CN113670956A - High vacuum environment optical mirror electron microscope associated imaging system - Google Patents
High vacuum environment optical mirror electron microscope associated imaging system Download PDFInfo
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Abstract
The invention relates to a high-vacuum environment photomicroscope electron microscope correlated imaging system which comprises a microscope main body, a high-vacuum chamber, a sample transmission rod and a structured light illumination system. Wherein, be provided with imaging system in the microscope main part, high vacuum chamber locates in the microscope main part, is equipped with on the apron of high vacuum chamber top and leads to the unthreaded hole, and the bottom is equipped with down leads to the unthreaded hole, goes up to lead to the unthreaded hole and leads to the vertical alignment of unthreaded hole down, link up each other. The objective lens of the imaging system is arranged in the high vacuum chamber and is positioned between the upper light through hole and the lower light through hole. The frozen sample is arranged at the object placing end of the sample transmission rod, and the object placing end penetrates through the high-vacuum chamber so that the frozen sample is arranged above the objective lens. The structured light illuminating system is arranged on the rear side of the microscope body and used for emitting structured light into the high-vacuum chamber, the structured light penetrates through the lower light through hole to irradiate on the frozen sample, the frozen structured light illuminating imaging is realized, and the problems of low resolution of frozen optical imaging and low optical lens electron microscope imaging correlation positioning precision in the prior art are solved.
Description
Technical Field
The invention relates to the technical field of microscopic imaging of frozen samples, in particular to a high-vacuum environment optical microscope and electron microscope associated imaging system.
Background
The three-dimensional reconstruction technology of the cryoelectron microscope has become the most important experimental means for the research of high-resolution structure biology. The rapid freezing preparation technology of the biological sample can freeze and fix the sample in a near physiological state, and avoids adverse effects of sample deformation, damaged ultrastructure and the like caused by a chemical fixing method, so that more real biological sample structure information can be obtained. Meanwhile, by combining fluorescence labeling, cryofluorescence Microscopy and cryoelectron Microscopy tomography imaging technologies, people can also perform specific identification and high-resolution ultrastructure analysis on molecular machines at the same position in the same cell by using fluorescence positioning and an Electron microscope, and the technology is called Cryo-relative Light and Electron Microscopy (Cryo-CLEM).
The cryo-optical microscope and electron microscope correlated microscopic imaging technology can integrate the positioning and structural information of target molecules, so as to analyze the high-resolution three-dimensional structure of the target molecules in situ in cells, and accurately count and analyze the in-situ dynamic change rule, the biological function, the action mechanism and the like of the target molecules. However, the existing frozen fluorescence imaging system is limited by the frozen imaging environment and the numerical aperture of the optical objective, the resolution ratio which can be achieved by the frozen optical imaging system is severely restricted, the use and the operation are inconvenient, the sample is easy to pollute and damage, and the associated positioning precision, the experimental success rate and the popularization and the application of the technology of the frozen optical microscope electron microscope associated imaging technology are further limited.
Disclosure of Invention
Technical problem to be solved
In view of the above disadvantages and shortcomings in the prior art, the invention provides a high vacuum environment photomicroscope electron microscope correlated imaging system, which solves the problems of low resolution and low success rate of experiments caused by poor freezing imaging environment in the prior art.
(II) technical scheme
In order to achieve the purpose, the invention provides a high vacuum environment optical microscope and electron microscope related imaging system, which has the following specific technical scheme:
a high vacuum environment optical mirror electron microscope correlation imaging system comprises:
a microscope body provided with an imaging system;
the high vacuum chamber is arranged on the microscope main body through a fixing frame, an upper light through hole is formed in the top cover plate, a lower light through hole is formed in the bottom of the top cover plate, and the upper light through hole and the lower light through hole are communicated with each other;
an objective lens of the imaging system is arranged in the high vacuum chamber and is positioned between the upper light through hole and the lower light through hole;
a sample transfer rod comprising a placement end and a withdrawal end and extending between the placement end and the withdrawal end;
the frozen sample is arranged at the object placing end which is arranged in the high vacuum chamber in a penetrating way so as to be arranged above the objective lens;
and the structured light illuminating system is arranged on the rear side of the microscope body and used for emitting structured light into the high-vacuum chamber, and the structured light penetrates through the lower light through hole to irradiate on the frozen sample.
Furthermore, a position adjusting device is also arranged on the side wall of the high vacuum chamber;
the object placing end of the sample transmission rod can penetrate through the position adjusting device and extend into the high vacuum chamber, and the drawing end is connected with the position adjusting device;
the position adjusting device is suitable for position adjustment and can drive the sample transmission rod to move so as to adjust the position of the frozen sample relative to the objective lens.
