CN117723784A - Freezing electron microscope sample preparation device and sample preparation method thereof - Google Patents
Freezing electron microscope sample preparation device and sample preparation method thereof Download PDFInfo
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Abstract
The invention discloses a freeze electron microscope sample preparation device and a sample preparation method thereof, and relates to the field of freeze electron microscopes. The sample preparation device can realize the real-time detection and control of the preparation experiment of the frozen electron microscope sample with the thickness of the surface sample of the carrier mesh, and the sample preparation method realizes the thinning of the surface liquid sample of the frozen electron microscope carrier mesh based on a centrifugal mode; carrying out reflection spectrum detection on a carrier surface sample in the centrifugation process, and calculating the liquid layer thickness of the sample in real time, so that the thickness of the finally prepared sample is accurately controlled, and high success rate and reproducibility of the frozen electron microscope sample preparation are realized; and because the sample thickness detection principle adopted by the invention is not influenced by the physical and chemical property change of the sample, the sample preparation method of the device has universality, is suitable for different sample solutions, and is favorable for popularization and application.
Description
Technical Field
The invention relates to the field of a refrigeration electron microscope, in particular to a refrigeration electron microscope sample preparation device and a sample preparation method thereof.
Background
The Cryo-electron microscopy (Cryo-EM) is a high resolution electron microscopy technique that is capable of obtaining high quality electron microscopy images of a sample in a frozen state. Unlike conventional electron microscopy, the freeze electron microscopy does not require staining and slicing of the sample, and thus the structure and assembly of the biomolecules can be more accurately observed. This technology has been widely used in the field of life sciences including cell biology, biochemistry, biophysics, and biomedicine.
Single particle technology is one of the mainstream technologies for resolving biological macromolecular structures by using a cryoelectron microscope. When the single particle technology of the frozen electron microscope is used for analyzing the structures of biomacromolecules such as proteins, the preparation of samples is a very critical step, and the success or failure of subsequent experiments is often determined. The thickness of a suitable glassy sample for use in a cryoelectron microscope is typically between 50-200nm, and in this range the sample is sufficiently thin that the electron beam can penetrate and form a high resolution image. If the sample is too thick, the electron beam is scattered and absorbed, the image quality is affected, and in the worst case, the electron beam cannot penetrate the sample layer, resulting in a black observed image, and such a sample cannot be used for the electron-freezing analysis. Therefore, the thickness of the frozen electron microscope sample needs to be controlled when the sample is prepared, so that high-quality electron microscope imaging can be obtained. How to prepare a frozen electron microscope sample with proper ice layer thickness, and improve the success rate of frozen electron microscope sample preparation and observation, is one of the hot spots of current academic research.
The current mainstream preparation method of the frozen electron microscope sample applied to the single-particle structure analysis technology uses special frozen sample preparation equipment or manual sample preparation (sample thickness control method: absorbing superfluous sample on the surface of a carrier net by filter paper), and the method is introduced as follows:
semi-automated cryoelectron microscope sampling is performed using a cryo-sampling device (e.g., a Vitrobot). The method mainly comprises the steps of manually injecting 3-5 mu L of sample onto a freezing electron microscope carrier net, then absorbing excessive sample (more than 99% of sample is absorbed by filter paper) on the carrier net by the instrument through the filter paper in a ballast net manner, and finally throwing the carrier net into a refrigerant (usually liquid ethane) by the instrument to quickly freeze the sample. If the instrument parameters (sample making temperature, humidity, filter paper pressing time, pressing force, waiting time before the cryogen is injected, etc.) are set correctly, a glassy sample with a desired thickness can be prepared.
The conventional main method has the advantages of simple sample preparation method, but has obvious defects, too many experimental parameters are required to be tried and optimized, and particularly, the filter paper is used for absorbing redundant samples to prepare samples with the thickness of only tens to hundreds of nanometers, and the reproducibility and success rate are too low.
