope body, is used to emit structured light into the high vacuum chamber. The structured light passes through the lower light aperture and illuminates the frozen sample.

[0015] Furthermore, a position adjustment device is also installed on the side wall of the high vacuum chamber;

[0016] The sample transfer rod's placement end can extend into the high vacuum chamber through the position adjustment device, and the pull-out end is connected to the position adjustment device;

[0017] The position adjustment device is suitable for position adjustment and can drive the sample transfer rod to adjust the position of the frozen sample relative to the objective lens.

[0018] Furthermore, the position adjustment device includes a bellows, a sample transfer tube, and a three-dimensional translation stage;

[0019] The three-dimensional translation stage is fixedly mounted on the high vacuum chamber.

[0020] One end of the bellows is connected to the high vacuum chamber, and the other end is connected to the sample transfer tube, which is connected to the three-dimensional translation stage.

[0021] The sample transfer rod's placement end passes through the sample transfer tube and the bellows, and its outer periphery is sealed to the sample transfer tube.

[0022] The pull-out end abuts against the sample transfer tube, and the operation of the three-dimensional translation stage can drive the corrugated tube connected to the sample transfer tube to extend and retract.

[0023] Furthermore, it also includes a vacuum system;

[0024] The vacuum system includes a low vacuum pump, a vacuum gauge, a first vacuum valve, a first bypass, and a second bypass.

[0025] The first vacuum valve is located between the bellows and the high vacuum chamber, and is sealed to both the bellows and the high vacuum chamber to form a sealed channel between the first vacuum valve, the bellows, the sample delivery tube and the sample transfer rod. The first vacuum valve is used to control the connection and closure of the high vacuum chamber and the sealed channel.

[0026] One end of the first bypass is connected to the sample transfer tube and communicates with the sealed channel, while the other end is connected to the low vacuum pump to evacuate the sealed channel to a low vacuum.

[0027] The second bypass is connected at one end to the side wall of the high vacuum chamber and at the other end to the low vacuum pump, and is used to evacuate the high vacuum chamber to a high vacuum.

[0028] The vacuum gauge is sealed to the side wall of the high vacuum chamber and is used to detect the vacuum level of the high vacuum chamber.

[0029] Furthermore, the first bypass includes a second vacuum valve connected via a gas line;

[0030] One end of the second vacuum valve is connected to the suction port of the low vacuum pump, and the other end of the second vacuum valve is connected to the sample transfer tube.

[0031] The second bypass includes a third vacuum valve and a molecular pump;

[0032] The third vacuum valve is connected to the pump port of the low vacuum pump and is connected to the molecular pump through a gas pipeline. The molecular pump is sealed to the side wall of the high vacuum chamber.

[0033] Furthermore, it also includes a pollution prevention system;

[0034] The anti-contamination system includes a Dewar flask, vacuum connecting tube, heat-conducting rod, flexible connecting strip, connector, and cold box;

[0035] The Dewar jar is connected to the high vacuum chamber via a vacuum connecting tube and placed outside the high vacuum chamber;

[0036] The connector is mounted in the high vacuum chamber by a support frame. One end is connected to the heat-conducting rod via a flexible connecting strip. The heat-conducting rod passes through the vacuum connecting tube and is connected to the Dewar jar. The other end is connected to the cold box, and the frozen sample is placed in the cold box.

[0037] Furthermore, the structured light illumination system includes a laser emitter, a first lens group, a beam splitter, a half-wave plate, a spatial light modulator, a second lens group, an aperture, a third lens group, and a dichroic mirror arranged sequentially along the optical path;

[0038] It also includes a light intensity sensor, located on the side of the beam splitter away from the second lens group.

[0039] Furthermore, the imaging system also includes a fluorescence filter, a fourth lens group, and a detector arranged sequentially along the optical path;

[0040] The detector communicates with a data processor, which processes the images of frozen samples acquired by the detector.

[0041] Furthermore, it also includes an objective lens mount;

[0042] The objective lens mount is detachably and sealed to the bottom of the high vacuum chamber and is located at the lower light passage.

[0043] A circular hole is provided at the center of the objective lens holder, which is connected to the upper and lower light-transmitting holes.

[0044] Furthermore, it also includes temperature sensors and controllers;

[0045] The temperature sensor is located inside the high vacuum chamber and is connected to the controller.