Further, the position adjusting device comprises a corrugated pipe, a sample transmission pipe and a three-dimensional translation table;
the three-dimensional translation stage is arranged on the high vacuum chamber through a translation stage fixing frame;
one end of the corrugated pipe is connected to the high vacuum chamber, the other end of the corrugated pipe is connected with a sample transmission pipe, and the sample transmission pipe is connected with the three-dimensional translation table;
the object placing end of the sample transmission rod is arranged in the sample transmission pipe and the corrugated pipe in a penetrating way, and the periphery of the sample transmission rod is hermetically connected with the sample transmission pipe;
the drawing end is abutted to the sample transmission pipe, and the three-dimensional translation table can drive the corrugated pipe connected with the sample transmission pipe to stretch.
Further, the device also comprises a vacuum pumping system;
the vacuumizing system comprises a low-vacuum pump, a vacuum gauge, a first vacuum valve, a first bypass and a second bypass;
the first vacuum valve is arranged between the corrugated pipe and the high vacuum chamber and is respectively connected with the corrugated pipe and the high vacuum chamber in a sealing way so as to form a sealing channel among the first vacuum valve, the corrugated pipe, the sample conveying pipe and the sample transmission rod, and the first vacuum valve is used for controlling the communication and the closing of the high vacuum chamber and the sealing channel;
one end of the first bypass is connected to the sample transmission pipe and communicated with the sealed channel, and the other end of the first bypass is connected with the low vacuum pump and used for pumping low vacuum to the sealed channel;
one end of the second bypass is connected to the side wall of the high vacuum chamber, and the other end of the second bypass is connected with the low vacuum pump and used for pumping high vacuum to the high vacuum chamber;
the vacuum gauge is connected to the side wall of the high vacuum chamber in a sealing mode and used for detecting the vacuum degree of the high vacuum chamber.
Further, the first bypass comprises a second vacuum valve connected by a gas line;
one end of the second vacuum valve is connected to the air exhaust port end of the low vacuum pump, and the other end of the second vacuum valve is connected to the sample transmission pipe;
the second bypass comprises a third vacuum valve and a molecular pump;
the third vacuum valve is connected with the air exhaust port end of the low vacuum pump and connected with the molecular pump through a gas pipeline, and the molecular pump is hermetically connected on the side wall of the high vacuum chamber.
Further, an anti-pollution system is also included;
the anti-pollution system comprises a Dewar tank, a vacuum connecting pipe, a heat conducting rod, a soft connecting belt, a connector and a cold box;
the dewar tank is connected to the high vacuum chamber through a vacuum connecting pipe and is arranged outside the high vacuum chamber;
the connector is arranged in the high-vacuum chamber through the support frame, one end of the connector is connected with the heat conducting rod through the flexible connecting belt, the heat conducting rod penetrates through the vacuum connecting pipe to be connected with the Dewar tank, the other end of the heat conducting rod is connected with the cold box, and the frozen sample is arranged in the cold box.
Further, the structured light illumination system comprises a laser transmitter, a first lens group, a beam splitter prism, a half-wave plate, a spatial light modulator, a second lens group, a diaphragm, a third lens group and a dichroic mirror which are sequentially arranged along a light path;
the light intensity sensor is arranged on one side, far away from the second lens group, of the light splitting prism.
Furthermore, the imaging system also comprises a fluorescence filter, a fourth lens group and a detector which are sequentially arranged along the light path;
the detector is in communication connection with the data processor, and the data processor is used for processing the frozen sample image collected by the detector.
Further, the device also comprises an objective lens fixing seat;
the objective lens fixing seat is detachably connected to the bottom of the high vacuum chamber in a sealing manner and is positioned at the position of the lower light through hole;
the center of the objective lens fixing seat is provided with a round hole which is communicated with the upper light through hole and the lower light through hole.
Further, the device also comprises a temperature sensor and a controller;
the temperature sensor is arranged in the high vacuum chamber and is in communication connection with the controller;
the controller is also in communication connection with the position adjusting device, the imaging system, the structured light illuminating system and the vacuum pumping system, and the controller is used for controlling the start and stop of the equipment.
(III) advantageous effects
The high-vacuum environment optical microscope and electron microscope correlated imaging system provided by the invention has the following beneficial effects.
According to the invention, the high vacuum chamber is arranged, so that the objective lens and the frozen sample are placed in the high vacuum chamber during use, and a high vacuum environment is provided for the frozen sample. Furthermore, a structured light illumination imaging system is arranged on the microscope main body, and light beams emitted by the structured light illumination system penetrate through the lower light through hole of the high vacuum chamber and irradiate on the surface of the frozen sample, so that structured light illumination imaging of the frozen sample in a vacuum environment is realized, and the optical resolution of the frozen optical imaging system is improved.