Therefore, a great deal of research work is carried out around the field of frozen electron microscope sample preparation by a plurality of research groups at home and abroad, and a plurality of methods for improving the success rate and the reproducibility of sample preparation are provided and mainly divided into the following categories:
1. and (3) chemically modifying the surface of the carrier surface of the frozen electron microscope, attaching hydrophilic materials, and absorbing redundant samples so as to achieve the purpose of thinning the liquid layer thickness of the samples and improve the success rate of frozen electron microscope sample preparation. ( The sample thickness control method comprises the following steps: absorbing the redundant sample by chemically modifying the surface of the carrier web with a hydrophilic material )
2. By adopting methods such as spraying, piezoelectric spraying and the like, the volume of sample liquid drops is reduced to nanoliter and skin upgrading, and simultaneously, the sample liquid drops are sprayed in different areas of the same piece of frozen electron microscope carrier net so as to reduce the thickness of a sample liquid layer, increase the available carrier net area containing the sample and improve the success rate of frozen electron microscope sample preparation. ( The sample thickness control method comprises the following steps: by reducing the sample loading volume and ejecting a large number of sample droplets at different positions on the surface of the carrier web, the probability of obtaining a region of suitable sample thickness is increased )
3. And a probe, a capillary tube and the like are adopted to coat an ultra-trace sample on the surface of the carrier net so as to reduce the thickness of a sample liquid layer. ( The sample thickness control method comprises the following steps: by reducing the sample loading volume and by spontaneous diffusion of the sample on the surface of the carrier web, the thickness of the sample is reduced )
4. And (5) thinning the excessive liquid sample on the carrier mesh by using modes of air blowing, evaporation and the like. ( The sample thickness control method comprises the following steps: by sample evaporation or the like to reduce the thickness of the sample )
Compared with a semi-automatic frozen sample preparation instrument or manual sample preparation, the frozen electron microscope sample preparation technology improves the success rate and reproducibility of single-particle frozen electron microscope sample preparation experiments to a certain extent, but the following defects still exist:
(1) In the process of preparing the sample by the frozen electron microscope, the thickness change condition of the liquid layer of the sample on the surface of the carrier web of the frozen electron microscope cannot be detected in real time, and the proper sample preparation condition can be searched only by experience and repeated experiments, and the time when the carrier web is put into the refrigerant for freezing can be determined. The thickness of the glass-state ice layer of the finally prepared frozen electron microscope sample is uncontrollable, randomness exists, and the success rate of sample preparation is low.
(2) The method has no versatility (basically, the method for controlling the thickness of the liquid layer of the sample is influenced by the physicochemical properties of the sample). For example, a suitable sample preparation condition of the sample A is found out through experience and repeated experiments, but if the sample is changed into the sample B with different physical and chemical properties such as concentration, buffer composition, viscosity and the like, the sample preparation condition of the sample with a suitable thickness is also changed, and the sample preparation condition of the sample A cannot be directly used.
Disclosure of Invention
In order to solve the defects, the invention provides a frozen electron microscope sample preparation device capable of detecting and controlling the thickness of a carrier web surface sample in real time and a sample preparation method thereof.
The specific embodiments of the present invention are as follows:
the invention provides a frozen electron microscope sample preparation device capable of detecting and controlling the thickness of a carrier web surface sample in real time, which comprises:
the centrifugal module comprises a centrifugal machine, a rotating shaft of the centrifugal machine is horizontally arranged, a net-carrying centrifugal bracket rotating in a vertical plane during centrifugation is arranged at the end part of the centrifugal machine, and a position for placing a net is arranged at the end part of the net-carrying centrifugal bracket;
the detection module comprises a spectrometer for detecting the thickness of a sample on the carrier network in real time and a spectrometer light source,
during detection, the light source of the spectrometer emits detection light to irradiate on the centrifugal path of the carrier net, and when a sample on the carrier net is irradiated by the detection light in the centrifugal rotation process, the detection light is reflected and detected by the spectrometer.