[0046] The controller is also connected to the position adjustment device, imaging system, structured light illumination system and vacuum system, and is used to control the start and stop of the above devices.

[0047] (III) Beneficial Effects

[0048] The high-vacuum environment optical-electron microscopy correlation imaging system provided by the present invention has the following beneficial effects.

[0049] In this invention, a high-vacuum chamber is provided, in which the objective lens and the frozen sample are placed, creating a high-vacuum environment for the frozen sample. Furthermore, a structured light illumination imaging system is incorporated into the microscope body. The beam emitted by this system passes through the lower light-passing aperture of the high-vacuum chamber and illuminates the surface of the frozen sample, achieving structured light illumination imaging of the frozen sample in a vacuum environment and improving the optical resolution of the cryogenic optical imaging system.

[0050] In this invention, the frozen sample is transferred to the high vacuum chamber via a frozen sample transfer rod, which can effectively avoid deformation, ice contamination, and displacement of the frozen sample during the handling process, greatly improving the success rate of the experiment and the accuracy of the optical and electron microscope correlation positioning. Attached Figure Description

[0051] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments of this application and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0052] Figure 1 This is a schematic diagram of the high-vacuum environment optical microscope-electron microscope correlation imaging system in a specific embodiment;

[0053] Figure 2This is a schematic diagram of a partial structure of the optical-electron-microscopy correlated imaging system for a high-vacuum environment in a specific embodiment.

[0054] Figure 3 This is a schematic diagram of the structure inside the high-vacuum chamber in a specific implementation method;

[0055] Figure 4 for Figure 3 Sectional view of section AA;

[0056] Figure 5 for Figure 4 Enlarged view of part B in the image;

[0057] Figure 6 This is an optical path diagram of the imaging system and the structured light illumination system in a specific implementation embodiment;

[0058] Figure 7 This is a schematic diagram of the vacuum system in a specific implementation.

[0059] [Explanation of Labels in the Attached Image]

[0060] 1. Microscope body;

[0061] 2. High vacuum chamber; 201. Cover plate; 202. Upper light-transmitting hole; 204. Observation window; 205. Support frame; 206. Lower light-transmitting hole;

[0062] 3. Sample transfer rod; 301. Placement end; 302. Pull-out end;

[0063] 4. Position adjustment device; 401. Three-dimensional translation stage; 402. Sample transfer tube; 403. Corrugated pipe;

[0064] 5. Anti-contamination system; 501. Dewar jar; 502. Vacuum connecting pipe; 503. Heat-conducting rod; 504. Flexible connecting strip; 505. Connector; 506. Cold box;

[0065] 6. Vacuum system; 601. Molecular pump; 602. Vacuum gauge; 603. First vacuum valve; 605. Second vacuum valve; 606. Low vacuum pump; 607. Third vacuum valve; 60A. First bypass; 60B. Second bypass;

[0066] 7. Imaging system; 701. Objective lens; 703. Fluorescent filter; 704. Fourth lens group; 705. Detector; 706. Data processor;

[0067] 8. Structured light illumination system; 801. Laser emitter; 802. First lens group; 803. Light intensity sensor; 804. Beam splitter; 805. Half-wave plate; 806. Spatial light modulator; 807. Second lens group; 808. Aperture stop; 809. Third lens group; 810. Dichroic mirror;

[0068] 9. Temperature sensor;

[0069] 10. Sensor vacuum socket; 11. Mounting bracket; 12. Controller; 13. Objective lens mount; 14. Translation stage mounting bracket. Detailed Implementation

[0070] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be described in more detail below with reference to the accompanying drawings of the preferred embodiments. In the drawings, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The described embodiments are some, but not all, embodiments of the present invention. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. The embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0071] In the description of this embodiment, it should be understood that the terms "center", "longitudinal", "lateral", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this embodiment and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the scope of protection of this embodiment.

[0072] See Figures 1 to 7 This embodiment provides a high-vacuum ambient light electron microscope-electron microscope correlation imaging system, including a microscope body 1, a high-vacuum chamber 2, a sample transfer rod 3, an imaging system 7, and a structured light illumination system 8.