According to the invention, the frozen sample is conveyed into the high vacuum chamber through the frozen sample conveying rod, so that the deformation, ice pollution and movement of the position of the frozen sample in the clamping process can be effectively avoided, and the experiment success rate and the correlation positioning precision of the optical microscope and the electron microscope are greatly improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application, and in which:
FIG. 1 is a schematic structural diagram of a high-vacuum environment photomicroscope electron microscope related imaging system in an embodiment;
FIG. 2 is a schematic view of a partial structure of a high-vacuum environment optical microscope-electron microscope-associated imaging system in an embodiment;
FIG. 3 is a schematic diagram of the structure in a high vacuum chamber in an embodiment;
FIG. 4 is a cross-sectional view taken along line A-A of FIG. 3;
FIG. 5 is a partial enlarged view B of FIG. 4;
FIG. 6 is an optical path diagram of an imaging system and a structured light illumination system in an embodiment;
figure 7 is a schematic diagram of an evacuation system in accordance with an embodiment.
[ description of reference ]
1. A microscope body;
2. a high vacuum chamber; 201. a cover plate; 202. an upper light through hole; 204. an observation window; 205. a support frame; 206. a lower light through hole;
3. a sample transfer rod; 301. a placing end; 302. drawing the end;
4. a position adjustment device; 401. a three-dimensional translation stage; 402. a sample transfer tube; 403. a bellows;
5. an anti-contamination system; 501. a dewar tank; 502. a vacuum connecting pipe; 503. a heat conducting rod; 504. a soft connecting belt; 505. a connector; 506. cooling the box;
6. a vacuum pumping system; 601. a molecular pump; 602. a vacuum gauge; 603. a first vacuum valve; 605. a second vacuum valve; 606. a roughing pump; 607. a third vacuum valve; 60A, a first bypass; 60B, a second bypass;
7. an imaging system; 701. an objective lens; 703. a fluorescence filter; 704. a fourth lens group; 705. a detector; 706. a data processor;
8. a structured light illumination system; 801. a laser transmitter; 802. a first lens group; 803. a light intensity sensor; 804. a beam splitter prism; 805. a half-wave plate; 806. a spatial light modulator; 807. a second lens group; 808. a diaphragm; 809. a third lens group; 810. a dichroic mirror;
9. a temperature sensor;
10. a sensor vacuum receptacle; 11. a fixed mount; 12. a controller; 13. an objective lens holder; 14. a translation stage fixing frame.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be described in more detail below with reference to the accompanying drawings in the preferred embodiments of the present invention. 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 only some, but not all embodiments of the invention. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
In the description of the present embodiment, it is to be understood that the terms "center", "longitudinal", "lateral", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplicity of description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and therefore, should not be construed as limiting the scope of the present embodiment.
Referring to fig. 1 to 7, the present embodiment provides a high vacuum environment photomicroscope-related imaging system, which includes a microscope body 1, a high vacuum chamber 2, a sample transmission rod 3, an imaging system 7, and a structured light illumination system 8.
Specifically, referring to fig. 1, 2 and 3, the high vacuum chamber 2 is mounted on the microscope body 1 through the fixing frame 11, the high vacuum chamber 2 is a box-packed structure with an upper end opening, a detachable cover plate 201 is arranged at the opening end of the box-packed structure, an upper light through hole 202 is arranged on the cover plate 201, a lower light through hole 206 is arranged at the box bottom of the high vacuum chamber 2, and the upper light through hole 202 and the lower light through hole 206 are communicated with each other. The bottom of the high vacuum chamber 2 is further provided with a support frame 205, the support frame 205 is made of heat insulation materials such as plastics, glass fibers and ceramics, the front side face of the high vacuum chamber 2 is further provided with an observation window 204, and the observation window 204 is an optical window sheet, so that the condition in the high vacuum chamber 2 can be observed conveniently.
Further, referring to fig. 4, the imaging system 7 is provided on the microscope body 1, the objective lens 701 of the imaging system 7 is mounted in the high vacuum chamber 2 through the objective lens holder 13, and the objective lens holder 13 is detachably attached to the bottom of the high vacuum chamber 2 and located at the position of the lower light-passing hole 206. The center of the objective lens holder 13 is correspondingly provided with a circular hole, and the circular hole is communicated with the upper light through hole 202 and the lower light through hole 206. In order to ensure the sealing environment in the high vacuum chamber 2, a sealing ring is arranged between the objective lens fixing seat 13 and the high vacuum chamber 2, and the round hole and the upper light-transmitting hole 202 are correspondingly provided with light-transmitting sheets so that light beams can enter the high vacuum chamber 2. In this embodiment, the objective lens 701 with different magnification factors can be replaced by detaching the objective lens fixing base 13, so as to adapt to different optical imaging requirements, for example, large-view preview under the low-power objective lens 701 and high-resolution imaging of a local area under the high-power objective lens 701 can be realized.