According to the invention, the centrifugal module can realize thinning of the surface liquid sample of the surface of the frozen electron microscope carrier, and the detection module detects the thickness of the surface liquid sample of the surface of the frozen electron microscope carrier according to the preset sampling interval, and can stop the operation of the centrifugal module when the thickness reaches the preset range.
Preferably, a freezing module is also included, the freezing module including a cryogen storage vessel for holding cryogen,
the freezing module is responsible for rapidly freezing the liquid sample on the surface of the freezing electron microscope carrier reaching the preset range.
Preferably, the freezing module stores the cryogen using foam dewar. Liquid ethane is used as the refrigerant. The periphery of the center of the foam Dewar is provided with liquid nitrogen, and a freezing electron microscope carrier net sample disk is immersed in the liquid nitrogen and used for storing the freezing electron microscope carrier net after sample preparation.
In the invention, the freezing module is positioned below the centrifugal module, and the freezing module and the centrifugal module can relatively move so that the centrifuged sample stretches into the cryogen storage container to soak the cryogen;
preferably, the case where the freezing module and the centrifugation module are relatively movable includes: when the freezing module is fixed, the centrifugal module can move towards the freezing module on the linear sliding table through the driving sliding block;
when the vertical arm is fixed, the freezing module moves towards the centrifugal module, and the freezing module can move through a lifting table or other moving devices, so that the centrifuged sample stretches into the cryogen storage container to soak the cryogen.
The moving device can select various structures with transferring and conveying functions, for example, a motor and a conveying arm structure driven by the motor can be adopted, or a synchronous belt sliding table structure driven by the motor and the motor can be adopted, or a cylinder, a hydraulic cylinder or the like can be used as a quick moving device driven by a driving piece, a lifting table or the like.
Preferably, the cryoelectron microscope sample preparation device further comprises an accessible sample preparation chamber, wherein the centrifugation module and the detection module complete the centrifugation and detection operations; the sample preparation chamber shell is provided with a circulating fan structure.
As a further preference, the sample preparation device further comprises a temperature and humidity control module for controlling the temperature and the humidity of the sample preparation chamber.
Preferably, the centrifugal support for carrying the net is provided with a clamping groove for placing the net at the end far away from the rotating shaft, the bottom surface of the clamping groove is provided with a diversion trench, the diversion trench extends to the outer surface of the centrifugal support for carrying the net,
the clamping groove is used for fixing the carrying net, the clamping groove can clamp the edge of the carrying net, the liquid sample on the upper surface of the carrying net is continuously thinned during centrifugation, and the liquid sample thinned in the thinning process flows out through the diversion trench. Alternatively, the screen-loaded centrifuge support may be positioned one opposite each, allowing for the preparation of multiple samples at a time.
Preferably, the surface of the clamping groove, which is opposite to the detection module, is provided with a window for the detection module to detect. On one hand, the non-contact thickness measuring device is beneficial to detecting the thickness of a sample liquid layer, and on the other hand, the sample after centrifugation is dipped with the refrigerant for convenience.
Preferably, the centrifuge should be selected from high rotational speed motors with absolute position fixes (position encoders), such as closed loop stepper motors, servo motors.
The carrying net is a copper net, a molybdenum net or a gold net;
before the frozen electron microscope carrier net is used, the hydrophilic treatment of glow discharge is needed, and the glow discharge parameters are as follows: current 15mA, time 25s. Through the surface treatment, the carrier surface is ensured to have better affinity to the sample to be detected.
The surface of the carrier net is covered with a supporting film, the thickness of the supporting film is 15-20nm, and the diameter of the pore diameter is 1 mu m.