[0073] Specifically, see Figure 1 , Figure 2 and Figure 3 The high-vacuum chamber 2 is mounted on the microscope body 1 via a mounting bracket 11. The high-vacuum chamber 2 is a box-shaped structure with an opening at the top. A removable cover plate 201 is provided at the opening of the box-shaped structure. The cover plate 201 has an upper light-transmitting hole 202, and the bottom of the high-vacuum chamber 2 has a lower light-transmitting hole 206. The upper light-transmitting hole 202 and the lower light-transmitting hole 206 are interconnected. The bottom of the high-vacuum chamber 2 is also provided with a support frame 205, which is made of heat-insulating materials such as plastic, fiberglass, and ceramic. An observation window 204 is also provided on the front side of the high-vacuum chamber 2. The observation window 204 is an optical window to facilitate observation of the situation inside the high-vacuum chamber 2.

[0074] Further, see Figure 4 The imaging system 7 is mounted on the microscope body 1. The objective lens 701 of the imaging system 7 is installed in the high vacuum chamber 2 via an objective lens holder 13. The objective lens holder 13 is detachably connected to the bottom of the high vacuum chamber 2 and is located at the position of the lower light-transmitting aperture 206. A circular hole is provided at the center of the objective lens holder 13, which communicates with the upper light-transmitting aperture 202 and the lower light-transmitting aperture 206. To ensure a sealed environment within the high vacuum chamber 2, a sealing ring is provided between the objective lens holder 13 and the high vacuum chamber 2. Light-transmitting plates are provided at the positions of the circular hole and the upper light-transmitting aperture 202 to facilitate the entry of the light beam into the high vacuum chamber 2. In this embodiment, the objective lens 701 with different magnifications can be replaced by disassembling the objective lens holder 13 to adapt to different optical imaging needs. For example, it can achieve a large field of view preview under low magnification objective lens 701 and high-resolution imaging of local areas under high magnification objective lens 701.

[0075] In this embodiment, see Figure 2 , Figure 3 , Figure 4 and Figure 5 The sample transfer rod 3 includes a placement end 301 and a pull-out end 302, extending between the placement end 301 and the pull-out end 302. The placement end 301 is used to fix the frozen sample and passes through the high vacuum chamber 2, positioning the frozen sample above the objective lens 701. The side wall of the high vacuum chamber 2 is provided with a corresponding adapter interface for the sample transfer rod 3, which can meet the usage requirements of different models of sample transfer rods 3. The placement end 301 of the sample transfer rod 3 can extend into the high vacuum chamber 2 through the position adjustment device 4. The pull-out end 302 is connected to the position adjustment device 4, which is adapted for position adjustment and can move the sample transfer rod 3 to adjust the position of the frozen sample relative to the objective lens 701.

[0076] Specifically, the position adjustment device 4 includes a bellows 403, a sample transfer tube 402, and a three-dimensional translation stage 401. The three-dimensional translation stage 401 is mounted on the high vacuum chamber 2 via a translation stage fixing bracket 14. One end of the bellows 403 is sealed to the high vacuum chamber 2, and the other end is connected to the sample transfer tube 402. The sample transfer tube 402 is connected to the three-dimensional translation stage 401, and the three-dimensional translation stage 401 is suitable for position adjustment in the X, Y, and Z directions. The placement end 301 of the sample transfer rod 3 passes through the bellows 403 and the sample transfer tube 402, and the pull-out end 302 abuts against the sample transfer tube 402. The outer periphery of the sample transfer rod 3 is sealed to the inner wall of the sample transfer tube 402 via a sealing ring. The bellows 403 is suitable for position adjustment in the length direction, horizontal direction and vertical direction. When the three-dimensional translation stage 401 is working, it can drive the bellows 403 connected to the sample transfer tube 402 to move accordingly. The position of the sample transfer tube 402 relative to the high vacuum chamber 2 is moved, and the insertion length of the sample transfer rod 3 is also changed accordingly. It can accurately adjust the position of the frozen sample relative to the objective lens 701, which facilitates fluorescence imaging of the frozen sample at different positions.