In this embodiment, referring to fig. 2, 3, 4 and 5, the sample transmission rod 3 includes a placement end 301 and a withdrawal end 302, and the sample transmission rod 3 extends between the placement end 301 and the withdrawal end 302. Put thing end 301 and be used for fixed freezing sample to wear to locate in high vacuum chamber 2, make freezing sample be located objective 701 top, correspond on the lateral wall of high vacuum chamber 2 and be provided with the 3 adaptation interfaces of sample transmission pole, can satisfy the user demand of different model sample transmission poles 3. The object placing end 301 of the sample transmission rod 3 can penetrate through the position adjusting device 4 to extend into the high vacuum chamber 2, the drawing end 302 is connected with the position adjusting device 4, and the position adjusting device 4 is suitable for position adjustment and can drive the sample transmission rod 3 to move so as to adjust the position of the frozen sample relative to the objective lens 701.
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 being mounted on the high vacuum chamber 2 by a translation stage mount 14, the bellows 403 being hermetically connected at one end to the high vacuum chamber 2 and at the other end to the sample transfer tube 402, the sample transfer tube 402 being connected to the three-dimensional translation stage 401, the three-dimensional translation stage 401 being adapted to X, Y, Z position adjustment in three directions. The object placing end 301 of the sample transmission rod 3 is arranged in the corrugated pipe 403 and the sample transmission pipe 402 in a penetrating way, the drawing end 302 is abutted against the sample transmission pipe 402, and the periphery of the sample transmission rod 3 is connected with the inner wall of the sample transmission pipe 402 in a sealing way through a sealing ring. The bellows 403 is suitable for position adjustment in the length direction, the horizontal direction and the vertical direction, and when the three-dimensional translation stage 401 works, the bellows 403 connected with the sample transmission pipe 402 can be driven to move correspondingly, the sample transmission pipe 402 moves relative to the position of the high vacuum chamber 2, the penetration length of the sample transmission rod 3 is correspondingly changed, the position of the frozen sample relative to the objective lens 701 can be accurately adjusted, and fluorescence imaging of the frozen sample at different positions is facilitated.
Further, referring to fig. 2, fig. 3 and fig. 7, the high vacuum environment optical microscope and electron microscope related imaging system in this embodiment further includes a vacuum pumping system 6, which includes a low vacuum pump 606, a first bypass 60A, a second bypass 60B, a first vacuum valve 603 and a vacuum gauge 602. Wherein, the first vacuum valve 603 is disposed between the high vacuum chamber 2 and the bellows 403, and is hermetically connected with the high vacuum chamber 2 and the bellows 403 respectively, so as to form a sealed passage between the high vacuum chamber 2, the bellows 403, the sample transmission pipe 402 and the sample transmission rod 3, and the first vacuum valve 603 is used for controlling the communication and closing of the high vacuum chamber 2 and the sealed passage. The first bypass 60A is a pre-vacuum system, and the outlet end is hermetically connected to the exhaust port end of the roughing pump 606, and the inlet end is hermetically connected to the sample transfer tube 402, for pre-evacuating the sealed channel. The second bypass 60B is a high vacuum pumping system, and the outlet end is hermetically connected to the pumping port end of the roughing pump 606, and the inlet end is connected to the sidewall of the high vacuum chamber 2, for pumping high vacuum to the high vacuum chamber 2. The vacuum gauge 602 is hermetically attached to a sidewall of the high vacuum chamber 2, and detects a degree of vacuum of the high vacuum chamber 2.
Specifically, the first bypass 60A includes a second vacuum valve 605, one end of the second vacuum valve 605 is hermetically connected to the sample transfer tube 402, and the other end is connected to the suction port end of the roughing pump 606, and the second vacuum valve 605 is used for controlling the communication and closing of the first bypass 60A and the sealed channel. When the low vacuum pumping of the sealed channel is needed, the low vacuum pump 606 is controlled to be started, the first vacuum valve 603 is closed, the second vacuum valve 605 is opened, the low vacuum pumping of the sealed channel is started, and when the air pressure of the sealed channel reaches a set value, the second vacuum valve 605 is controlled to be closed.
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 suction port of the roughing pump 606 and connected to the molecular pump 601 through a gas pipeline, the molecular pump 601 is disposed on the sidewall of the high vacuum chamber 2, the third vacuum valve 607 is used for controlling the connection and the disconnection between the second bypass 60B and the roughing pump 606, and the molecular pump 601 is used for evacuating the high vacuum chamber 2.
Based on the specific structure of the vacuum pumping system, the high vacuum chamber 2 is pumped high. First, the first vacuum valve 603 and the second vacuum valve 605 are kept closed, the roughing pump 606 is controlled to be started, the third vacuum valve 607 is opened, the roughing vacuum starts to be performed on the molecular pump 601 and the high vacuum chamber 2, when a set value is reached, the molecular pump 601 is controlled to be started, the roughing vacuum starts to be performed on the high vacuum chamber 2, and the vacuum gauge 602 is used for monitoring the vacuum degree of the high vacuum chamber 2.