The invention also provides a frozen electron microscope sample preparation method capable of detecting and controlling the thickness of the surface sample of the carrier web in real time, which comprises the following steps:
(1) Placing the carrier net into a centrifugal module, placing the sample solution on the carrier net, fully soaking,
the centrifugal module comprises a centrifugal machine, a rotating shaft of the centrifugal machine is horizontally arranged, a net-carrying centrifugal bracket rotating in a vertical plane during centrifugation is arranged at the end part of the centrifugal machine, and a position for placing a net is arranged at the end part of the net-carrying centrifugal bracket;
(2) The centrifugal machine in the step (1) rotates to drive the carrying net to carry out centrifugal motion;
(3) Measuring the thickness of the sample liquid layer in the centrifugal motion in the step (2) by using a detection module, and stopping the centrifugation when the thickness reaches a preset range;
the detection module comprises a spectrometer for detecting the thickness of a sample on the carrier network in real time and a spectrometer light source,
when the sample on the carrier net is irradiated by the detection light in the centrifugal rotation process, the detection light is reflected and detected by the spectrometer;
there are 2 detection modes available for centrifugal rotation detection:
(1) The centrifugation is stopped briefly during detection: when the spectrometer detects the thickness of a sample according to a preset detection time interval (for example, once for 5 seconds), the centrifugal module controls the carrier net to be in a downward position for more than 20ms (depending on the sampling speed of the spectrometer, the integration time of the spectrometer preset by a user and the sampling times), so that the spectrometer can collect the reflection spectrum of the sample at one time.
(2) Centrifugation was not suspended at the time of detection: the carrier net continuously rotates and centrifugates, the motor is not suspended when the spectrometer collects, the spectrometer collects the sample reflection spectrum when the carrier net passes through for a plurality of times in the time of >20ms, but the signal to noise ratio of the spectrum is poor. In order to further improve the signal-to-noise ratio of the spectrum, the sampling can be repeated for 10 times, the spectrum is accumulated and measured, the signal-to-noise ratio of the spectrum is improved, and the collection of the reflection spectrum of the sample is completed.
(4) And freezing the centrifuged sample to prepare the sample for the frozen electron microscope.
In the invention, the initial centrifugal position, the spectrum detection position and the final centrifugal position of the screen-carrying centrifugal support are required to be controlled to be at the lowest position. The initial centrifugal position of the grid-loaded centrifugal bracket is controlled to be at the lowest position, so that an experimenter can conveniently put the frozen electron microscope grid into the bracket; the stop centrifugal position of the centrifugal support of the screen is controlled to be at the lowest position, so that the linear sliding table can drive the freezing electron microscope screen at the tail end of the centrifugal support to be immersed into the refrigerant downwards and rapidly, and rapid freezing of the sample is realized.
Preferably, in the step (1), before the carrier web is placed in the centrifugal module, the humidity of the centrifugal module is controlled to be 100%, the temperature is 4 ℃,
the frozen electron microscope sample preparation device also comprises a sample preparation chamber which can be accessed, wherein the centrifugation module and the detection module complete the centrifugation and detection operation in the sample preparation chamber; and the temperature and humidity control module is used for controlling the temperature and the humidity of the sample preparation chamber.
The temperature control and humidity control module comprises a humidity control module and a temperature control module, wherein the humidity control module consists of a humidifying/dehumidifying device (the humidifying can be realized by adopting the existing humidifying components such as a spraying component, a desorption humidifying component and the like, the dehumidifying can also be realized by adopting the existing components such as adsorption dehumidifying and the like, and the humidity and temperature control mechanism commonly adopted in the current detection field can be directly adopted to realize the control of humidity or temperature in the simplest way of course), a high-precision humidity sensor and a humidity controller (which can be a computer or a control chip, a control circuit and the like) and can maintain the humidity in a sample preparing chamber to be between 0 and 100 percent. The temperature control module consists of a refrigerating/heating device, a temperature sensor and a temperature controller (which can be a computer or a control chip, a control circuit and the like) and can maintain the temperature in the chamber to be between-20 ℃ and 100 ℃.
According to the invention, the temperature in the sample preparation chamber is controlled to slow down the evaporation rate of the sample droplets and to maintain the protein activity. Considering the influence of temperature on protein stability and evaporation rate, the temperature in the sample preparing chamber is set to be 2-25 ℃, the temperature value can be set to be 15-25 ℃ for samples with stable properties, and the temperature value can be set to be 4 ℃ for unstable samples. The lower sample preparation chamber temperature is selected, which is beneficial to reducing the evaporation speed of liquid drops. On the other hand, the humidity in the sample preparing chamber is accurately controlled (1% -0.1%) in the range of high humidity (90% -100%), so that the success rate of sample preparing can be further improved.
