WO2023015930A1 - 一种实时荧光监测冷冻聚焦离子束加工装置及方法 - Google Patents

一种实时荧光监测冷冻聚焦离子束加工装置及方法 Download PDF

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WO2023015930A1
WO2023015930A1 PCT/CN2022/087612 CN2022087612W WO2023015930A1 WO 2023015930 A1 WO2023015930 A1 WO 2023015930A1 CN 2022087612 W CN2022087612 W CN 2022087612W WO 2023015930 A1 WO2023015930 A1 WO 2023015930A1
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sample
ion beam
frozen
objective lens
platform
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PCT/CN2022/087612
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English (en)
French (fr)
Inventor
季刚
李硕果
孙飞
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中国科学院生物物理研究所
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Priority claimed from CN202110920369.4A external-priority patent/CN113670964A/zh
Priority claimed from CN202121877669.0U external-priority patent/CN216144726U/zh
Application filed by 中国科学院生物物理研究所 filed Critical 中国科学院生物物理研究所
Publication of WO2023015930A1 publication Critical patent/WO2023015930A1/zh
Priority to US18/437,697 priority Critical patent/US20240288370A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/02—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material
    • G01N23/04—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material and forming images of the material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
    • G01N23/223—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material by irradiating the sample with X-rays or gamma-rays and by measuring X-ray fluorescence

Definitions

  • the invention relates to the technical field of microscopic imaging, in particular to a real-time fluorescence monitoring cryo-focused ion beam processing device and method.
  • Cryo-electron microscopy technology freezes biological samples in a near-physiological state through rapid freezing or high-pressure freezing technology to preserve high-resolution structures.
  • Electron tomography is an important technical means of cryo-electron microscopy.
  • TEM imaging technology can only observe samples with a thickness of tens to hundreds of nanometers, it is impossible to directly observe cell tissue samples with a thickness of several microns or even more, and slices are required.
  • This technique requires the use of a diamond knife to cut the sample, which can be cut into slices about 100nm thick.
  • this technology is difficult and not easy to popularize, and the section is prone to wrinkle deformation, ice crystal pollution and other problems, and the success rate of the experiment is very low.
  • the other is to use cryo-focused ion beam technology for sectioning, and use a high-energy ion beam focused to a diameter of about several nanometers to process and thin the sample, so that a biological thin-section sample with a very flat cut surface and a thickness of about 200nm can be obtained.
  • the slices obtained by this technique are of high quality, so it has become a very important technical means in the field of biological slice sample preparation.
  • cryo-dual-beam scanning electron microscope is a sample processing equipment, and the cell structure that requires precise positioning is usually inside the cell.
  • the cryo-dual-beam scanning electron microscope which can only observe the surface topography of the sample, cannot observe and capture the intracellular structure.
  • the current common technical solution is to fluorescently label the cell structure to be observed in advance, pre-position through fluorescence imaging, and then perform cryofocused ion beam processing with the guidance of fluorescence positioning.
  • the first method is that the fluorescence imaging device and the cryo-focused ion beam device are two independent devices, and the pre-labeled frozen biological samples need to be transferred to the fluorescence imaging device to complete the cryo-fluorescence.
  • the cryo-focused ion beam device to obtain the scanning electron microscope image, and then the fluorescence image and the scanning electron microscope image are precisely aligned through the optical microscope electron microscope image correlation software, and the positioning information of the fluorescently labeled target object inside the cell is obtained to guide the focusing
  • the ion beam thins the target position; the second way is to integrate a fluorescence microscope in the chamber of the cryo-dual-beam scanning electron microscope, and the objective lens of the fluorescence microscope and the ion gun of the ion beam system of the scanning electron microscope are arranged side by side on the same side of the sample stage , from the hardware to realize the integration of the two imaging technologies.
  • the frozen sample needs to be moved repeatedly between the fluorescence objective lens and the ion gun along with the sample stage, first moved to the position of the fluorescence microscope to realize the positioning of the fluorescence imaging, and then moved to the position of the ion beam system.
  • position for ion beam cutting processing first roughly cut into thicker slices, and then transfer to the position of the fluorescence microscope, and observe whether the fluorescence signal on the slice is there again. After realizing the fluorescence positioning again, return to the ion beam position to conduct Cutting, and so on, until it cuts into a thin slice with a thickness of about 200nm or even thinner.
  • the above two methods have their own advantages and disadvantages.
  • the fluorescence microscope and the cryo-double-beam scanning electron microscope equipment are separated and do not affect each other, the two equipment can image separately, and the throughput is high.
  • the correlation alignment accuracy is relatively poor, and the success rate of capturing tiny structures is low.
  • fluorescence in situ imaging can be realized inside the scanning electron microscope, and the position of the fluorescence signal in the thick slice can be repeatedly monitored by moving the sample, so the success rate of capturing small targets is greatly improved.
  • the present invention provides a real-time fluorescence monitoring cryofocused ion beam processing device and method, which solves the problems of long processing time, low processing efficiency and poor positioning accuracy during ion beam processing of frozen samples in the prior art. , and the problem of low success rate.
  • the present invention provides a real-time fluorescence monitoring cryofocused ion beam processing device and method, and the specific technical scheme is as follows:
  • a cryo-focused ion beam processing device for real-time fluorescence monitoring comprising:
  • the ion beam system is set on the vacuum chamber and pointed to the frozen sample for ion beam processing on the frozen sample;
  • the fluorescence imaging system is arranged on the vacuum chamber and below the frozen sample, and is used for emitting excitation light to the frozen sample and performing real-time optical imaging.
  • the fluorescence imaging system includes a laser, an objective lens, a mirror, a tube lens, a dichroic mirror, a filter, and an image detector arranged sequentially along the optical path;
  • a fluorescent window is provided on the vacuum chamber, and the position of the fluorescent window is sealed and connected with a vacuum flange;
  • One end of the vacuum flange is connected to an optical connection cylinder, and the other end is connected to a base, the optical connection cylinder is located outside the vacuum chamber, and the base is located inside the vacuum chamber;
  • Both the objective lens and the mirror are arranged on the base;
  • the barrel lens, dichroic mirror, and filter are all set in the optical connection barrel, and the image detector is set on the side of the optical connection barrel away from the vacuum flange;
  • the center of the vacuum flange is provided with a light-transmitting sheet, which is used to seal the vacuum chamber;
  • a laser is also provided on a branch of the wall of the optical connection cylinder, and the laser is located on a side perpendicular to the dichroic mirror and the optical filter.
  • the base is provided with a linear translation platform
  • Both the objective lens and the reflective mirror are arranged on the linear translation stage, and the work of the linear translation stage can move the objective lens and the reflective mirror towards and/or away from the frozen sample.
  • linear translation stage is also equipped with an objective lens adjustment device and a mirror adjustment device;
  • the objective lens is arranged on the objective lens adjusting device, and the objective lens adjusting device is suitable for driving the objective lens to move and/or rotate in three dimensions;
  • the reflective mirror is arranged on the reflective mirror adjusting device, and the reflective mirror adjusting device is suitable for driving the reflective mirror to move and/or rotate in a plane.
  • the objective lens adjusting device includes an objective lens tilting stage and an objective lens translation stage;
  • the objective lens is arranged on the objective lens translation stage, the objective lens translation stage is arranged on the objective lens tilting stage, the objective lens tilting stage is arranged on the linear translation stage, the objective lens translation stage can drive the three-dimensional movement of the objective lens, and the objective lens tilting stage can drive the objective lens to tilt.
  • the mirror adjusting device includes a mirror translation platform and a mirror tilting platform;
  • the mirror is set on the mirror tilting platform, the mirror tilting platform is set on the mirror translation platform, the mirror translation platform is set on the linear translation platform, the mirror tilting platform can drive the mirror to tilt, and the mirror translation platform works It can drive the mirror plane to move.
  • the vacuum chamber is also equipped with an electron beam system for electron beam imaging of frozen samples
  • the electron beam system is arranged obliquely relative to the ion beam system, the electron beam emitted by the electron beam system and the ion beam emitted by the ion beam system can be converged and intersect at one point, and the frozen sample is located at the intersection point.
  • the cold table is arranged on the position adjustment device, and the position adjustment device is suitable for driving the cold table to perform position adjustment, including:
  • the first sample translation platform, the cold stage is set on the first sample translation platform
  • the second sample translation platform is arranged below the first sample translation platform
  • the sample lifting platform is located below the second sample translation platform
  • the sample tilting table is set on the door of the vacuum chamber, and is connected with the sample lifting platform through the L-shaped bracket.
  • the sample tilting table can drive the L-shaped bracket, the sample lifting platform, the second sample translation platform, the first sample translation platform and
  • the cooling table rotates synchronously.
  • the cold stage is one end of the frozen sample transmission device, and the frozen sample transmission device includes:
  • the sample transmission tube is provided with a sample transmission window on one side of the vacuum chamber door, and the sample transmission tube is softly connected to the position of the sample transmission window through the bellows assembly;
  • Frozen sample transmission rod with a Dewar bottle at one end and a fixed end for frozen samples at the other end;
  • the fixed end of the frozen sample is pierced in the sample transmission tube, and the outer circumference is dynamically sealed with the inner wall of the sample transmission tube, and can pass through the sample transmission window to transport the frozen sample into the vacuum chamber;
  • the end of the Dewar bottle can be clamped and/or unclamped with the sample transfer tube;
  • the three-dimensional translation platform for freezing transmission is connected with the sample transmission tube.
  • the three-dimensional translation platform for freezing transmission can drive the three-dimensional movement of the frozen sample transmission rod clamped with the sample transmission tube.