[0077] Further, see Figure 2 , Figure 3 and Figure 7 The high-vacuum environment optical microscope-electron microscope correlation imaging system in this embodiment also includes a vacuum system 6, comprising a low-vacuum pump 606, a first bypass 60A, a second bypass 60B, a first vacuum valve 603, and a vacuum gauge 602. The first vacuum valve 603 is located between the high-vacuum chamber 2 and the bellows 403, and is sealed to both the high-vacuum chamber 2 and the bellows 403 to form a sealed channel between the high-vacuum chamber 2, the bellows 403, the sample transfer tube 402, and the sample transfer rod 3. The first vacuum valve 603 controls the connection and closure of the high-vacuum chamber 2 and the sealed channel. The first bypass 60A is a pre-vacuum system, with its outlet sealed to the exhaust port of the low-vacuum pump 606 and its inlet sealed to the sample transfer tube 402, used to pre-evacuate the sealed channel to a low vacuum. The second bypass 60B is a high-vacuum extraction system. Its outlet end is sealed to the pump port of the low-vacuum pump 606, and its inlet end is connected to the side wall of the high-vacuum chamber 2. It is used to evacuate the high-vacuum chamber 2 to a high vacuum. The vacuum gauge 602 is sealed to the side wall of the high-vacuum chamber 2 and is used to detect the vacuum level of the high-vacuum chamber 2.

[0078] Specifically, the first bypass 60A includes a second vacuum valve 605. One end of the second vacuum valve 605 is sealed and connected to the sample transfer tube 402, and the other end is connected to the suction port of the low vacuum pump 606. The second vacuum valve 605 is used to control the connection and closure of the first bypass 60A and the sealed channel. When it is necessary to evacuate the sealed channel to a low vacuum, the low vacuum pump 606 is started, the first vacuum valve 603 is closed, and the second vacuum valve 605 is opened to begin evacuating the sealed channel to a low vacuum. When the gas pressure in the sealed channel reaches the set value, the second vacuum valve 605 is closed.

[0079] Specifically, the second bypass 60B includes a third vacuum valve 607 and a molecular pump 601. The third vacuum valve 607 is connected to the evacuation port of the low vacuum pump 606 and is connected to the molecular pump 601 through a gas pipeline. The molecular pump 601 is located on the side wall of the high vacuum chamber 2. The third vacuum valve 607 is used to control the connection and disconnection between the second bypass 60B and the low vacuum pump 606, and the molecular pump 601 is used to evacuate the high vacuum chamber 2 to a high vacuum.

[0080] Based on the specific structure of the vacuum system described above, a high vacuum chamber 2 is evacuated to a high vacuum. First, the first vacuum valve 603 and the second vacuum valve 605 are kept closed, the low vacuum pump 606 is started, and the third vacuum valve 607 is opened to begin evacuating the molecular pump 601 and the high vacuum chamber 2 to a low vacuum. When the set value is reached, the molecular pump 601 is started to begin evacuating the high vacuum chamber 2 to a high vacuum. The vacuum gauge 602 is used to monitor the vacuum level of the high vacuum chamber 2.

[0081] In this embodiment, the frozen sample is transferred to the high vacuum chamber 2 via the sample transfer rod 3. The frozen sample is pre-placed at the placement end 301 of the sample transfer rod 3, and the sample transfer rod 3 is pre-inserted into the sample transfer tube 402. The first vacuum valve 603 is kept closed, the third vacuum valve 607 is closed, and the second vacuum valve 605 is opened. The low vacuum pump 606 begins to evacuate the sealed channel to a low vacuum. When the set value is reached, the second vacuum valve 605 is closed, the first vacuum valve 603 is opened, and the third vacuum valve 607 is opened, connecting the sealed channel to the high vacuum chamber 2. The sample transfer rod 3 is then drawn into the high vacuum chamber 2 under negative pressure, realizing the transfer of the frozen sample from the atmosphere to a high vacuum environment. This embodiment, by using the sample transfer rod 3 to transport the frozen sample to the high vacuum chamber 2, avoids the deformation and ice contamination that can damage the structure of the frozen sample during handling in existing frozen sample transfer methods, greatly improving the experimental success rate and obtaining more realistic structural information of the biological sample. Different models of sample transfer rods 3 can be replaced to meet the imaging needs of different frozen samples, making it highly versatile and suitable for a wide range of applications.