The volume of sample solution placed on the carrier web was 1-10. Mu.L. In the present example, the loading volume was 5. Mu.L.
Preferably, in the step (2), the rotating shaft is driven by a centrifugal motor, and the centrifugal rotating speed of the centrifugal motor is 100-5000rpm;
the length of the net-carrying centrifugal bracket is 20cm,
and the centrifugal acceleration of the carrier net during centrifugation is 1000G.
The centrifugal motor of the device can be controlled to rotate by computer software to drive the carrier net to perform centrifugal motion at a specific speed. In the centrifugation process, the sample on the surface of the carrier net is continuously thrown away through the diversion trench at the tail end of the centrifugal support of the carrier net under the action of centrifugal force, and the thickness of the sample on the surface of the carrier net is continuously thinned.
The control of the centrifugal acceleration can be realized by adjusting the rotation speed of a motor and changing the length of a grid-loaded centrifugal bracket, and the specific formula is as follows:
wherein the diameter is the length of the centrifugal support of the carrying net, and the unit is mm;
the rotation speed is the number of times of finishing rotation per minute, and the unit is 1/min 2 ；
Acceleration of gravity of 9.8m/s 2 ；
G is the centrifugal acceleration.
In step (2), when the light emitted by the spectrometer light source is incident into the sample on the surface of the frozen electron microscope carrier, multiple reflection phenomena occur, the multiple reflection light can be enhanced or reduced due to the phase difference between the multiple reflection light and the sample, and the phase difference depends on the refractive index and the optical path of the sample, so that the reflection spectrum from the sample has a direct relation with the thickness of the sample. After the spectrometer detects the reflection spectrum, the thickness of the sample can be analyzed by utilizing a reflection spectrum fitting method.
In the invention, the measuring wavelength range of the spectrometer is 200-1100nm, the resolution is less than 1nm, the spectrum acquisition time is less than 20 ms/time, the thickness range of the liquid layer of the detection sample is 2nm-200 mu m,
the spectrometer light source adopts a tungsten halogen lamp, and the diameter of a light spot focused on the surface of the carrier net is 1mm.
The frozen electron microscope sample preparation method uses the frozen electron microscope sample preparation device capable of detecting and controlling the thickness of the carrier web sample in real time to prepare samples.
The invention has the beneficial effects that:
the sample preparation device can realize the real-time detection and control of the preparation experiment of the frozen electron microscope sample with the thickness of the surface sample of the carrier mesh, and the sample preparation method realizes the thinning of the surface liquid sample of the frozen electron microscope carrier mesh based on a centrifugal mode; carrying out reflection spectrum detection on a carrier surface sample in the centrifugation process, and calculating the liquid layer thickness of the sample in real time, so that the thickness of the finally prepared sample is accurately controlled, and high success rate and reproducibility of the frozen electron microscope sample preparation are realized; and because the sample thickness detection principle adopted by the invention is not influenced by the physical and chemical property change of the sample, the sample preparation method of the device has universality, is suitable for different sample solutions, and is favorable for popularization and application.
Drawings
FIG. 1 is a schematic diagram showing the overall structure of a cryo-electron microscope sample preparation device capable of detecting and controlling the thickness of a carrier web sample in real time.
Fig. 2 is a front view of the core area structure of the cryo-electron microscope sample preparation device.
FIG. 3 is a rear view of the core area structure of the cryo-electron microscope sample preparation device.
Fig. 4 is a schematic view of a screen-loaded centrifugal stand without a screen placed.
Fig. 5 is a grid-loaded and grid-loaded centrifugal support of the cryo-electron microscope.
FIG. 6 is a 300kV cryoelectron micrograph of a apoferritin sample prepared by the apparatus of the present invention.