  • the frozen transfer device also includes an angle adjustment device, including:
  • the shaft sleeve vacuum plate valve housing assembly one end is sealed and connected to the freezing transmission window, the other end is sealingly connected to the bellows assembly, one end of the bellows assembly is sealingly connected to the position of the sample transmission tube, and the other end is fixedly connected to the cryogenic transmission three-dimensional translation platform bracket ;
  • the worm gear is arranged on the shaft disk, the shaft disk is rotatably sleeved on the outer periphery of the sleeve vacuum plate valve shell assembly, and the shaft disk is fixedly connected with the support of the three-dimensional translation platform for freezing transmission;
  • the worm is rotatably connected to the worm bracket, the worm bracket is connected to the door of the vacuum chamber, one end of the worm is connected to the motor and meshed with the worm wheel,
  • the motor can drive the worm gear meshed with the worm to rotate, and then drive the shaft plate, the three-dimensional translation platform for frozen transmission, the sample transmission tube, the bellows assembly and the frozen sample transmission rod to rotate synchronously.
  • the motor can drive the worm gear meshed with the worm to rotate, and then drive the shaft plate, the three-dimensional translation platform for frozen transmission, the sample transmission tube, the bellows assembly and the frozen sample transmission rod to rotate synchronously.
  • a cryofocused ion beam processing method for real-time fluorescence monitoring comprising the following steps:
  • the start of the ion beam system is controlled, the ion beam processing is performed on the frozen sample, and the processing process of the frozen sample is monitored in real time through a fluorescence imaging system.
  • the real-time fluorescence monitoring cryo-focused ion beam processing device and method provided by the present invention have the following beneficial effects.
  • the frozen sample is set on the cold stage, the required structure in the frozen sample is marked with a fluorescent marker, guided by the fluorescent signal, and then the frozen sample is processed by an ion beam system.
  • the fluorescence imaging system emits excitation light to the frozen sample, and the excitation light irradiates the frozen sample to excite the fluorescent markers in the frozen sample to emit fluorescence, and the fluorescence signal is received by the fluorescence imaging system and forms an image.
  • the present invention by performing real-time imaging on the frozen sample, by acquiring changes in the fluorescence image and the intensity of the fluorescence signal in real time, the structure of the frozen sample cut off by the ion beam and the remaining structure are monitored in real time, and the further processing position of the ion beam is guided. Until the final thin-section sample is obtained, the precise processing of the frozen sample under the guidance of in-situ real-time fluorescence is realized, thereby improving the processing accuracy, shortening the processing process of the frozen sample, and improving the processing efficiency and success rate.
  • Fig. 1 is a schematic structural view of the real-time fluorescence monitoring cryofocused ion beam processing device in Example 1 of the specific implementation;
  • Fig. 2 is a schematic structural view of the fluorescent imaging system, the vacuum chamber, the ion beam system and the electron beam system in the first and second embodiments of the specific implementation;
  • Fig. 3 is a schematic structural view of the fluorescent imaging system of Embodiment 1 and Embodiment 2 in the specific embodiment;
  • Fig. 4 is the structural schematic diagram of the cooling table and the position adjusting device of the first embodiment in the specific embodiment
  • Fig. 5 is a schematic structural view of the real-time fluorescence monitoring cryofocused ion beam processing device in Example 2 of the specific implementation;
  • FIG. 6 is a schematic structural view of the frozen transport device and the cold stage device of Example 2 in the specific embodiment
  • Fig. 7 is a schematic structural view of the angle adjustment device of Example 2 in the specific embodiment
  • Fig. 8 is A-A sectional view among Fig. 7;
  • Fig. 9 is a schematic structural view of the vacuum chamber hatch of Example 2 in the specific embodiment.
  • Fig. 10 is a schematic structural view of the bellows assembly of Example 2 in the specific embodiment
  • Fig. 11 is an optical path diagram of the fluorescence imaging system of Embodiment 1 and Embodiment 2 in the specific implementation manner.
  • Vacuum chamber 110. Fluorescence window;
  • Fluorescence imaging system 201, objective lens; 202, mirror; 203, lens tube lens; 204, dichroic mirror; 205, filter; 206, image detector; 207, laser; 208, optical connection tube; 209, vacuum flange; 210, base; 211, linear translation stage; 212, objective lens translation stage; 213, objective lens tilting stage; 214, mirror translation stage; 215, mirror tilting stage;
  • Position adjustment device 410, first sample translation platform; 420, second sample translation platform; 430, sample lifting platform; 440, sample tilting platform; 450, L-shaped bracket;
  • Electron beam system 6. Ion beam system;
  • Freezing transmission three-dimensional translation platform 740. Freezing transmission three-dimensional translation platform; 750. Freezing transmission three-dimensional translation platform support;
  • angle adjustment device 761, shaft sleeve vacuum plate valve shell assembly; 762, shaft disc; 763, shaft disc bearing; 764, worm gear; 765, worm; 766, worm support; 767, motor;
  • Vacuum conveying system 771. Vacuum plate valve; 772. Pre-vacuum valve;
  • this embodiment provides a cryogenic focused ion beam processing device for real-time fluorescence monitoring, including a vacuum chamber 1 , an ion beam system 6 and a fluorescence imaging system 2 .
  • the vacuum chamber 1 is provided with a cold stage 3, and the cold stage 3 is used to store a frozen sample 8.
  • a fluorescent marker is arranged in the frozen sample 8. When the excitation light is irradiated on the frozen sample 8, the fluorescent marker is excited. capable of emitting fluorescence.
  • the ion beam system 6 is arranged on the vacuum chamber 1 and directed to the frozen sample 8 for emitting a focused ion beam to perform ion beam cutting on the frozen sample 8 .
  • the fluorescence imaging system 2 is set on the vacuum chamber 1 and below the frozen sample 8, and is used for emitting excitation light to the frozen sample 8 and performing real-time optical imaging on the frozen sample 8 at the same time.
  • the fluorescence imaging system 2 emits excitation light to the frozen sample 8, the excitation light is irradiated on the frozen sample 8, the fluorescent markers in the frozen sample 8 are excited to emit fluorescence, and the fluorescence is received by the fluorescence imaging system 2 to form a frozen sample 8 Images of fluorescently labeled structures in .
  • the processing status of the frozen sample 8 is monitored in real time through the fluorescence imaging system 2, realizing in-situ ion beam processing under the guidance of real-time fluorescence monitoring of the frozen sample 8, without transferring the frozen sample 8, and further
  • the processing accuracy of the frozen sample 8 is greatly improved, the processing process is simplified, the processing time is shortened, and the processing efficiency is improved.
  • the fluorescence imaging system 2 in this embodiment includes a laser 207 , an objective lens 201 , a mirror 202 , a barrel lens 203 , a dichroic mirror 204 , and a filter arranged in sequence along the optical path.
  • Light sheet 205 and image detector 206 are provided with a fluorescent window 110 , and the fluorescent window 110 is connected with a vacuum flange 209 in a sealed position.
  • the objective lens 201 is located on the optical path of the fluorescence emitted by the fluorescent marker of the frozen sample 8, and is located below the frozen sample 8, the objective lens 201 and the mirror 202 are all arranged on the base 210, the lens tube lens 203, the dichroic mirror 204 and the optical filter 205 are sequentially arranged in the optical connection barrel 208 along the optical path, and the image detector 206 is arranged at the end of the optical connection barrel 208 away from the vacuum flange 209 .
  • a light-transmitting sheet is arranged in the center of the vacuum flange 209 to seal the vacuum chamber 1 and transmit excitation light and fluorescence.
  • a laser 207 is also provided on the wall branch of the optical connection cylinder 208 , and the laser 207 is located on the side of the dichroic mirror 204 perpendicular to the filter 205 for emitting excitation light.
  • the dichroic mirror 204 has reflection and transmission functions, and can reflect excitation light (short wavelength) and transmit fluorescent light (long wavelength).
  • the excitation light (short wavelength) emitted by the laser 207 is reflected by the dichroic mirror 204 to the lens barrel lens 203, then expanded into parallel light and enters the reflector 202, and then reflected by the reflector 202 and then passed through the objective lens 201 is irradiated on the frozen sample 8 .
  • the fluorescence imaging system in this system can also be a wide-field imaging mode, or a laser confocal imaging mode, or a different imaging mode such as a structured light illumination imaging mode, and these imaging modes are all within the protection scope of the present invention. Experiments need to be determined.
  • the base 210 is further provided with a linear translation platform 211 , and the linear translation platform 211 is provided with an objective lens adjusting device and a mirror adjusting device.
  • the objective lens 201 is arranged on the objective lens adjustment device, and the objective lens adjustment device is suitable for driving the three-dimensional movement or tilting of the objective lens 201
  • the reflector 202 is arranged on the reflector adjustment device, and the reflector adjustment device is suitable for driving the reflector 202 to move or tilt change.
  • the linear translation stage 211 is suitable for moving in a single direction, and can move the objective adjusting device, objective lens 201 , reflective mirror adjusting device and reflective mirror 202 away from or close to the frozen sample 8 during operation.
  • the linear translation stage 211 is controlled to start, and the objective lens 201 and the mirror 202 are moved to the position of the frozen sample 8, and then the objective lens 201 is adjusted by the objective lens adjustment device. position, so that the objective lens 201 is located on the optical path of the fluorescence emitted by the frozen sample 8, and the mirror 202 is adjusted accordingly to ensure that the optical path of the fluorescence does not change.
  • the linear translation stage 211 is controlled to start, and the objective lens 201 and the mirror 202 are moved to a position away from the frozen sample 8, so as to facilitate the frozen transport of the frozen sample 8 , imaging and other operations.