[0082] Further, see Figure 3The high-vacuum environment light and electron microscope correlation imaging system in this embodiment also includes an anti-contamination system 5, specifically comprising a Dewar jar 501, a vacuum connecting tube 502, a heat-conducting rod 503, a flexible connecting strap 504, a cold box 506, and a temperature sensor 9. The Dewar jar 501 is connected to the side wall of the high-vacuum chamber 2 via the vacuum connecting tube 502. A connector 505 is fixed to a support frame 205 inside the high-vacuum chamber 2, with one end connected to the heat-conducting rod 503 via the flexible connecting strap 504. The heat-conducting rod 503 passes through the vacuum connecting tube 502 and connects to the Dewar jar 501. The other end of the connector 505 is connected to the cold box 506. The cold box 506 has a slot, and the placement end 301 of the sample transfer rod 3 is placed within the slot of the cold box 506, isolating the frozen sample from the vacuum environment of the high-vacuum chamber 2. This provides an anti-contamination imaging environment for the frozen sample, significantly reducing the deposition of contaminants on the sample during the imaging process. The liquid nitrogen Dewar flask 501, filled with liquid nitrogen, provides a cooling source for the low-temperature environment within the high-vacuum chamber 2. The corresponding vacuum connecting pipe 502, heat-conducting rod 503, flexible connecting strip 504, connector 505, and cold box 506 are all made of copper and other excellent thermal conductors. The cold box 506 within the high-vacuum chamber 2 is cooled through heat conduction, ensuring it remains close to liquid nitrogen temperature and providing excellent protection against contamination of frozen samples. A temperature sensor 9, mounted on the support frame 205, is used to detect the temperature of the cold box 506 within the high-vacuum chamber 2.

[0083] Furthermore, in this embodiment, the structured light illumination system 8 is an external structured light illumination system 8, which is set on the microscope body 1 and located behind the high vacuum chamber 2, and is used to emit structured light into the high vacuum chamber 2 to provide structured light illumination for the objective lens 701 and the frozen sample.

[0084] Specifically, see Figure 6The structured light illumination system 8 in this embodiment includes an illumination module, a first lens group 802, a beam splitter 804, a half-wave plate 805, a spatial light modulator 806, a second lens group 807, an aperture 808, a third lens group 809, and a dichroic mirror 810 arranged sequentially along the optical path. The illumination module is a laser emitter 801, which emits excitation light beams of different wavelengths. The emitted beam is expanded into a parallel beam by the first lens group 802. The parallel beam is converted into a linearly polarized beam with a specific polarization direction by the beam splitter 804. The linearly polarized beam is modulated into structured light with a specific direction and spatial frequency by the half-wave plate 805 and the spatial light modulator 806. The modulated structured light is deflected by 90 degrees after passing through the beam splitter 804 again. It is then focused onto the aperture 808 by the second lens group 807. The aperture 808 filters the focused beam according to the usage requirements, allowing only the positive and negative first-order and zero-order diffraction light to pass through and enter the subsequent optical path. The positive and negative first-order and zero-order diffraction light is expanded and focused by the third lens group 809, and then reflected by the dichroic mirror 810 and passes through the lower light-passing aperture 206 to illuminate the rear pupil of the objective lens 701. After being focused onto the sample by the objective lens 701, it provides structured light illumination for fluorescence imaging of the frozen sample. Furthermore, the first lens group 802 includes three cemented doublet lenses arranged sequentially along the optical path. These three lenses sequentially expand, focus, and expand the emitted beam, transforming the point source illumination beam emitted from the laser emitter 801 into a uniform illumination spot, increasing the uniform illumination range of the beam. The second cemented doublet lens group includes one cemented doublet lens used to focus the modulated structured light onto the aperture 808, filtering the positive and negative first-order and zero-order diffracted light. The third lens group 809 includes two cemented doublet lenses arranged along the optical path, used for beam expansion and focusing, respectively. The focusing cemented doublet lens and the dichroic mirror 810 are respectively mounted on the microscope body 1. The dichroic mirror 810 is located below the lower light aperture 206 and has both reflection and transmission functions, reflecting excitation light (short wavelength) and transmitting emitted light (long wavelength). Furthermore, the structured light illumination system 8 also includes a light intensity sensor 803, which is located on the side of the beam splitter 804 away from the second lens group 807, and is used to sense the intensity of the structured light. In practical use, when two-dimensional structured light illumination imaging is required, only the positive and negative first-order diffracted light at the back pupil of the objective lens 701 is allowed to enter the illumination path to illuminate the sample. The spatial light modulator 806 controls the acquisition of a total of 9 original illumination imaging data points from three illumination angles (each illumination angle shifts three illumination phases). When three-dimensional structured light illumination imaging is required, only the positive and negative first-order and zero-order diffracted light at the back pupil of the objective lens 701 is allowed to enter the illumination path to illuminate the sample. The spatial light modulator 806 controls the acquisition of a total of 15 original illumination imaging data points from three illumination angles at each optical level (each illumination angle shifts five illumination phases).In this embodiment, the spatial frequency of the illumination structured light is changed by the spatial light modulator 806 to adapt to the numerical aperture requirements of the objective lens 701 with different magnification, so as to obtain the corresponding image resolution.