In this embodiment, the frozen sample is transferred into the high vacuum chamber 2 through the sample transfer rod 3. The frozen sample is placed in the placing end 301 of the sample transmission rod 3 in advance, and the sample transmission rod 3 is inserted into the sample transmission tube 402 in advance. Keeping the first vacuum valve 603 closed, controlling the third vacuum valve 607 closed, controlling the second vacuum valve 605 open, and the roughing pump 606 starting to evacuate the sealed channel. When the set value is reached, the second vacuum valve 605 is controlled to be closed, the first vacuum valve 603 is controlled to be opened, the third vacuum valve 607 is controlled to be opened, the sealing channel is communicated with the high vacuum chamber 2, and the sample transmission rod 3 is sucked into the high vacuum chamber 2 under the action of negative pressure, so that the transmission of the frozen sample from the atmosphere to the high vacuum environment is realized. In this embodiment will freeze the sample and carry to high vacuum chamber 2 through sample transmission pole 3, can avoid the frozen sample among the current freezing transmission mode to get the in-process and take place deformation and the damage of ice pollution to the structure of freezing sample, improved the experiment success rate greatly to obtain more real biological sample's structural information. For the imaging requirements of different frozen samples, the sample transmission rods 3 of different models can be replaced, the universality is strong, and the application range is wide.
Further, referring to fig. 3, the high vacuum environment optical mirror and electron microscope related imaging system in the present embodiment further includes an anti-contamination system 5, specifically including a dewar tank 501, a vacuum connection pipe 502, a heat conduction rod 503, a flexible connection belt 504, a cold box 506, and a temperature sensor 9. Wherein, dewar jar 501 passes through vacuum connecting pipe 502 to be connected on the lateral wall of high vacuum chamber 2, connector 505 is fixed in on the support frame 205 in high vacuum chamber 2, one end is passed through flexible coupling strip 504 and is connected with heat conduction stick 503, heat conduction stick 503 passes vacuum connecting pipe 502 and is connected with dewar jar 501, the connector 505 other end is connected with cold box 506, be provided with the notch on the cold box 506, the thing end 301 of putting of sample transmission pole 3 is arranged in the notch of cold box 506, make frozen sample and high vacuum chamber 2's vacuum environment keep apart, provide an anti-pollution imaging environment for frozen sample, the deposit of the pollutant in the imaging process on the sample has significantly reduced. Liquid nitrogen is filled in the liquid nitrogen dewar 501 to provide a cold source for the low temperature environment in the high vacuum chamber 2, and the corresponding vacuum connecting pipe 502, heat conduction rod 503, flexible connecting belt 504, connector 505 and cold box 506 are made of good conductor materials of heat such as red copper, cools the cold box 506 in the high vacuum chamber 2 through the mode of heat conduction, makes the cold box 506 be in and is close to the liquid nitrogen temperature all the time, realizes the good anti-pollution effect to freezing the sample. The temperature sensor 9 is provided on the support frame 205 and detects the temperature of the cold box 506 in the high vacuum chamber 2.
Further, the structured light illumination system 8 in this embodiment is an external structured light illumination system 8, which is disposed on the microscope body 1 and behind the high vacuum chamber 2, and is used for emitting structured light into the high vacuum chamber 2 to provide structured light illumination for the objective lens 701 and the frozen sample.
Specifically, referring to fig. 6, the structured light illumination system 8 in the present embodiment includes an illumination module, a first lens group 802, a beam splitting prism 804, a half-wave plate 805, a spatial light modulator 806, a second lens group 807, a stop 808, a third lens group 809, and a dichroic mirror 810, which are sequentially arranged along an optical path. The illumination module is a laser emitter 801 and is used for emitting exciting light beams with different wavelengths, the emitted light beams are expanded into parallel light beams through a first lens group 802, the parallel light beams are converted into linearly polarized light beams with a specific polarization direction through a beam splitter 804, the linearly polarized light beams are modulated into structured light with a specific direction and a spatial frequency through a half-wave plate 805 and a spatial light modulator 806, the modulated structured light is deflected by 90 degrees in the transmission direction after passing through the beam splitter 804 again, and is focused onto a diaphragm 808 through a second lens group 807, the diaphragm 808 filters the focused light beams according to use requirements, only positive and negative first-order and zero-order diffracted lights are allowed to pass through and enter a subsequent light path, the positive and negative first-order and zero-order diffracted lights are expanded and focused through a third lens group 809, and then are reflected by a dichroic mirror 810, pass through a lower light-passing hole 206 and irradiate to a rear pupil of an objective 701, and are converged on a sample through the objective 701, structured light illumination is provided for fluorescence imaging of frozen samples. Further, first lens group 802 includes 3 double-cemented lenses that set gradually along the light path, and 3 double-cemented lenses expand the beam, gather, expand the beam to the transmission light beam in proper order, expand the pointolite illumination beam of laser emitter 801 outgoing into even illumination facula, increase the even illumination scope of light beam. The second cemented doublet group comprises 1 cemented doublet for focusing the