  • the objective lens adjustment device in this embodiment includes an objective lens translation platform 212 and an objective lens tilting platform 213 , the objective lens translation platform 212 is arranged on the objective lens tilting platform 213 , and the objective lens tilting platform 213 is arranged on the linear translation platform 211 .
  • the objective lens translation stage 212 can drive the objective lens 201 to move along the horizontal or height direction when working.
  • the objective lens tilting table 213 can drive the objective lens 201 to tilt around the frozen sample 8 when working.
  • the objective lens 201 is aligned with the ion beam cutting position on the frozen sample 8, and the focus of the image is realized.
  • Reflective mirror adjustment device comprises reflective mirror translation platform 214 and reflective mirror tilting platform 215, reflective mirror 202 is located on the reflective mirror tilting platform 215, reflective mirror tilting platform 215 is located on the reflective mirror translation platform 214, and reflective mirror translation platform 214 is located at On the linear translation stage 211, the reflector tilting stage 215 can drive the reflector 202 to tilt when it works, and the reflector translation stage 214 can drive the reflector 202 to adjust the position along the horizontal direction when it is in operation.
  • the reflective mirror translation stage 214 and the reflective mirror tilting stage 215 drive the reflective mirror 202 to be adjusted correspondingly, so that the excitation light is accurately irradiated on the frozen sample 8, and at the same time, the fluorescence emitted by the fluorescent marker on the frozen sample 8 is converged by the objective lens 201 , accurately reflected to the imaging optical path.
  • an electron beam system 5 is also provided on the vacuum chamber 1 , and the electron beam system 5 is used for performing electron beam imaging on a frozen sample 8 .
  • the electron beam system 5 is vertically arranged on the top of the vacuum chamber 1, and the ion beam system 6 is arranged obliquely relative to the electron beam system 5.
  • the electron beams emitted by the electron beam system 5 and the ion beams emitted by the ion beam system 6 can converge and cross at one point, and freeze Sample 8 was placed at the intersection.
  • the cold stage 3 in the present embodiment is arranged on the position adjusting device 4, and is suitable for driving the cold stage 3 and the frozen sample 8 arranged on the cold stage 3 to move and/or rotate.
  • the position adjustment device 4 drives the frozen sample 8 to move, so that the frozen sample 8 is located at the converging point of the electron beam and the ion beam, and then controls the position adjustment device 4 to drive the frozen sample 8 to tilt, so that the frozen sample 8 is aligned with the
  • the ion beam emitted by the ion beam system 6 forms a certain angle, usually 10°-20°, which is convenient for ion beam cutting and processing.
  • the position adjustment device 4 in this embodiment includes a first sample translation stage 410 , a second sample translation stage 420 , a sample lifting stage 430 and a sample tilting stage 440 .
  • the cold stage 3 is arranged on the first sample translation platform 410, the first sample translation platform 410 is arranged on the top of the second sample translation platform 420, the second sample translation platform 420 is arranged on the top of the sample lifting platform 430, and the sample lifting platform 430 is set on the L-shaped bracket 450, and the L-shaped bracket 450 is connected to the sample tilting platform 440, the sample tilting platform 440 is fixed on the vacuum chamber hatch 120, and the vacuum chamber hatch 120 is sealed and connected to the vacuum chamber 1.
  • the moving directions of the first sample translation platform 410, the second sample translation platform 420 and the sample lifting platform 430 are perpendicular to each other, which can drive the cold platform 3 to adjust the position in three mutually perpendicular directions in space, so as to precisely adjust the freezing temperature.
  • the sample 8 is positioned such that the frozen sample 8 is at the intersection of the electron beam and the ion beam.
  • the sample tilting table 440 can drive the L-shaped support 450, the sample lifting table 430, the second sample translation table 420, the first sample translation table 410 and the cold table 3 to rotate synchronously, so that the frozen samples 8 placed on the cold table 3 It forms a certain angle with the ion beam emitted by the ion beam system 6 or the electron beam emitted by the electron beam system 5.
  • the frozen sample 8 and the ion beam emitted by the ion beam system 6 form an angle of 10° to 20°, it can be used to freeze samples
  • the ion beam cutting process of 8 when the frozen sample 8 is perpendicular to the electron beam emitted by the electron beam system 5, can be used for electron beam imaging of the frozen sample 8, meeting the requirements of different scenarios.
  • a controller 9 is also included, and the controller 9 is connected in communication with the linear translation stage 211, the objective lens translation stage 212, the mirror translation stage 214, the first sample translation stage 410 and the second sample translation stage 420, for controlling the above-mentioned equipment The start and stop of the machine and the adjustment of the moving distance.
  • the controller 9 is also connected in communication with the objective lens tilting platform 213, the mirror tilting platform 215 and the sample tilting platform 440, and is used to control the start and stop of the above equipment and the adjustment of the rotation angle.
  • the controller 9 is also communicatively connected with the laser 207 and the image detector 206 for controlling the emission and stop of the excitation light, and controlling the image detector 206 to acquire fluorescence images and signals in real time.
  • the controller 9 controls the start and stop of the equipment connected with the communication, realizes the automatic or semi-automatic adjustment of the optical path of the real-time fluorescence system, the real-time acquisition of the fluorescence image, and improves the automation of the cryo-focused ion beam processing device for fluorescence monitoring, which is convenient and quick.
  • ion beam processing and electron beam imaging can be performed on the frozen sample 8 .
  • An ion beam processing method for real-time fluorescence monitoring of a frozen sample comprising the following steps:
  • ion beam system 6 and the fluorescence imaging system 2 together to determine whether there is a structure that needs to be cut in the frozen sample 8 . If so, confirm the specific cutting position by analyzing the fluorescence image, set the cutting parameters of the ion beam system 6, cut the frozen sample 8, and monitor the cutting position in real time in a continuous or intermittent manner while cutting the ion beam and progress until a flake with a thickness of 200nm is obtained and the target structure carrying the fluorescent signal is preserved;
  • Electron beam imaging of frozen samples including the following steps:
  • the real-time fluorescence monitoring cryofocused ion beam processing device provided in this embodiment differs only in the structure of the vacuum chamber hatch 120' and the structure of the cold stage 3 in this embodiment. different form of cooling.
  • the vacuum chamber hatch 120 ′ in this embodiment is a concave structure, and is provided with a sample transmission window 121 ′.
  • the cold stage 3 is one end of the cryo-transfer device 7 , which includes a cryo-transfer rod 710 , a sample transfer tube 720 , a bellows assembly 730 , a cryo-transfer three-dimensional translation stage 740 and an angle adjustment device 760 .
  • the sample transmission tube 720 is sealed and connected to the position of the sample transmission window 121 ′ through the bellows assembly 730 .
  • One end of the frozen sample transmission rod 710 is a frozen sample fixing end 713 for fixing the frozen sample 8 , and the other end is provided with a Dewar bottle 711 .
  • the fixed end 713 of the frozen sample is penetrated in the sample transmission tube 720 and the bellows assembly 730, the outer circumference of the frozen sample transmission rod 710 is slidably connected with the inner wall of the sample transmission tube 720, and the frozen sample transmission rod 710 is along the sample transmission tube 720.
  • the inner wall slides, and the frozen sample fixing end 713 can pass through the sample transmission window 121 ′ to transport the frozen sample 8 into the vacuum chamber 1 .
  • the Dewar bottle 711 is used to provide a cold source for the frozen sample 8 through heat conduction, so that the frozen sample 8 is always in the frozen form of glass.
  • the frozen sample transmission rod 710 can be used as a transmission device, and the frozen sample fixed end 713 can also be used as a cold stage 3, which greatly simplifies the structure of the real-time fluorescence monitoring cryofocused ion beam processing device.
  • the frozen sample transmission rod 710 on the side of the Dewar bottle 711 is provided with a card slot 712
  • the sample transmission tube 720 is correspondingly provided with a card needle 721.
  • the cryo-transport three-dimensional translation stage 740 is arranged on the cryo-transport three-dimensional translation stage support 750 and connected to the sample transfer tube 720 .
  • the bellows assembly 730 is suitable for telescopic adjustment.
  • the cryotransfer three-dimensional translation stage 740 can drive the bellows assembly 730 connected to the sample transfer tube 720 to expand and contract accordingly, and the sample transfer tube
  • the position of 720 relative to the vacuum chamber 1 is changed, and the position of the frozen sample 8 arranged at the fixed end 713 of the frozen sample is also changed correspondingly.
  • the position of the frozen sample 8 can be precisely adjusted through the cryotransmission three-dimensional translation stage 740, so that the frozen sample 8 is located at the focal point of the ion beam emitted by the ion beam system 6 and the electron beam emitted by the electron beam system 5.
  • the angle adjustment device 760 includes a shaft sleeve vacuum plate valve housing assembly 761 , a shaft disc 762 , a worm wheel 764 and a worm screw 765 .
  • one end of the sleeve vacuum plate valve housing assembly 761 is sealed and connected to the sample transmission window 121 ′, and is sleeved on the outer circumference of the bellows assembly 730 , the inner wall of the sleeve vacuum plate valve housing assembly 761 is in contact with the outer circumference of the bellows assembly 730 .
  • the bellows assembly 730 is fixedly connected with the cryogenic transmission three-dimensional translation platform support 750 by bolts
  • the worm gear 764 is fixed on the shaft disk 762 by bolts
  • the shaft disk 762 is rotatably sleeved on the shaft sleeve vacuum plate valve through the shaft disk bearing 763
  • the outer periphery of the shell assembly 761 is fixedly connected with the three-dimensional cryotransfer platform support 750 by bolts.
  • Both ends of the worm 765 are rotatably connected to the worm bracket 766 through bearings, and the worm bracket 766 is arranged on the vacuum chamber hatch 120' through bolts.