The marks in the figure: the device comprises a 1-sliding block, a 2-centrifugal machine, a 3-screen, a 4-screen centrifugal support, a 5-spectrometer, a 6-spectrometer light source, a 7-optical filter, an 8-cryogen storage container, 9-cryogen, a 10-diversion trench, an 11-temperature and humidity control module, a 12-sample preparation chamber shell, a 13-sample preparation chamber door, a 14-circulating fan, a 15-linear sliding table and a 16-rotating shaft.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Please refer to fig. 1-5. It should be noted that, the illustrations provided in the present embodiment merely illustrate the basic concept of the present invention by way of illustration, and only the components related to the present invention are shown in the drawings and are not drawn according to the number, shape and size of the components in actual implementation, and the form, number and proportion of the components in actual implementation may be arbitrarily changed, and the layout of the components may be more complex. The present invention will be further illustrated by the following examples, but the scope of the present invention is not limited thereto.
Example 1
As shown in fig. 1 to 5, the invention provides a frozen electron microscope sample preparation device capable of detecting and controlling the thickness of a sample on the surface of a carrier web in real time, which comprises a centrifugal module and a detection module, wherein the centrifugal module comprises a centrifugal machine 2, a rotating shaft 16 of the centrifugal machine 2 is horizontally arranged, a carrier web centrifugal bracket 4 rotating in a vertical plane during centrifugation is arranged at the end part of the centrifugal machine 2, and a position for placing a carrier web 3 is arranged at the end part of the carrier web centrifugal bracket 4; the detection module comprises a spectrometer 5 and a spectrometer light source 6, wherein the spectrometer 5 and the spectrometer light source 6 are used for detecting the thickness of a sample on the carrier net 3 in real time, during detection, the spectrometer light source 6 emits detection light to irradiate on a centrifugal path of the carrier net 3, and when the sample on the carrier net 3 is irradiated by the detection light in the centrifugal rotation process, the detection light is reflected and detected by the spectrometer 5. The centrifugal module can realize thinning of the liquid sample on the surface of the carrier of the frozen electron microscope, and a sample with ideal thickness is prepared through the detection module.
Preferably, the system further comprises a freezing module, wherein the freezing module comprises a refrigerant storage container 8 for placing a refrigerant 9, and the freezing module is responsible for rapidly freezing the surface liquid sample of the frozen electron microscope carrier reaching a preset range.
In the invention, the freezing module is positioned below the centrifugal module, and the freezing module and the centrifugal module can relatively move so that the centrifuged sample stretches into the cryogen storage container to soak the cryogen; as an example, with the freezing module fixed, the centrifugal module is moved on the linear sliding table 15 by the driving sliding block 1 toward the freezing module;
preferably, the cryoelectron microscope sample preparation device further comprises a sample preparation chamber capable of entering and exiting, wherein the sample preparation chamber comprises a sample preparation chamber shell 12 and a sample preparation chamber door 13, and the centrifugation module and the detection module complete centrifugation and detection operation in the sample preparation chamber; a circulation fan 14 is provided in the sample preparation chamber housing 12 to regulate the flow of air between the sample preparation chamber and the sample preparation chamber. Further preferably, the sample preparation chamber further comprises a temperature and humidity control module 11 for controlling the temperature and humidity of the sample preparation chamber.
Preferably, the screen-carrying centrifugal support 4 is provided with a clamping groove for placing the screen 3 at the end far away from the rotating shaft 16, the bottom surface of the clamping groove is provided with a diversion groove 10, the diversion groove 10 extends to the outer surface of the screen-carrying centrifugal support 4, the clamping groove is used for fixing the screen-carrying centrifugal support, the liquid sample on the upper surface of the screen is continuously thinned during centrifugation, and the liquid sample thinned in the thinning process flows out through the diversion groove. The surface of the clamping groove, which is opposite to the detection module, is provided with a window for the detection module to detect, so that the spectrometer 5 and the spectrometer light source 6 can detect the thickness of the sample liquid layer. A light filter is also arranged in front of the spectrometer light source 6.