modulated structure light to the diaphragm 808, thereby realizing the filtration of positive and negative first-order and zero-order diffracted light. The third lens group 809 includes 2 double cemented lenses disposed along the optical path for expanding beam and focusing respectively. The focusing double-cemented lens and the dichroic mirror 810 are respectively disposed on the microscope body 1, the dichroic mirror 810 is disposed below the lower light-passing hole 206, and the dichroic mirror 810 has reflection and transmission functions, and can reflect excitation light (with a short wavelength) and transmit emission light (with a long wavelength). Further, the structured light illumination system 8 further includes a light intensity sensor 803, and the light intensity sensor 803 is disposed on a side of the beam splitter prism 804 away from the second lens group 807 for sensing the intensity of the structured light. When the two-dimensional structured light illumination imaging is needed, only positive and negative first-order diffracted lights at the back pupil of the objective lens 701 are allowed to enter an illumination light path to illuminate a sample, and the spatial light modulator 806 controls and acquires 9 original illumination imaging data in total from three illumination angles (each illumination angle moves by three illumination phases). When three-dimensional structured light illumination imaging is required, only plus-minus first-order and zero-order diffracted lights at the back pupil of the objective lens 701 are allowed to enter an illumination light path to illuminate a sample, and the spatial light modulator 806 controls and acquires 15 original illumination imaging data in total from three illumination angles (each illumination angle moves by five illumination phases) of each optical layer. The spatial frequency of the illumination structure light is changed by the spatial light modulator 806 in the present embodiment to adapt to the numerical aperture requirements of the objective lens 701 with different magnification factors, so as to obtain the corresponding image resolution.
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, which are disposed in the high vacuum chamber 2, the objective lens 701, the dichroic mirror 810, the fluorescence filter 703, the fourth lens group 704 and the detector 705 are sequentially disposed along an optical path, and the detector 705 is in communication connection with the data processor 706. Specifically, in this embodiment, the fourth lens group 704 is a double-cemented lens, a fluorescent substance is marked on the frozen sample in advance, when the structured light irradiates the surface of the frozen sample, the fluorescent substance is excited to emit an emission light beam with a wavelength longer than that of the excitation light (structured light), the emission light beam (long-wavelength light beam) penetrates through the dichroic mirror 810, stray light is filtered by the fluorescence filter 703, the fourth lens group 704 is focused on the detector 705 to perform imaging data acquisition, and the imaging data is transmitted to the data processor 706, and the data processor 706 performs analysis processing and high-resolution information reconstruction on the imaging data of the structured light.
Further, the high-vacuum environment optical microscope and electron microscope related imaging system 7 in this embodiment further includes a controller 12, and the controller 12 is in communication connection with the position adjusting device 4, the imaging system 7, the vacuum pumping system 6, the structured light illumination system 8, and the temperature sensor 9, respectively. The temperature sensor 9 is connected with the controller 12 through the sensor vacuum socket 10, and transmits the temperature information of the cold box 506 in the high vacuum chamber 2 to the controller 12 for display, so as to monitor that the temperature of the cold box 506 is always lower than a safety value of-160 ℃, thereby preventing the temperature from rising, weakening the pollution resistance and leading the frozen sample to be polluted by impurities.
Based on the high vacuum environment optical mirror electron microscope correlation imaging system, the specific using process comprises the following steps:
1) controlling the starting of the structured light illuminating system 8, leading structured light into the high vacuum chamber 2 from a light inlet at the rear side of the microscope host, and performing system light path collimation and illumination light spatial frequency modulation parameter optimization setting;
2) and high vacuum pumping of the high vacuum chamber 2:
the first vacuum valve 603 is controlled to be closed, the roughing pump 606, the molecular pump 601 and the third vacuum valve 607 are controlled to be started, the high vacuum chamber 2 is vacuumized, and when the vacuum gauge 602 detects that the vacuum degree in the high vacuum chamber 2 is better than a set value (for example, 5 × 10)-3Pa), the system can be used;
3) filling Dewar 501 with liquid nitrogen to cool cold box 506, and when controller 12 detects that the temperature of cold box 506 is lower than a set value (e.g., -160 ℃) through temperature sensor 9, preparing frozen sample for transmission;
4) the pre-frozen sample is fixed at the placing end 301 of the sample transmission rod 3;
5) and vacuumizing the sealing channel:
inserting the sample transmission rod 3 filled with the frozen sample into the sample transmission pipe 402, controlling the third vacuum valve 607 to be closed, controlling the second vacuum valve 605 to be opened, starting the low vacuum pump 606 to vacuumize the sealed channel, controlling the second vacuum valve 605 to be closed, controlling the first vacuum valve 603 to be opened, controlling the third vacuum valve 607 to be opened when the vacuum degree reaches a set value (for example, 10Pa), and sucking the sample transmission rod 3 into the high vacuum chamber 2 under the action of negative pressure;