  • Worm screw 765 is meshed with worm wheel 764, and the end of worm screw 765 is connected with motor 767, and motor 767 work can drive worm screw 765 and worm wheel 764 meshing transmission.
  • angle adjustment confirm that the frozen sample transmission rod 710 is clamped with the sample transmission tube 720, control the motor 767 to start, and the motor 767 drives the worm 765 and the worm gear 764 to engage and drive, and then drives the shaft plate 762, the frozen transmission three-dimensional translation platform support 750,
  • the sample transmission tube 720, the bellows assembly 730, the frozen sample transmission rod 710 and the frozen sample 8 rotate synchronously, so that the frozen sample 8 forms an included angle of 10°-20° with the ion beam emitted by the ion beam system 6, or with the electron beam system 5
  • the emitted electron beams are perpendicular to meet the needs of frozen samples 8 ion beam processing or electron beam imaging.
  • the bellows assembly 730 includes a bellows 732 and a synchronous sleeve 731 sleeved on the outer periphery of the bellows 732.
  • the synchronous sleeve 731 is sleeved on the outer circumference of the bellows 732 and placed in a vacuum In the plate valve shell assembly 761 , the outer circumference of the synchronous sleeve 731 is dynamically and sealingly connected with the inner wall of the bushing vacuum plate valve shell assembly 761 .
  • one end of the synchronous sleeve 731 is fixedly connected to the end of the bellows 732 by welding, and the other end is detachably connected to the cryotransfer three-dimensional translation platform bracket 750 through bolts.
  • the three-dimensional translation platform bracket 750 for freezing transmission drives the synchronous sleeve 731 and the bellows 732 to rotate synchronously.
  • a vacuum conveying system 770 is also included, which is used to drive the frozen sample transfer rod 710 to slide along the sample transfer tube 720 through the working principle of negative pressure, and transfer the frozen sample 8 from the atmospheric environment to the high vacuum environment.
  • the vacuum delivery system 770 includes a vacuum plate valve 771 and a pre-vacuum valve 772.
  • the vacuum plate valve 771 is sealingly connected to the shaft sleeve vacuum plate valve housing assembly 761, so that the frozen sample transmission rod 710, the sample transmission tube 720, A sealed channel is formed between the bellows 732 , the bushing vacuum plate valve housing assembly 761 and the vacuum plate valve 771 , and the vacuum plate valve 771 is used to control the connection and closure of the sealed channel and the vacuum chamber 1 .
  • the pre-vacuum valve 772 is sealingly connected to the sample transmission tube 720 and communicated with the sealed channel, and is used for drawing a low vacuum to the sealed channel.
  • the frozen sample 8 is fixed on the frozen sample transmission rod 710 in advance, the vacuum plate valve 771 is kept closed, the frozen sample fixed end 731 is inserted into the sample transmission tube 720, and then the pre-vacuum valve 772 is controlled to open, and the vacuum plate valve 771 is kept closed.
  • the vacuum pump starts to evacuate the sealed channel.
  • the pre-vacuum valve 772 is controlled to close, and the vacuum plate valve 771 is controlled to open. Since the vacuum degree of the vacuum chamber 1 is far lower than the external atmospheric pressure, manual control and freezing
  • the sample transmission rod 710 slides along the sample transmission tube 720 under the action of negative pressure to transport the frozen sample 8 into the vacuum chamber 1 .
  • the transfer process of the frozen sample 8 is simple, convenient and fast. Then control the cryo-transport three-dimensional translation stage 740 to start, and adjust the position of the frozen sample 8 so that the frozen sample 8 is located at the intersection of the ion beam emitted by the ion beam system 6 and the electron beam emitted by the electron beam system 5 .
  • the control angle adjustment device 760 is activated to adjust the frozen sample 8 to be perpendicular to the electron beam emitted by the electron beam system 5 , so as to meet the electron beam imaging requirements of the frozen sample 8 .
  • the angle adjustment device 760 is activated to adjust the angle between the frozen sample 8 and the ion beam emitted by the ion beam system 6 to be 10°-20°, so as to meet the ion beam processing requirements of the frozen sample 8 .
  • the controller 9 communicates with the vacuum plate valve 771 and the pre-vacuum valve 772, and controls the opening and closing of the vacuum plate valve 771 and the pre-vacuum valve 772 through the controller 9, so as to realize the freezing of the sample 8. Vacuum delivery. Further, the controller 9 is also connected in communication with the three-dimensional translation platform 740 for freezing transmission and the angle adjustment device 760, and is used to control the start-up, adjustment of the moving distance and the adjustment of the rotation angle of the three-dimensional translation platform 740 for freezing transmission, so as to realize precise adjustment of the position of the frozen sample 8 The purpose is to meet the usage needs of different scenarios. In this embodiment, the automatic control of the real-time fluorescence monitoring cryo-focused ion beam processing device is realized through the controller 9, which improves the degree of automation and is convenient and fast.
  • ion beam processing and electron beam imaging can be performed on the frozen sample 8 .
  • the frozen sample 8 is placed on the frozen sample fixed end 713 of the frozen sample transmission rod 710, and the frozen sample 8 is transported into the vacuum chamber 1 through the frozen sample transmission rod 710;
  • ion beam system 6 and the fluorescence imaging system 2 together to determine whether there is a structure that needs to be cut in the frozen sample 8 . If so, confirm the specific cutting position by analyzing the fluorescence image, set the cutting parameters of the ion beam system 6, cut the frozen sample 8, and monitor the cutting position in real time in a continuous or intermittent manner while cutting the ion beam and progress until a flake with a thickness of 200nm is obtained and the target structure carrying the fluorescent signal is preserved;
  • Electron beam imaging of frozen samples 8 including the following steps:
  • the real-time fluorescence monitoring frozen focused ion beam processing device provided in Embodiment 1 and Embodiment 2, by setting the fluorescence imaging system 2 and the ion beam system 6, the in-situ real-time fluorescence monitoring and real-time ion beam processing of the frozen sample 8 are realized.
  • the positioning is accurate, and in the process of positioning and monitoring the fluorescence, the processing of the ion beam does not need to be stopped, the speed is fast and the flux is high, which greatly improves the working efficiency and precision of the ion beam processing.
  • the device can also realize electron beam-fluorescence two-beam joint imaging, ion beam-fluorescence two-beam joint imaging, electron beam-ion beam-fluorescence three-beam joint imaging and other functions.
  • the cold stage 3 can also be replaced with different sample rods or sample stages as required, which is flexible and convenient to use.
  • the vacuum chamber hatch 120 and the vacuum chamber hatch 120 ′ can be adapted to different types of cryo-double-beam scanning electron microscopes by changing their dimensions, and have strong versatility.
  • the real-time fluorescence monitoring cryofocused ion beam processing device provided by the present invention is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto.