[0085] Specifically, the imaging system 7 in this embodiment includes an objective lens 701, a dichroic mirror 810, a fluorescence filter 703, a fourth lens group 704, a detector 705, and a data processor 706, all disposed within the high-vacuum chamber 2. The objective lens 701, dichroic mirror 810, fluorescence filter 703, fourth lens group 704, and detector 705 are arranged sequentially along the optical path. The detector 705 is communicatively connected to the data processor 706. In this embodiment, the fourth lens group 704 is a cemented doublet lens. A fluorescent substance is pre-marked on the frozen sample. When structured light irradiates the surface of the frozen sample, the fluorescent substance is excited, emitting an emission beam with a longer wavelength than the excitation light (structured light). The emission beam (long-wavelength beam) passes through the dichroic mirror 810, and after stray light is filtered out by the fluorescence filter 703, it is focused by the fourth lens group 704 onto the detector 705 for imaging data acquisition and transmitted to the data processor 706. The data processor 706 analyzes and processes the structured light imaging data and reconstructs high-resolution information.

[0086] Furthermore, the high-vacuum ambient light electron microscope-electron microscope-correlated imaging system 7 in this embodiment also includes a controller 12. The controller 12 is communicatively connected to the position adjustment device 4, the imaging system 7, the vacuum system 6, the structured light illumination system 8, and the temperature sensor 9. The temperature sensor 9 is connected to the controller 12 via a sensor vacuum socket 10 and transmits the temperature information of the cold box 506 inside the high-vacuum chamber 2 to the controller 12 for display. This monitors the temperature of the cold box 506 to ensure it remains below a safe value of -160°C, preventing temperature rise, weakening of anti-contamination performance, and contamination of frozen samples by impurities.

[0087] Based on the above-mentioned high-vacuum environment optical-electron microscopy correlation imaging system, its specific usage process includes the following steps:

[0088] 1) The structured light illumination system 8 is started, and the structured light is introduced from the light inlet at the rear of the microscope host and then into the high vacuum chamber 2. The system optical path collimation and illumination light spatial frequency modulation parameters are optimized and set.

[0089] 2) Evacuate the high-vacuum chamber to a high vacuum level:

[0090] The first vacuum valve 603 is closed, the low vacuum pump 606, the molecular pump 601, and the third vacuum valve 607 are activated to begin evacuating the high vacuum chamber 2. When the vacuum gauge 602 detects that the vacuum level in the high vacuum chamber 2 is better than a set value (e.g., 5 × 10⁻⁶), the evacuation continues. -3 The system can be used when Pa) is reached;

[0091] 3) Fill the Dewar jar 501 with liquid nitrogen to cool the cold box 506. When the controller 12 detects through the temperature sensor 9 that the temperature of the cold box 506 is lower than the set value (e.g., -160°C), the frozen sample transfer can be prepared.

[0092] 4) The pre-frozen sample is fixed to the placement end 301 of the sample transfer rod 3;

[0093] 5) Evacuate a low vacuum in the sealed channel:

[0094] Insert the sample transfer rod 3 containing the frozen sample into the sample transfer tube 402, control the third vacuum valve 607 to close, the second vacuum valve 605 to open, and the low vacuum pump 606 to start drawing a low vacuum into the sealed channel. When the vacuum level reaches the set value (e.g., 10 Pa), control the second vacuum valve 605 to close, the first vacuum valve 603 to open, and the third vacuum valve 607 to open. The sample transfer rod 3 is then sucked into the high vacuum chamber 2 under negative pressure.