The rotating shaft 16 is driven by the centrifugal motor 2; as an example, a servo motor is used in the present invention.
Correspondingly, the invention also provides a frozen electron microscope sample preparation method capable of detecting and controlling the thickness of the surface sample of the carrier web in real time, which at least comprises the following steps:
(1) The sample preparation chamber door 13 is closed and the internal temperature and humidity control module 11 (in this embodiment, the humidity is adjusted to 100% and the temperature is adjusted to 4 ℃) is opened to protect the activity of the biological sample in the subsequent sample preparation process and to avoid sample evaporation.
(2) The copper mesh is selected as a carrier mesh 3, a carbon film of a supporting film is coated on the surface of the copper mesh, the thickness of the carbon film is 15-20nm, micropores on the carbon film are uniformly and orderly arranged, the diameter of the pore is 1 mu m, the surface of the copper mesh is subjected to glow discharge hydrophilic treatment, the current of glow discharge is 15mA, and the time is 25s.
(3) The sample preparation chamber door 13 is opened, and the carrier net 3 is put into the clamping groove of the carrier net centrifugal bracket 4 of the centrifugal module by using tweezers.
(4) mu.L of the apoferritin sample solution was injected onto the surface of the copper mesh with a pipette, allowed to wet and spread sufficiently, and the sample preparation chamber door 13 was closed.
(5) The control software automatically controls the centrifugal machine 2 according to the preset centrifugal acceleration of 1000G, and drives the copper net to rotate and centrifuge through the rotating shaft 16.
(6) According to a preset spectrum sampling interval (5 s/time), a spectrometer light source is started, a reflection spectrum of a sample is detected by a spectrometer 5, and the liquid layer thickness of the sample at the moment is calculated; when the thickness reaches within a preset range (< 200 nm), the software controls the centrifuge 2 to stop centrifugation.
(7) And the copper mesh is rotated to the bottommost part, then the copper mesh is quickly driven by the linear sliding table 15 through the sliding block 1 to be inserted into the liquid ethane refrigerant 9 at the bottom part, and the liquid ethane refrigerant instantaneously freezes the sample on the surface of the copper mesh into a glass state, so that the preparation of the sample thickness-controllable frozen electron microscope sample is completed.
The prepared frozen electron microscope sample is subjected to 300kV frozen electron microscope observation, and as can be seen from fig. 6, the system can be used for efficiently completing the frozen sample preparation experiment of the protein sample.
Claims (10)
1. A cryo-electron microscope sample preparation device capable of detecting and controlling the thickness of a surface sample of a carrier web in real time, comprising:
the centrifugal module comprises a centrifugal machine, a rotating shaft of the centrifugal machine is horizontally arranged, a net-carrying centrifugal bracket rotating in a vertical plane during centrifugation is arranged at the end part of the centrifugal machine, and a position for placing a net is arranged at the end part of the net-carrying centrifugal bracket;
the detection module comprises a spectrometer for detecting the thickness of a sample on the carrier network in real time and a spectrometer light source,
during detection, the light source of the spectrometer emits detection light to irradiate on the centrifugal path of the carrier net, and when a sample on the carrier net is irradiated by the detection light in the centrifugal rotation process, the detection light is reflected and detected by the spectrometer.
2. The device for preparing a sample by a cryoelectron microscope capable of detecting and controlling a thickness of a surface sample of a carrier web in real time according to claim 1, further comprising a freezing module including a cryogen storage container for placing a cryogen,
the freezing module is positioned below the centrifugal module, and the freezing module and the centrifugal module can relatively move so that the centrifuged sample stretches into the cryogen storage container to soak the cryogen;
the frozen electron microscope sample preparation device also comprises a sample preparation chamber and a temperature and humidity control module for controlling the temperature and the humidity of the sample preparation chamber.
3. The device of claim 2, wherein the freezing module and the centrifugation module are capable of moving relative to each other, comprising: when the freezing module is fixed, the centrifugal module moves towards the freezing module on the linear sliding table through the driving sliding block;
when the vertical arm is fixed, the freezing module moves toward the centrifugal module.