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Abstract

一种实时荧光监测冷冻聚焦离子束加工装置及方法，包括真空腔室（1）、离子束系统（6）和荧光成像系统（2）。其中，真空腔室（1）内设置有冷台（3），用于放置冷冻样品（8），冷冻样品（8）内设有荧光标记物。离子束系统（6）设于真空腔室（1）上，指向冷冻样品（8）设置，用于对冷冻样品（8）进行离子束加工。荧光成像系统（2）设于真空腔室（1）上，位于冷冻样品（8）的下方，用于对冷冻样品（8）进行实时光学成像。加工时，通过荧光成像系统（2）对冷冻样品（8）发射激发光，激发样品中的荧光标记物发射荧光，荧光被荧光成像系统（2）接收形成冷冻样品（8）中目标结构的图像。通过对荧光图像和信号的监测分析，实时监控冷冻样品（8）的加工过程，实现了冷冻样品（8）的原位实时监测加工，无需转移，提高了目标结构的加工准确度，缩短了加工时间。

Description

一种实时荧光监测冷冻聚焦离子束加工装置及方法
相关申请的交叉引用
本申请要求2021年8月11日提交的中国专利申请第202110920369.4号和第202121877669.0号的优先权，该申请的全部内容通过引用并入本文用于所有目的。
技术领域
本发明涉及显微成像的技术领域，尤其涉及一种实时荧光监测冷冻聚焦离子束加工装置及方法。
背景技术
冷冻电镜技术通过快速冷冻或高压冷冻技术把生物样品冷冻在近生理状态，保存高分辨结构。电子断层成像技术是冷冻电镜的重要技术手段，通过对冷冻样品的系列倾转成像以及三维重构，得到生物样品超微结构的高分辨三维结构。由于透射电镜成像技术只能观察几十至几百纳米厚度的样品，对于几微米甚至更厚的细胞组织样品，无法直接观察，需要进行切片。对冷冻的生物样品切片的技术主要有两类：一类是使用冷冻超薄切片机对样品进行切片，该技术需要使用钻石刀切割样品，可以切到约100nm厚的切片。但该技术难度大，不易普及，且切片容易产生皱褶形变、冰晶污染等问题，实验成功率很低。另一种是使用冷冻聚焦离子束技术进行切片，利用聚焦到直径约几纳米的高能离子束对样品进行加工减薄，可以得到切割面非常平整的、厚度在200nm左右的生物薄片样品。该技术所获得的切片质量高，故而已经成为生物切片样品制备领域中非常重要的技术手段。
在冷冻聚焦离子束技术的操作中，对冷冻样品精确的定位切割加工是非常重要的技术环节。冷冻双束扫描电镜是样品加工设备，而需要精准定位的细胞结构通常在细胞内部，仅能观察样品表面形貌结构的冷冻双束扫描电镜无法实现对胞内结构的观察和捕获。为了解决这一难题，目前比较普遍的技术方案是提前对需要观察的细胞结构进行荧光标记，通过荧光成像进行预定位，借助荧光定位的指导，再进行冷冻聚焦离子束加工。目前荧光成像定位通常有两种方式：第一种方式是，荧光成像装置和冷冻聚焦离子束装置是两台独立设备，预先做好荧光标记的冷冻生物样品需要先转移至荧光成像装置完成冷冻荧光成像，之后再被转移至冷冻聚焦离子束装置获得扫描电镜图像，然后通过光镜电镜图像关联软件把荧光图像和扫描电镜图像精确对齐，获得荧光标记的目标物在细胞内部的定位信息，指导聚焦离子束对目标位置进行减薄加工；第二种方式是在冷冻双束扫描电镜腔室内，集成一个荧光显微镜，荧光显微镜的物镜和扫描电镜离子束系统的离子枪并排设置在样品台的同侧，从硬件上实现两种成像技术的整合。在这种集成型光镜电镜关联成像系 统中，冷冻样品需要随着样品台在荧光物镜和离子枪之间反复移动，先移动到荧光显微镜的位置实现荧光成像定位，再移动至离子束系统的位置进行离子束切割加工，先粗切割成较厚的切片，然后再转移至荧光显微镜位置，再次观察切片上的荧光信号是否在，实现再次荧光定位后，再回到离子束位置对目标区域进行切割，如此反复，直到切成一个厚度在200nm左右甚至更薄的薄片。
上述两种方法各有优缺点，第一种方式中，由于荧光显微镜和冷冻双束扫描电镜设备分离、互不影响，两台设备可以分别成像，通量高。但由于该技术流程中仅进行一次对齐，关联对齐精度相对较差，对微小结构的捕获成功率较低。第二种方式，可以在扫描电镜内部实现荧光原位成像，通过样品移动可以反复监测荧光信号在厚切片内的位置，故而对小目标物捕获的成功率大大提高。但在整个加工过程中，由于荧光成像和离子束加工不能同时进行，需要把样品来回移动或倾转，故实验时间长，效率低，不利于高通量样品制备，而且关联定位精度也有限，对于较小的目标物容易在切割过程中突然丢失，不能满足高准确性的离子束加工需求。
发明内容
(一)要解决的技术问题
鉴于现有技术的上述缺点、不足，本发明提供一种实时荧光监测冷冻聚焦离子束加工装置及方法，解决了现有技术中冷冻样品离子束加工时加工过程长、加工效率低，定位精度差，且成功率低的问题。
(二)技术方案
为了达到上述目的，本发明提供了实时荧光监测冷冻聚焦离子束加工装置及方法，具体技术方案如下：
一种实时荧光监测冷冻聚焦离子束加工装置，包括：
真空腔室，内部设置有冷台，用于存放冷冻样品，冷冻样品内设有荧光标记物；
离子束系统，设于真空腔室上，并指向冷冻样品设置，用于对冷冻样品进行离子束加工；
荧光成像系统，设于真空腔室上，并位于冷冻样品的下方，用于对冷冻样品发射激发光并进行实时光学成像。
进一步，荧光成像系统包括沿光路依次设置的激光器、物镜、反光镜、镜筒透镜、二向色镜、滤光片和图像探测器；其中，
真空腔室上设有荧光窗口，荧光窗口位置密封连接有真空法兰；
真空法兰一端连接有光学连接筒，另一端连接有底座，光学连接筒位于真空腔室外侧，底座位于真空腔室内侧；
物镜和反光镜均设于底座上；
镜筒透镜、二向色镜、滤光片均设于光学连接筒内，图像探测器设于光学连接筒远离真空法兰的一侧；
真空法兰的中心设置有透光片，透光片用于对真空腔室进行密封；
光学连接筒的筒壁的支路上还设有激光器，激光器位于二向色镜与滤光片垂直的一侧。
进一步，底座上设置有线性平移台；
物镜和反光镜均设于线性平移台上，线性平移台工作能够带动物镜和反光镜向靠近和/或远离冷冻样品的方向移动。
进一步，线性平移台上还设有物镜调节装置和反光镜调节装置；
物镜设于物镜调节装置上，物镜调节装置适于带动物镜三维移动和/或转动；
反光镜设于反光镜调节装置上，反光镜调节装置适于带动反光镜平面移动和/或转动。
进一步，物镜调节装置包括物镜倾转台和物镜平移台；
物镜设于物镜平移台上，物镜平移台设于物镜倾转台上，物镜倾转台设于线性平移台上，物镜平移台工作能够带动物镜三维移动，物镜倾转台工作能够带动物镜倾转。
进一步，反光镜调节装置包括反光镜平移台和反光镜倾转台；
反光镜设于反光镜倾转台上，反光镜倾转台设于反光镜平移台上，反光镜平移台设于线性平移台上，反光镜倾转台工作能够带动反光镜倾转，反光镜平移台工作能够带动反光镜平面移动。
进一步，真空腔室上还设有电子束系统，用于对冷冻样品进行电子束成像；
电子束系统相对于离子束系统倾斜设置，电子束系统发射的电子束和离子束系统发射的离子束的能够汇聚交叉于一点，冷冻样品位于交叉点处。
进一步，冷台设于位置调节装置上，位置调节装置适于带动冷台进行位置调节，包括：
第一样品平移台，冷台设于第一样品平移台上；
第二样品平移台，设于第一样品平移台的下方，
样品升降台，设于第二样品平移台的下方；
样品倾转台，设于真空腔室舱门上，通过L型支架与样品升降台相连，样品倾转台工作能够带动L型支架、样品升降台、第二样品平移台、第一样品平移台和冷台同步转动。
进一步，冷台为冷冻样品传输装置的一端，冷冻样品传输装置包括：
样品传输管，真空腔室舱门的一侧设置有样品传输窗口，样品传输管通过波纹管组件软连接于样品传输窗口位置；
冷冻样品传输杆，一端设有杜瓦瓶，另一端为冷冻样品固定端；
冷冻样品固定端穿设于样品传输管内，外周与样品传输管内壁动密封连接，能够穿过样品传输窗口以将冷冻样品输送至真空腔室内；
杜瓦瓶所在端能够与样品传输管卡接和/或解除卡接；
冷冻传输三维平移台，与样品传输管连接，当冷冻样品传输杆与样品传 输管卡接时，冷冻传输三维平移台工作能够带动与样品传输管卡接的冷冻样品传输杆三维移动。
进一步，冷冻传输装置还包括角度调节装置，包括：
轴套真空板阀外壳组合体，一端密封连接于冷冻传输窗口位置，另一端与波纹管组件密封连接，波纹管组件一端密封连接于样品传输管位置，另一端与冷冻传输三维平移台支架固定连接；
蜗轮，设于轴盘上，轴盘可转动地套设于轴套真空板阀外壳组合体的外周，轴盘与冷冻传输三维平移台支架固定连接；
蜗杆，转动连接于蜗杆支架上，蜗杆支架连接于真空腔室舱门上，蜗杆一端与电机相连，并与蜗轮相啮合，
当冷冻样品传输杆与样品传输管卡接时，电机工作能够带动与蜗杆啮合的蜗轮转动，进而带动轴盘、冷冻传输三维平移台、样品传输管、波纹管组件及冷冻样品传输杆同步旋转，以使冷冻样品在电子束系统和/或离子束系统之间进行调节。
一种实时荧光监测冷冻聚焦离子束加工方法，包括如下步骤：
将冷冻样品输送至真空腔室内，并置于冷台上；
控制荧光成像系统启动，向冷冻样品发送激发光并进行实时成像；
控制离子束系统启动，对冷冻样品进行离子束加工，并通过荧光成像系统实时监测所述冷冻样品的加工过程。
(三)有益效果
本发明提供的实时荧光监测冷冻聚焦离子束加工装置及方法，具有以下有益效果。
本发明中，将冷冻样品设于冷台上，在冷冻样品内需要的结构上标记荧光标记物，通过荧光信号的指导，然后通过离子束系统对冷冻样品进行离子束加工。加工过程中，通过荧光成像系统对冷冻样品发射激发光，激发光照射于冷冻样品，激发冷冻样品内的荧光标记物发射荧光，荧光信号被荧光成像系统接收并形成图像。本发明通过对冷冻样品进行实时成像，通过实时获取荧光图像的变化和荧光信号强弱的变化，实时监测冷冻样品被离子束切掉的结构和保留的结构情况，指导离子束的进一步加工位置，直至得到最终需要的薄片样品，实现了冷冻样品的原位实时荧光指导下的精确加工，进而提高了加工精度，缩短了冷冻样品加工过程，提高了加工效率和成功率。