[0095] 6) Control the three-dimensional translation stage 401 to precisely adjust the horizontal position and height of the sample transfer rod 3 so that the frozen sample is positioned above the objective lens 701;

[0096] 7) Once the vacuum level in the vacuum chamber returns to the specified value, open the metal baffle and activate structured light illumination imaging: For two-dimensional structured light illumination imaging, use the spatial light modulator 806 to control the acquisition of 9 original illumination imaging data points from three illumination angles (each illumination angle moves three illumination phases); for three-dimensional structured light illumination imaging, use the spatial light modulator 806 to control the acquisition of 15 original illumination imaging data points from three illumination angles at each optical layer (each illumination angle moves five illumination phases), and acquire the original three-dimensional data by controlling the up-and-down movement of the three-dimensional translation stage 401. After data acquisition, the high-frequency information containing high-resolution information is restored by frequency shifting using a structured light illumination reconstruction algorithm, thereby obtaining a frozen structured light illumination fluorescence image with twice the resolution of frozen wide-field illumination fluorescence imaging.

[0097] 8) After optical imaging is completed, the sample transfer rod 3 is pulled out of the high vacuum chamber 2 by the opposite operation of inserting the sample transfer rod 3 into the high vacuum chamber 2, and transferred to the transmission electron microscope (or scanning electron microscope). Based on the obtained fluorescence image, the target area of ​​interest is selected for high-resolution electron microscope data acquisition, thereby obtaining cryo-optical-electron microscope correlated imaging data.

[0098] The above describes the specific structure and usage of the high-vacuum ambient light-electron microscope correlation imaging system of this embodiment. It can be widely applied in cryo-optical imaging and photoelectric correlation imaging technologies, enabling structured light illumination imaging of frozen samples under vacuum conditions, thereby improving the optical resolution and photoelectric correlation alignment accuracy of the imaging system 7. This high-vacuum ambient light-electron microscope correlation imaging system can be used with different types of sample transfer rods 3 and system parameter settings to achieve imaging under different conditions such as freezing, high temperature, atmosphere, and liquid. In this embodiment, different magnification objectives 701 can be replaced according to optical imaging requirements, and a motorized multi-lens mount can be placed to allow free switching of objectives 701 at different magnifications during experiments. The structured light illumination system 8 adjusts the spatial frequency of the structured light through the spatial light modulator 806 to adapt to the usage requirements of objectives 701 at different magnifications, thus meeting the imaging needs of different frozen samples. It has a wide range of applications and strong versatility.

[0099] In summary, the high-vacuum environment optical microscopy-electron microscopy correlation imaging system provided by the present invention is only a preferred embodiment of the present invention. However, the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. A high-vacuum environment optical-electron microscopy correlation imaging system, characterized in that, include: The microscope body (1) is equipped with an imaging system (7); A high vacuum chamber (2) is mounted on the microscope body (1) via a mounting bracket (11). The top cover plate (201) is provided with an upper light-transmitting hole (202) and the bottom is provided with a lower light-transmitting hole (206). The upper light-transmitting hole (202) and the lower light-transmitting hole (206) are interconnected. The objective lens (701) of the imaging system (7) is located in the high vacuum chamber (2) and between the upper light-transmitting hole (202) and the lower light-transmitting hole (206); The sample transfer rod (3) includes a placement end (301) and a pull-out end (302) and extends between the placement end (301) and the pull-out end (302); The frozen sample is placed in the placement end (301), which passes through the high vacuum chamber (2) so that the frozen sample is placed above the objective lens (701); A structured light illumination system (8) is located on the rear side of the microscope body (1) and is used to emit structured light, which passes through the lower light aperture (206) and illuminates the frozen sample. A position adjustment device (4) is also provided on the side wall of the high vacuum chamber (2); the placement end (301) of the sample transfer rod (3) can pass through the position adjustment device (4) and extend into the high vacuum chamber (2), and the pull-out end (302) is connected to the position adjustment device (4); the position adjustment device (4) is adapted for position adjustment and can drive the sample transfer rod (3) to move in order to adjust the position of the frozen sample relative to the objective lens (701); The position adjustment device (4) includes a bellows (403), a sample transfer tube (402), and a three-dimensional translation stage (401); the three-dimensional translation stage (401) is mounted on the high vacuum chamber (2) via a translation stage fixing frame (14); one end of the bellows (403) is connected to the high vacuum chamber (2), and the other end is connected to the sample transfer tube (402), and the sample transfer tube (402) is connected to the three-dimensional translation stage (401); the placement end (301) of the sample transfer rod (3) passes through the sample transfer tube (402) and the bellows (403), and its outer periphery is sealed to the sample transfer tube (402); the pull end (302) abuts against the sample transfer tube (402), and the operation of the three-dimensional translation stage (401) can drive the bellows (403) connected to the sample transfer tube (402) to extend and retract.