6) controlling the three-dimensional translation stage 401 to accurately adjust the horizontal position and the height of the sample transmission rod 3 so that the frozen sample is positioned above the objective lens 701;
7) and when the vacuum degree in the vacuum chamber recovers to the specified value, the metal separation blade is opened, and the structured light is opened for lighting imaging: if two-dimensional structured light illumination imaging is required, the spatial light modulator 806 controls and acquires 9 original illumination imaging data in total from three illumination angles (each illumination angle is shifted by three illumination phases); if three-dimensional structured light illumination imaging is required, the collection of 15 original illumination imaging data in total is controlled by the spatial light modulator 806 to obtain three illumination angles (each illumination angle is moved by five illumination phases) of each optical layer, and the original three-dimensional data is obtained by controlling the up-and-down movement of the three-dimensional translation stage 401. After data acquisition is finished, restoring high-frequency information containing high-resolution information in a frequency shift mode through a structured light illumination reconstruction algorithm, and thus obtaining a frozen structured light illumination fluorescence image with resolution doubled compared with that of frozen wide-field illumination fluorescence imaging;
8) after optical imaging is finished, the sample transmission rod 3 is pulled out from the high vacuum chamber 2 through the operation opposite to the operation that the sample transmission rod 3 is inserted into the high vacuum chamber 2, and is transferred into a transmission electron microscope (or a scanning electron microscope), and an interested target area is selected for carrying out electron microscope high-resolution data acquisition according to the obtained fluorescence image navigation and positioning, so that the associated imaging data of the freezing optical mirror and the electron microscope are obtained.
The specific structure and the using method of the high-vacuum environment optical mirror electron microscope correlation imaging system can be widely applied to the application fields of the freezing optical imaging and photoelectric correlation imaging technology, so that the structured light illumination imaging is performed on a frozen sample in a vacuum environment, and the optical resolution and the photoelectric correlation alignment precision of the imaging system 7 are further improved. The high-vacuum environment optical mirror and electron microscope associated imaging system can be matched with the sample transmission rods 3 of different types and system parameter setting to realize imaging under different conditions of freezing, high temperature, atmosphere, liquid and the like, and the objective lenses 701 with different magnification factors can be replaced according to optical imaging requirements and an electric multi-lens holder is placed in the high-vacuum environment optical mirror and electron microscope associated imaging system to realize free switching of the objective lenses 701 with different magnification factors in experiments. The structured light illumination system 8 correspondingly adjusts the spatial frequency of the structured light through the spatial light modulator 806 so as to adapt to the use requirements of the objective lens 701 with different magnification factors, and further meets the imaging requirements of different frozen samples, and the structured light illumination system is wide in application range and high in universality.
In summary, the high vacuum environment photo-mirror electron microscope related imaging system provided by the present invention is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can be covered by the scope of the present invention by equivalent replacement or change according to the technical solution and the inventive concept of the present invention.
Claims (10)
1. The utility model provides a high vacuum environment light mirror electron microscope correlation imaging system which characterized in that includes:
a microscope body (1) provided with an imaging system (7);
the high vacuum chamber (2) is arranged on the microscope main body (1) through a fixing frame (11), an upper light through hole (202) is formed in the top cover plate (201), a lower light through hole (206) is formed in the bottom of the top cover plate, and the upper light through hole (202) and the lower light through hole (206) are communicated with each other;
the objective lens (701) of the imaging system (7) is arranged in the high vacuum chamber (2) and is positioned between the upper light through hole (202) and the lower light through hole (206);
a sample transmission rod (3) comprising a deposit end (301) and a withdrawal end (302) and extending between said deposit end (301) and said withdrawal end (302);
the frozen sample is arranged at the object placing end (301), and the object placing end (301) is arranged in the high vacuum chamber (2) in a penetrating way, so that the frozen sample is arranged above the objective lens (701);
and the structured light illuminating system (8) is arranged at the rear side of the microscope body (1) and is used for emitting structured light, and the structured light passes through the lower light through hole (206) and irradiates on the frozen sample.
2. High vacuum environment photomicroscope electron microscopy correlated imaging system according to claim 1, characterized in that the high vacuum chamber (2) is further provided with a position adjusting device (4) on the side wall;
the object placing end (301) of the sample transmission rod (3) can penetrate through the position adjusting device (4) and extend into the high vacuum chamber (2), and the drawing end (302) is connected with the position adjusting device (4);
the position adjusting device (4) is suitable for position adjustment and can drive the sample transmission rod (3) to move so as to adjust the position of the frozen sample relative to the objective lens (701).