附图说明
此处所说明的附图用来提供对本申请的进一步理解，构成本申请的一部分，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定，在附图中：
图1为具体实施方式中实施例一的实时荧光监测冷冻聚焦离子束加工装置的结构示意图；
图2为具体实施方式中实施例一和实施例二荧光成像系统、真空腔室及离子束系统和电子束系统的结构示意图；
图3为具体实施方式中实施例一和实施例二的荧光成像系统的结构示意图；
图4为具体实施方式中实施例一的冷台及位置调节装置结构示意图；
图5为具体实施方式中实施例二的实时荧光监测冷冻聚焦离子束加工装置的结构示意图；
图6为具体实施方式中实施例二的冷冻传输装置和冷台装置的结构示意图；
图7为具体实施方式中实施例二的角度调节装置的结构示意图；
图8为图7中的A-A剖视图；
图9为具体实施方式中实施例二的真空腔室舱门的结构示意图；
图10为具体实施方式中实施例二的波纹管组件的结构示意图；
图11为具体实施方式中实施例一和实施例二荧光成像系统的光路图。
【附图标记说明】
1、真空腔室；110、荧光窗口；
120、真空腔室舱门；120'、真空腔室舱门；121'、样品传输窗口；
2、荧光成像系统；201、物镜；202、反光镜；203、镜筒透镜；204、二向色镜；205、滤光片；206、图像探测器；207、激光器；208、光学连接筒；209、真空法兰；210、底座；211、线性平移台；212、物镜平移台；213、物镜倾转台；214、反光镜平移台；215、反光镜倾转台；
3、冷台；
4、位置调节装置；410、第一样品平移台；420、第二样品平移台；430、样品升降台；440、样品倾转台；450、L型支架；
5、电子束系统；6、离子束系统；
7、冷冻传输装置；
710、冷冻样品传输杆；711、杜瓦瓶；712、卡槽；713、冷冻样品固定端；
720、样品传输管；721、卡针；
730、波纹管组件；731、同步套管；732、波纹管；
740、冷冻传输三维平移台；750、冷冻传输三维平移台支架；
760、角度调节装置；761、轴套真空板阀外壳组合体；762、轴盘；763、轴盘轴承；764、蜗轮；765、蜗杆；766、蜗杆支架；767、电机；
770、真空输送系统；771、真空板阀；772、预抽真空阀；
8、冷冻样品；9、控制器。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明的优选实施例中的附图，对本发明实施例中的技术方案进行更加详细的描述。在附图中，自始至终相同或类似的标号表示相同或类似的元件或具有相同或类 似功能的元件。所描述的实施例是本发明一部分实施例，而不是全部的实施例。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。下面结合附图对本发明的实施例进行详细说明。
在本实施例的描述中，需要理解的是，术语“中心”、“纵向”、“横向”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本实施例和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本实施例保护范围的限制。
实施例一
参见图1至图4及图11，本实施例提供了一种实时荧光监测冷冻聚焦离子束加工装置，包括真空腔室1、离子束系统6和荧光成像系统2。其中，真空腔室1内设置有冷台3，冷台3用于存放冷冻样品8，冷冻样品8内设置有荧光标记物，当把激发光照射于冷冻样品8上时，荧光标记物被激发能够发射荧光。离子束系统6设于真空腔室1上，并指向冷冻样品8设置，用于发射聚焦离子束，以对冷冻样品8进行离子束切割加工。荧光成像系统2设于真空腔室1上，并位于冷冻样品8的下方，用于对冷冻样品8发射激发光，同时对冷冻样品8进行实时光学成像。具体使用时，通过荧光成像系统2对冷冻样品8发射激发光，激发光照射于冷冻样品8，冷冻样品8内的荧光标记物被激发发射荧光，荧光被荧光成像系统2接收并形成冷冻样品8中荧光标记结构的图像。对冷冻样品8进行离子束切割时，通过荧光成像系统2实时监测冷冻样品8的加工情况，实现了冷冻样品8实时荧光监测指导下的原位离子束加工，无需对冷冻样品8进行转移，进而大大提高了冷冻样品8的加工准确度，简化了加工过程，缩短了加工时间，提高了加工效率。
具体地，参见图2、图3及图11，本实施例中的荧光成像系统2包括沿光路依次设置的激光器207、物镜201、反光镜202、镜筒透镜203、二向色镜204、滤光片205和图像探测器206。真空腔室1的一侧设置有荧光窗口110，荧光窗口110位置密封连接有真空法兰209，真空法兰209一侧与光学连接筒208相连，另一侧与底座210相连。进一步，物镜201设于冷冻样品8的荧光标记物发射的荧光的光路上，并位于冷冻样品8的下方，物镜201和反光镜202均设于底座210上，镜筒透镜203、二向色镜204和滤光片205沿光路依次设于光学连接筒208内，图像探测器206设于光学连接筒208远离真空法兰209一侧的端部。真空法兰209的中心设置透光片，对真空腔室1进行密封，并能够透射激发光和荧光。光学连接筒208的筒壁支路上还设有激光器207，激光器207位于二向色镜204垂直于滤光片205的一侧，用于发射激发光。其中，二向色镜204具有反射和透射功能，可以反射激发光(波长短)，透射荧光(波长长)。具体到本实施例中，激光器207发射的激发光(波长短)经二向色镜204反射至镜筒透镜 203后，扩束成平行光进入反光镜202，再经反光镜202反射后经物镜201照射于冷冻样品8上。冷冻样品8上的荧光标记物被激发后发射荧光(波长长)，荧光经物镜201汇聚接收后成为平行光由反光镜202反射至镜筒透镜203汇聚后通过二向色镜204，经滤光片205过滤掉杂散光后，聚焦到图像探测器206上形成冷冻样品8的荧光标记结构的图像。该系统中的荧光成像系统还可以是宽场成像模式，也可以是激光共聚焦成像模式，或者结构光照明成像模式等不同的成像模式，这些成像方式均在本发明的保护范围内，可根据实验需要确定。
进一步，参见图3，底座210上还设置有线性平移台211，线性平移台211上设置有物镜调节装置和反光镜调节装置。其中，物镜201设于物镜调节装置上，物镜调节装置适于带动物镜201三维移动或倾转，反光镜202设于反光镜调节装置上，反光镜调节装置适于带动反光镜202平面移动或倾转。本实施例中，线性平移台211适于单一方向的移动，工作时能够带动物镜调节装置、物镜201、反光镜调节装置和反光镜202向远离或靠近冷冻样品8的方向移动。当冷冻样品8需要在荧光成像系统2实时监测下进行离子束切割时，控制线性平移台211启动，带动物镜201和反光镜202移动至冷冻样品8所在位置，然后通过物镜调节装置调节物镜201的位置，以使物镜201位于冷冻样品8发射的荧光的光路上，对应调节反光镜202，确保荧光的光路不发生改变。当冷冻样品8在荧光成像系统2实时监测下进行离子束切割完毕时，控制线性平移台211启动，带动物镜201和反光镜202移动至远离冷冻样品8所在位置，以方便冷冻样品8的冷冻传输、成像等其他操作。
具体地，本实施例中物镜调节装置包括物镜平移台212和物镜倾转台213，物镜平移台212设于物镜倾转台213上，物镜倾转台213设于线性平移台211上。物镜平移台212工作时能够带动物镜201沿水平或高度方向移动。物镜倾转台213工作时能够带动物镜201绕冷冻样品8倾转。通过物镜平移台212和物镜倾转台213的联合调节，使物镜201对正冷冻样品8上离子束切割的位置，并实现图像的聚焦。反光镜调节装置包括反光镜平移台214和反光镜倾转台215，反光镜202设于反光镜倾转台215上，反光镜倾转台215设于反光镜平移台214上，反光镜平移台214设于线性平移台211上，反光镜倾转台215工作时能够带动反光镜202倾转，反光镜平移台214工作时能够带动反光镜202沿水平方向的位置调节，具体使用时，根据物镜201的调节情况，反光镜平移台214和反光镜倾转台215带动反光镜202对应调节，以使激发光准确的照到冷冻样品8上，同时冷冻样品8上的荧光标记物发射的荧光，经物镜201汇聚后，准确的反射至成像光路。
进一步，参见图1及图2，真空腔室1上还设有电子束系统5，电子束系统5用于对冷冻样品8进行电子束成像。电子束系统5垂直设于真空腔室1的顶部，离子束系统6相对电子束系统5倾斜设置，电子束系统5发射的电子束与离子束系统6发射的离子束能够汇聚交叉于一点，冷冻样品8置于交叉点处。进一步，本实施例中的冷台3设于位置调节装置4上，适于带动冷 台3及设于冷台3上冷冻样品8移动和/或转动。具体使用时，位置调节装置4带动冷冻样品8移动，以使冷冻样品8位于电子束和离子束的汇聚点上，然后控制位置调节装置4启动带动冷冻样品8倾转，以使冷冻样品8与离子束系统6发射的离子束形成一定的角度，通常为10°～20°，方便离子束切割加工。