2. The high-vacuum environment optical-electron microscopy correlation imaging system according to claim 1, characterized in that, It also includes a vacuum system (6); The vacuum system (6) includes a low vacuum pump (606), a vacuum gauge (602), a first vacuum valve (603), a first bypass (60A), and a second bypass (60B); The first vacuum valve (603) is disposed between the bellows (403) and the high vacuum chamber (2), and is sealed to the bellows (403) and the high vacuum chamber (2) respectively, so as to form a sealed channel between the first vacuum valve (603), the bellows (403), the sample transfer tube (402) and the sample transfer rod (3). The first vacuum valve (603) is used to control the connection and closure of the high vacuum chamber (2) and the sealed channel. One end of the first bypass (60A) is connected to the sample transfer tube (402) and communicates with the sealed channel, and the other end is connected to the low vacuum pump (606) for drawing a low vacuum in the sealed channel; One end of the second bypass (60B) is connected to the side wall of the high vacuum chamber (2), and the other end is connected to the low vacuum pump (606) for pumping a high vacuum into the high vacuum chamber (2); The vacuum gauge (602) is sealed to the side wall of the high vacuum chamber (2) and is used to detect the vacuum level of the high vacuum chamber (2).

3. The high-vacuum environment optical-electron microscopy correlation imaging system according to claim 2, characterized in that, The first bypass (60A) includes a second vacuum valve (605); One end of the second vacuum valve (605) is sealed and connected to the sample transfer tube (402), and the other end is connected to the gas extraction port of the low vacuum pump (606) through a gas pipeline; The second bypass (60B) includes a third vacuum valve (607) and a molecular pump (601). The third vacuum valve (607) is connected to the pump port of the low vacuum pump (606) and is connected to the molecular pump (601) through a gas pipeline. The molecular pump (601) is sealed to the side wall of the high vacuum chamber (2).

4. The high-vacuum environment optical-electron microscopy correlation imaging system according to claim 1, characterized in that, It also includes a pollution prevention system (5); The anti-pollution system (5) includes a Dewar jar (501), a vacuum connecting tube (502), a heat-conducting rod (503), a flexible connecting strip (504), a connector (505), and a cold box (506). The Dewar jar (501) is connected to the high vacuum chamber (2) via the vacuum connecting pipe (502) and is placed outside the high vacuum chamber (2); The connector (505) is located in the high vacuum chamber (2) via a support frame (205). One end is connected to the heat-conducting rod (503) via the flexible connecting strip (504). The heat-conducting rod (503) passes through the vacuum connecting tube (502) and is connected to the Dewar jar (501). The other end of the connector (505) is connected to the cold box (506). The frozen sample is placed in the cold box (506).

5. The high-vacuum environment optical-electron microscopy correlation imaging system according to claim 1, characterized in that, The structured light illumination system (8) includes a laser emitter (801), a first lens group (802), a beam splitter (804), a half-wave plate (805), a spatial light modulator (806), a second lens group (807), an aperture (808), a third lens group (809), and a dichroic mirror (810) arranged sequentially along the optical path. It also includes a light intensity sensor (803) located on the side of the beam splitter (804) away from the second lens group (807).

6. The high-vacuum environment optical-electron microscopy correlation imaging system according to claim 1, characterized in that, The imaging system (7) also includes a fluorescence filter (703), a fourth lens group (704) and a detector (705) arranged sequentially along the optical path. The detector (705) is communicatively connected to the data processor (706), which is used to process the frozen sample images acquired by the detector (705).

7. The high-vacuum environment optical-electron microscopy correlation imaging system according to claim 1, characterized in that, It also includes an objective lens mount (13); The objective lens mount (13) is detachably and sealed to the bottom of the high vacuum chamber (2) and located at the lower light-transmitting hole (206); The objective lens holder (13) has a circular hole at its center, which is connected to the upper light-transmitting hole (202) and the lower light-transmitting hole (206).

8. The high-vacuum environment optical-electron microscopy correlation imaging system according to any one of claims 2 to 7, characterized in that, It also includes a temperature sensor (9) and a controller (12); The temperature sensor (9) is located in the high vacuum chamber and is communicatively connected to the controller (12); The controller (12) is also communicatively connected to the position adjustment device (4), the imaging system (7), the structured light illumination system (8) and the vacuum system (6), and the controller (12) is used to control the start and stop of the equipment.

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