3. The high vacuum environment photomirror electron microscope related imaging system of claim 2, characterized in that, the position adjusting device (4) comprises a bellows (403), a sample transmission tube (402) and a three-dimensional translation stage (401);
the three-dimensional translation stage (401) is arranged on the high vacuum chamber (2) through a translation stage fixing frame (14);
one end of the corrugated pipe (403) is connected to the high vacuum chamber (2), the other end of the corrugated pipe is connected with the sample transmission pipe (402), and the sample transmission pipe (402) is connected with the three-dimensional translation stage (401);
the object placing end (301) of the sample transmission rod (3) is arranged in the sample transmission tube (402) and the corrugated tube (403) in a penetrating way, and the periphery of the sample transmission rod is hermetically connected with the sample transmission tube (402);
the drawing end (302) is abutted to the sample transmission pipe (402), and the three-dimensional translation table (401) can drive the corrugated pipe (403) connected with the sample transmission pipe (402) to stretch and retract.
4. The high-vacuum environment photomirror electron microscope related imaging system of claim 3, characterized by further comprising an evacuation system (6);
the evacuation system (6) comprises a roughing 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 arranged between the corrugated pipe (403) and the high vacuum chamber (2) and is respectively connected with the corrugated pipe (403) and the high vacuum chamber (2) in a sealing way so as to form a sealing channel among the first vacuum valve (603), the corrugated pipe (403), the sample conveying pipe (402) and the sample transmission rod (3), and the first vacuum valve (603) is used for controlling the communication and the closing of the high vacuum chamber (2) and the sealing channel;
one end of the first bypass (60A) is connected to the sample transmission tube (402) and communicated with the sealed channel, and the other end of the first bypass is connected with the low vacuum pump (606) and used for pumping low vacuum to 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 with the low vacuum pump (606) and is used for pumping high vacuum to the high vacuum chamber (2);
the vacuum gauge (602) is hermetically connected to the side wall of the high vacuum chamber (2) and used for detecting the vacuum degree of the high vacuum chamber (2).
5. The high vacuum ambient photoeye microscopy correlated imaging system of claim 4, wherein said first bypass (60A) comprises a second vacuum valve (605);
one end of the second vacuum valve (605) is hermetically connected to the sample transmission pipe (402), and the other end of the second vacuum valve is connected with the air exhaust port end of the low vacuum pump (606) through a gas pipeline;
the second bypass (60B) comprises a third vacuum valve (607) and a molecular pump (601);
the third vacuum valve (607) is connected to the pumping port end of the low vacuum pump (606) and is connected with the molecular pump (601) through a gas pipeline, and the molecular pump (601) is hermetically connected to the side wall of the high vacuum chamber (2).
6. The high vacuum environment photomirror electron microscope related imaging system of claim 1, characterized by further comprising an anti-contamination system (5);
the anti-pollution system (5) comprises a Dewar tank (501), a vacuum connecting pipe (502), a heat conducting rod (503), a soft connecting belt (504), a connector (505) and a cold box (506);
the Dewar flask (501) is connected to the high vacuum chamber (2) through the vacuum connecting pipe (502) and is arranged outside the high vacuum chamber (2);
the connector (505) is arranged in the high vacuum chamber (2) through a support frame (205), one end of the connector is connected with the heat conducting rod (503) through the flexible connecting belt (504), the heat conducting rod (503) penetrates through the vacuum connecting pipe (502) to be connected with the Dewar tank (501), the other end of the connector (505) is connected with the cold box (506), and the frozen sample is placed in the cold box (506).
7. The high-vacuum environment optical mirror electron microscope related imaging system according to claim 1, wherein the structured light illumination system (8) comprises a laser emitter (801), a first lens group (802), a beam splitter prism (804), a half wave plate (805), a spatial light modulator (806), a second lens group (807), a diaphragm (808), a third lens group (809) and a dichroic mirror (810) which are arranged in sequence along an optical path;
the lens further comprises a light intensity sensor (803) which is arranged on one side of the beam splitting prism (804) far away from the second lens group (807).
8. The high vacuum environment photomirror electron microscope related imaging system of claim 1, characterized in that, the imaging system (7) further comprises a fluorescence filter (703), a fourth lens group (704) and a detector (705) arranged in sequence along the light path;
the detector (705) is communicatively connected to a data processor (706), and the data processor (706) is configured to process images of the frozen sample acquired by the detector (705).
9. The high vacuum environment photomirror electron microscope related imaging system of claim 1, further comprising an objective lens holder (13);
the objective lens fixing seat (13) is detachably connected to the bottom of the high vacuum chamber (2) in a sealing manner and is positioned at the position of the lower light through hole (206);
the center of the objective lens fixing seat (13) is provided with a round hole, and the round hole is communicated with the upper light through hole (202) and the lower light through hole (206).
10. The high vacuum environment photomirror electron microscope related imaging system of any one of claims 1 to 9, further comprising a temperature sensor (9) and a controller (12);
the temperature sensor (9) is arranged in the high vacuum chamber and is in communication connection with the controller (12);
the controller (12) is also in communication connection with the position adjusting device (401), the imaging system (7), the structured light illuminating system (8) and the vacuum pumping system (6), and the controller (12) is used for controlling the start and stop of the equipment.
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