具体地，参见图4，本实施例中的位置调节装置4包括第一样品平移台410、第二样品平移台420、样品升降台430和样品倾转台440。冷台3设于第一样品平移台410上，第一样品平移台410设于第二样品平移台420的上方，第二样品平移台420设于样品升降台430的上方，样品升降台430设于L型支架450上，L型支架450连接于样品倾转台440上，样品倾转台440固定于真空腔室舱门120上，真空腔室舱门120密封连接于真空腔室1上。其中，第一样品平移台410、第二样品平移台420和样品升降台430的移动方向相互垂直，能够带动冷台3在空间中的三个相互垂直的方向进行位置调节，以精确调节冷冻样品8的位置，使冷冻样品8位于电子束和离子束的交叉点上。样品倾转台440工作时能够带动L型支架450、样品升降台430、第二样品平移台420、第一样品平移台410和冷台3同步转动，以使设于冷台3上冷冻样品8与离子束系统6发射的离子束或电子束系统5发射的电子束形成一定的角度，当冷冻样品8与离子束系统6发射的离子束成10°～20°夹角时，可用于冷冻样品8的离子束切割加工，当冷冻样品8与电子束系统5发射的电子束相垂直时，可用于冷冻样品8的电子束成像，满足不同场景的使用需求。
进一步，还包括控制器9，控制器9与线性平移台211、物镜平移台212、反光镜平移台214、第一样品平移台410和第二样品平移台420通讯连接，用于控制上述设备的启停及移动距离的调节。控制器9还与物镜倾转台213、反光镜倾转台215和样品倾转台440通讯连接，用于控制上述设备的启停及旋转角度的调节。控制器9还与激光器207和图像探测器206通讯连接，用于控制激发光的发射和停止，控制图像探测器206实时获取荧光图像和信号。通过控制器9控制与其通讯连接设备的启停，实现了实时荧光系统的光路的自动或半自动调节，荧光图像的实时获取，提高了实施荧光监测冷冻聚焦离子束加工装置的自动化程度，方便快捷。
基于本实施例实时荧光监测冷冻聚焦离子束加工装置，可以对冷冻样品8进行离子束加工和电子束成像。
冷冻样品实时荧光监测的离子束加工方法，包括如下步骤：
1)、把冷冻样品8传输至冷台3上；
2)、精确控制位置调节装置4启动，调节冷冻样品8至离子束系统6位置，并将冷冻样品置于离子束视野中，使冷冻样品和离子束的夹角为10°～20°；
3)、控制线性平移台211启动，带动物镜201及反光镜202移动至冷冻样品8所在位置；
4)、精确调节物镜平移台212、物镜倾转台213、反光镜平移台214、反光镜倾转台215，使物镜201正对冷冻样品8并实现图像聚焦，能够通过图像探测器206呈现清晰的冷冻样品8中荧光标记的结构的像；
5)、通过离子束系统6和荧光成像系统2共同使用，判断冷冻样品8中是否有需要切割的结构。如果有，通过分析荧光图像确认具体的切割位置，设定离子束系统6的切割参数，对冷冻样品8进行切割，在离子束切割的同时，通过连续或间歇式的方式，实时监控切割的位置和进度，直到获得厚度为200nm的薄片，并且保留了携带荧光信号的目标结构；
冷冻样品电子束成像，包括如下步骤：
1)、控制线性平移台211启动，带动物镜201及反光镜202移动至远离冷冻样品8所在位置；
2)、控制位置调节装置4启动，调节冷冻样品8倾转至垂直于电子束系统5发射的电子束位置，对冷冻样品8进行电子束成像。
实施例二
基于实施例一，本实施例提供的实时荧光监测冷冻聚焦离子束加工装置与实施例一相比，不同之处仅在于本实施例中真空腔室舱门120'的结构、冷台3的结构和致冷形式不同。
参见图5至图10，本实施例中的真空腔室舱门120'为凹型结构，设置有样品传输窗口121'。而冷台3为冷冻传输装置7的一端，冷冻传输装置7包括冷冻样品传输杆710、样品传输管720、波纹管组件730、冷冻传输三维平移台740和角度调节装置760。样品传输管720通过波纹管组件730密封连接于样品传输窗口121＇位置。冷冻样品传输杆710一端为冷冻样品固定端713，用于固定冷冻样品8，另一端设有杜瓦瓶711。其中，冷冻样品固定端713穿设于样品传输管720和波纹管组件730内，冷冻样品传输杆710的外周与样品传输管720内壁可滑动地密封连接，冷冻样品传输杆710沿样品传输管720内壁滑动，冷冻样品固定端713能够穿过样品传输窗口121＇以将冷冻样品8输送至真空腔室1内。杜瓦瓶711用于通过热传导的方式为冷冻样品8提供冷源，使冷冻样品8始终处于玻璃态冷冻形式。冷冻样品传输杆710可作为传输装置使用，冷冻样品固定端713还可作为冷台3使用，大大简化了实时荧光监测冷冻聚焦离子束加工装置的结构。
进一步，杜瓦瓶711所在侧的冷冻样品传输杆710上设置有卡槽712，样品传输管720上对应设置有卡针721，当卡针721卡入卡槽712时，冷冻样品传输杆710与样品传输管720卡接，当卡针721拔出卡槽712时，冷冻样品传输杆710与样品传输管720解除卡接。冷冻传输三维平移台740设于冷冻传输三维平移台支架750上，并与样品传输管720相连。波纹管组件730适于伸缩调节，当冷冻样品传输杆710与样品传输管720卡接时，冷冻传输三维平移台740工作能够带动与样品传输管720连接的波纹管组件730对应伸缩，样品传输管720相对于真空腔室1的位置发生改变，设于冷冻样品固定端713的冷冻样品8的位置也对应改变。本实施例通过冷冻传输三维平移 台740可精确调节冷冻样品8的位置，以使冷冻样品8位于离子束系统6发射的离子束和电子束系统5发射的电子束的聚焦点上。
具体地，参见图7，角度调节装置760包括轴套真空板阀外壳组合体761、轴盘762、蜗轮764和蜗杆765。其中，轴套真空板阀外壳组合体761一端密封连接于样品传输窗口121＇位置，并套设于波纹管组件730的外周，轴套真空板阀外壳组合体761内壁与波纹管组件730外周动密封连接，波纹管组件730与冷冻传输三维平移台支架750通过螺栓固定连接，蜗轮764通过螺栓固定于轴盘762上，轴盘762通过轴盘轴承763可转动地套设于轴套真空板阀外壳组合体761的外周，并通过螺栓与冷冻传输三维平移台支架750固定连接。蜗杆765的两端通过轴承转动连接于蜗杆支架766上，蜗杆支架766通过螺栓设于真空腔室舱门120'上。蜗杆765与蜗轮764相啮合，蜗杆765端部与电机767相连，电机767工作能够带动蜗杆765和蜗轮764啮合传动。当需要角度调节时，确认冷冻样品传输杆710与样品传输管720卡接，控制电机767启动，电机767带动蜗杆765和蜗轮764啮合传动，进而带动轴盘762、冷冻传输三维平移台支架750、样品传输管720、波纹管组件730、冷冻样品传输杆710和冷冻样品8同步转动，使冷冻样品8与离子束系统6发射的离子束成10°～20°的夹角，或与电子束系统5发射的电子束相垂直，以满足冷冻样品8离子束加工或电子束成像的需求。
本实施例中，参见图10，波纹管组件730包括波纹管732及套设于波纹管732外周的同步套管731，同步套管731套设于波纹管732的外周，并置于轴套真空板阀外壳组合体761内，同步套管731外周与轴套真空板阀外壳组合体761内壁动密封连接。进一步，同步套管731一端与波纹管732的端部采用焊接的方式固定连接，另一端通过螺栓与冷冻传输三维平移台支架750可拆卸连接。转动时，冷冻传输三维平移台支架750带动同步套管731和波纹管732同步转动。
进一步，还包括真空输送系统770，用于通过负压工作原理带动冷冻样品传输杆710沿样品传输管720滑动，并把冷冻样品8从大气环境传输至高真空环境。具体地，真空输送系统770包括真空板阀771和预抽真空阀772，真空板阀771密封连接于轴套真空板阀外壳组合体761上，以在冷冻样品传输杆710、样品传输管720、波纹管732、轴套真空板阀外壳组合体761和真空板阀771之间形成一密封通道，真空板阀771用于控制密封通道与真空腔室1的连通和关闭。预抽真空阀772密封连接于样品传输管720上，并与密封通道相连通，用于对密封通道抽低真空。
具体使用时，预先将冷冻样品8固定于冷冻样品传输杆710上，保持真空板阀771关闭，将冷冻样品固定端731穿设于样品传输管720内，然后控制预抽真空阀772开启，用真空泵开始对密封通道抽低真空，当到达设定值时，控制预抽真空阀772关闭、控制真空板阀771开启，由于真空腔室1的真空度远低于外界大气压，手动控制并使冷冻样品传输杆710在负压的作用下沿样品传输管720滑动，以将冷冻样品8输送至真空腔室1内。本实施例 中，冷冻样品8的传输过程操作简单，方便快捷。然后控制冷冻传输三维平移台740启动，调节冷冻样品8的位置，以使冷冻样品8位于离子束系统6发射的离子束和电子束系统5发射的电子束的交叉点上。当需要电子束成像时，控制角度调节装置760启动，调节冷冻样品8与电子束系统5发射的电子束相垂直，以实现冷冻样品8的电子束成像需求。当需要离子束加工时，控制角度调节装置760启动，调节冷冻样品8与离子束系统6发射的离子束成10°～20°的夹角，以实现冷冻样品8离子束加工需求。
具体到本实施例中，控制器9与真空板阀771和预抽真空阀772通讯连接，通过控制器9控制真空板阀771和预抽真空阀772的开启和关闭，以实现冷冻样品8的真空输送。进一步，控制器9还与冷冻传输三维平移台740和角度调节装置760通讯连接，用于控制冷冻传输三维平移台740的启动、移动距离及旋转角度的调节，以实现精确调节冷冻样品8位置的目的，以满足不同场景的使用需求。本实施例通过控制器9实现了实时荧光监测冷冻聚焦离子束加工装置的自动化控制，提高了自动化程度，方便快捷。
基于本实施例实时荧光监测冷冻聚焦离子束加工装置，可以对冷冻样品8进行离子束加工和电子束成像。
冷冻样品8离子束加工方法，包括如下步骤：
1)、把冷冻样品8置于冷冻样品传输杆710的冷冻样品固定端713上，通过冷冻样品传输杆710将冷冻样品8输送至真空腔室1内；
2)、精确控制冷冻传输三维平移台740和角度调节装置760启动，调节冷冻样品8至离子束系统6位置，并将冷冻样品置于离子束视野中，使冷冻样品和离子束的夹角为10°～20°；
3)、控制线性平移台211启动，带动物镜201及反光镜202移动至冷冻样品8所在位置；