4. The frozen electron microscope sample preparation device capable of detecting and controlling the thickness of a surface sample of a carrier web in real time according to claim 1, wherein the carrier web centrifugal support is provided with a vertical clamping groove for placing the carrier web at a position far away from a rotating shaft end, the bottom surface of the clamping groove is provided with a diversion trench, the diversion trench extends to the outer surface of the carrier web centrifugal support,
the surface of the clamping groove, which is opposite to the detection module, is provided with a window for the detection module to detect.
5. The frozen electron microscope sample preparation device capable of detecting and controlling the thickness of a surface sample of a carrier web in real time according to claim 1, wherein the carrier web is a copper web, a molybdenum web or a gold web;
the surface of the carrier net is covered with a supporting film, the thickness of the supporting film is 15-20nm, and the diameter of the pore diameter is 1 mu m.
6. A method for preparing a sample by a frozen electron microscope capable of detecting and controlling the thickness of a surface sample of a carrier web in real time is characterized by comprising the following steps:
(1) Placing the carrier net into a centrifugal module, placing the sample solution on the carrier net, fully soaking,
the centrifugal module comprises a centrifugal machine, a rotating shaft of the centrifugal machine is horizontally arranged, a net-carrying centrifugal bracket rotating in a vertical plane during centrifugation is arranged at the end part of the centrifugal machine, and a position for placing a net is arranged at the end part of the net-carrying centrifugal bracket;
(2) The centrifugal machine in the step (1) rotates to drive the carrying net to carry out centrifugal motion;
(3) Measuring the thickness of the sample liquid layer in the centrifugal motion in the step (2) by using a detection module, and stopping the centrifugation when the thickness reaches a preset range;
the detection module comprises a spectrometer for detecting the thickness of a sample on the carrier network in real time and a spectrometer light source,
when the sample on the carrier net is irradiated by the detection light in the centrifugal rotation process, the detection light is reflected and detected by the spectrometer;
(4) And freezing the centrifuged sample to prepare the sample for the frozen electron microscope.
7. The method according to claim 6, wherein in the step (1), the humidity of the centrifugal module is controlled to be 100% and the temperature is controlled to be 4 ℃ before the carrier web is placed on the centrifugal module,
the volume of sample solution placed on the carrier web was 1-10. Mu.L.
8. The method according to claim 6, wherein in the step (2), the centrifugal speed of the centrifuge is 100 to 5000rpm;
the length of the net-carrying centrifugal bracket is 20cm,
and the centrifugal acceleration of the carrier net during centrifugation is 1000G.
9. The method of preparing a sample by a cryoelectron microscope according to claim 6, wherein in the step (2), the measuring wavelength of the spectrometer is 200-1100nm, the resolution is less than 1nm, the spectrum acquisition time is less than 20 ms/time, the thickness of the liquid layer of the detection sample is 2nm-200 μm,
the spectrometer light source adopts a tungsten halogen lamp, and the diameter of a light spot focused on the surface of the carrier net is 1mm.
10. The method for preparing a sample by using a freeze electron microscope according to claim 6, wherein the sample is prepared by using the freeze electron microscope sample preparation device capable of detecting and controlling the thickness of a surface sample of a carrier according to any one of claims 1 to 5 in real time.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311654790.0A CN117723784A (en) | 2023-12-05 | 2023-12-05 | Freezing electron microscope sample preparation device and sample preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311654790.0A CN117723784A (en) | 2023-12-05 | 2023-12-05 | Freezing electron microscope sample preparation device and sample preparation method thereof |
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| CN117723784A true CN117723784A (en) | 2024-03-19 |
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| CN202311654790.0A Pending CN117723784A (en) | 2023-12-05 | 2023-12-05 | Freezing electron microscope sample preparation device and sample preparation method thereof |
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| CN (1) | CN117723784A (en) |
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2023
- 2023-12-05 CN CN202311654790.0A patent/CN117723784A/en active Pending
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