4)、精确调节物镜平移台212、物镜倾转台213、反光镜平移台214、反光镜倾转台215，使物镜201正对冷冻样品8并实现图像聚焦，能够通过图像探测器206呈现清晰的冷冻样品8中荧光标记的结构的像；
5)、通过离子束系统6和荧光成像系统2共同使用，判断冷冻样品8中是否有需要切割的结构。如果有，通过分析荧光图像确认具体的切割位置，设定离子束系统6的切割参数，对冷冻样品8进行切割，在离子束切割的同时，通过连续或间歇式的方式，实时监控切割的位置和进度，直到获得厚度为200nm的薄片，并且保留了携带荧光信号的目标结构；
冷冻样品8电子束成像，包括如下步骤：
1)、控制线性平移台211启动，带动物镜201及反光镜202移动至远离冷冻样品8所在位置；
2)、控制冷冻传输三维平移台740和角度调节装置760启动，调节冷冻样品8倾转至垂直于电子束系统5发射的电子束位置，对冷冻样品8进行电子束成像。
基于实施例一和实施例二提供的实时荧光监测冷冻聚焦离子束加工装 置，通过设置荧光成像系统2和离子束系统6，实现对冷冻样品8原位实时荧光监测，实时离子束加工的方式，定位准确，且在荧光的定位监测过程中，离子束的加工不用停止，速度快、通量高，大大提高了离子束加工的工作效率和精度。该装置还可实现电子束-荧光两束联合成像，离子束-荧光两束联合成像，电子束-离子束-荧光三束联合成像等多种功能。冷台3也可根据需要更换成不同的样品杆或样品台的形式，使用灵活方便。真空腔室舱门120和真空腔室舱门120'可通过尺寸的改变适用于不同型号的冷冻双束扫描电镜，通用性强。
综上所述，本发明提供的实时荧光监测冷冻聚焦离子束加工装置，仅为本发明的较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都涵盖在本发明的保护范围内。

Claims (11)

  1. 一种实时荧光监测冷冻聚焦离子束加工装置，其特征在于，包括：
    真空腔室(1)，内部设置有冷台(3)，用于存放冷冻样品(8)，所述冷冻样品(8)内设有荧光标记物；
    离子束系统(6)，设于所述真空腔室(1)上，并指向所述冷冻样品(8)设置，用于对所述冷冻样品(8)进行离子束加工；
    荧光成像系统(2)，设于所述真空腔室(1)上，并位于所述冷冻样品(8)的下方，用于对所述冷冻样品(8)发射激发光并进行实时光学成像。
  2. 根据权利要求1所述的实时荧光监测冷冻聚焦离子束加工装置，其特征在于，所述荧光成像系统(2)包括沿光路依次设置的激光器(207)、物镜(201)、反光镜(202)、镜筒透镜(203)、二向色镜(204)、滤光片(205)和图像探测器(206)；其中，
    所述真空腔室(1)上设有荧光窗口(110)，所述荧光窗口(110)位置密封连接有真空法兰(209)；
    所述真空法兰(209)一端连接有光学连接筒(208)，另一端连接有底座(210)，所述光学连接筒(208)位于所述真空腔室(1)外侧，所述底座(210)位于所述真空腔室(1)内侧；
    所述物镜(201)和所述反光镜(202)均设于所述底座(210)上；
    所述镜筒透镜(203)、所述二向色镜(204)、所述滤光片(205)均设于所述光学连接筒(208)内，所述图像探测器(206)设于所述光学连接筒(208)远离所述真空法兰(209)的一侧；
    所述真空法兰(209)的中心设置有透光片，所述透光片用于对所述真空腔室(1)进行密封；
    所述光学连接筒(208)的筒壁的支路上还设有激光器(207)，所述激光器(207)位于所述二向色镜(204)与滤光片(205)垂直的一侧。
  3. 根据权利要求2所述的实时荧光监测冷冻聚焦离子束加工装置，其特征在于，所述底座(210)上设置有线性平移台(211)；
    所述物镜(201)和所述反光镜(202)均设于所述线性平移台(211)上，所述线性平移台(211)工作能够带动所述物镜(201)和所述反光镜(202)向靠近和/或远离所述冷冻样品(8)的方向移动。
  4. 根据权利要求3所述的实时荧光监测冷冻聚焦离子束加工装置，其特征在于，所述线性平移台(211)上还设有物镜调节装置和反光镜调节装置；
    所述物镜(201)设于所述物镜调节装置上，所述物镜调节装置适于带动所述物镜(201)三维移动和/或倾转；
    所述反光镜(202)设于所述反光镜调节装置上，所述反光镜调节装置适于带动所述反光镜(202)平面移动和/或倾转。
  5. 根据权利要求4所述的实时荧光监测冷冻聚焦离子束加工装置，其特 征在于，所述物镜调节装置包括物镜倾转台(213)和物镜平移台(212)；
    所述物镜(201)设于所述物镜平移台(212)上，所述物镜平移台(212)设于所述物镜倾转台(213)上，所述物镜倾转台(213)设于所述线性平移台(211)上，所述物镜平移台(212)工作能够带动所述物镜(201)三维移动，所述物镜倾转台(213)工作能够带动所述物镜(201)倾转。
  6. 根据权利要求4所述的实时荧光监测冷冻聚焦离子束加工装置，其特征在于，所述反光镜调节装置包括反光镜平移台(214)和反光镜倾转台(215)；
    所述反光镜(202)设于所述反光镜倾转台(215)上，所述反光镜倾转台(215)设于所述反光镜平移台(214)上，所述反光镜平移台(214)设于所述线性平移台(211)上，所述反光镜倾转台(215)工作能够带动所述反光镜(202)倾转，所述反光镜平移台(214)工作能够带动所述反光镜(202)平面移动。
  7. 根据权利要求1所述的实时荧光监测冷冻聚焦离子束加工装置，其特征在于，所述真空腔室(1)上还设有电子束系统(5)，用于对所述冷冻样品(8)进行电子束成像；
    所述电子束系统(5)相对于所述离子束系统(6)倾斜设置，所述电子束系统(5)发射的电子束和所述离子束系统(6)发射的聚焦离子束能够汇聚交叉于一点，所述冷冻样品(8)位于所述交叉点处。
  8. 根据权利要求7所述的实时荧光监测冷冻聚焦离子束加工装置，其特征在于，所述冷台(3)设于位置调节装置(4)上，所述位置调节装置(4)适于带动所述冷台(3)进行位置调节，包括：
    第一样品平移台(410)，所述冷台(3)设于所述第一样品平移台(410)上；
    第二样品平移台(420)，设于所述第一样品平移台(410)的下方，
    样品升降台(430)，设于所述第二样品平移台(420)的下方；
    样品倾转台(440)，设于真空腔室舱门(120)上，通过L型支架(450)与所述样品升降台(430)相连，所述倾转台(440)工作能够带动所述L型支架(450)、所述样品升降台(430)、所述第二样品平移台(420)、所述第一样品平移台(410)和所述冷台(3)同步转动。
  9. 根据权利要求7所述的实时荧光监测冷冻聚焦离子束加工装置，其特征在于，所述冷台(3)为冷冻传输装置(7)的一端，包括：
    样品传输管(720)，真空腔室舱门(120＇)的一侧设置有样品传输窗口(121＇)，所述样品传输管(720)通过波纹管组件(730)软连接于所述样品传输窗口(121＇)位置；
    冷冻样品传输杆(710)，一端设有杜瓦瓶(711)，另一端为冷冻样品固定端(713)；
    所述冷冻样品固定端(713)穿设于所述样品传输管(720)内，外周与所述样品传输管(720)内壁动密封连接，能够穿过所述样品传输窗口(121＇)以将所述冷冻样品(8)输送至真空腔室(1)内；
    所述杜瓦瓶(711)所在端能够与所述样品传输管(720)卡接和/或解除卡接；
    冷冻传输三维平移台(740)，与所述样品传输管(720)连接，当所述冷冻样品传输杆(710)与所述样品传输管(720)卡接时，所述冷冻传输三维平移台(740)工作能够带动与所述样品传输管(720)卡接的所述冷冻样品传输杆(710)三维移动。
  10. 根据权利要求9所述的实时荧光监测冷冻聚焦离子束加工装置，其特征在于，所述冷冻传输装置(7)还包括角度调节装置(760)，包括：
    轴套真空板阀外壳组合体(761)，一端密封连接于所述冷冻传输窗口位置(121＇)，另一端与所述波纹管组件(730)动密封连接，所述波纹管组件(730)一端密封连接于所述样品传输管(720)位置，另一端与所述冷冻传输三维平移台支架(750)固定连接；
    蜗轮(764)，设于轴盘(762)上，所述轴盘(762)可转动地套设于所述轴套真空板阀外壳组合体(761)的外周，所述轴盘(762)与所述冷冻传输三维平移台支架(750)固定连接；
    蜗杆(765)，端部转动连接于蜗杆支架(766)上，所述蜗杆支架(766)设于所述真空腔室舱门(121＇)上，所述蜗杆(765)一端与电机(767)相连，并与所述蜗轮(764)相啮合，
    当所述冷冻样品传输杆(710)与所述样品传输管(720)卡接时，所述电机(767)工作能够带动与所述蜗杆(765)啮合的所述蜗轮(764)转动，进而带动所述轴盘(762)、所述冷冻传输三维平移台(740)、所述样品传输管(720)、所述波纹管组件(730)及所述冷冻样品传输杆(710)同步旋转，以使所述冷冻样品(8)在所述电子束系统(5)和/或所述离子束系统(6)之间的进行调节。
  11. 一种实时荧光监测冷冻聚焦离子束加工方法，其特征在于，包括如下步骤：
    将冷冻样品输送至真空腔室内，并置于冷台上；
    控制荧光成像系统启动，向所述冷冻样品发送激发光并进行实时成像；
    控制离子束系统启动，对所述冷冻样品进行离子束加工，并通过所述荧光成像系统实时监测所述冷冻样品的加工过程。
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