CN115406914A - Device and method for preparing attached frozen sample - Google Patents

Abstract

The invention relates to a device for preparing an attached frozen sample, comprising: the sample freezing unit is used for providing an environment for sample freezing operation; the cooling unit is used for storing cooling liquid and providing the cooling liquid for the sample freezing unit; the sample processing unit is arranged in the sample freezing unit and is used for carrying out atomization processing on the sample; the sample bearing unit is arranged in the frozen sample unit and corresponds to the sample processing unit, and comprises a metal substrate for containing a metal carrier net and a pipeline unit, wherein the pipeline unit is connected with the frozen sample unit and is used for implementing a cooling unit and guiding cooling liquid to the frozen sample unit from the cooling unit. The device replaces traditional input type freezing technical scheme with the attached type quick freezing technical scheme after sample solution atomization, and greatly improves the quality of frozen samples and the efficiency of the whole sample freezing process.

Description

Apparatus and method for preparing attached frozen samples

technical field

The present invention generally relates to the field of frozen sample preparation for cryo-transmission electron microscopy, in particular to a device and method for preparing an attached frozen sample.

Background technique

In recent years, thanks to the innovation of hardware and software technology, cryo-electron microscopy has become an indispensable technical means in the field of structural biology. The information limit resolution of current high-end cryo-electron microscopes has reached about 1 angstrom (such as Titan krios 300KV), but the resolution of a large number of electron microscope data hovers around 6 angstroms. The main reason is that the current frozen sample preparation technology still has a series of bottleneck problems.

The ultimate goal of frozen sample preparation is to provide ultra-thin liquid film and glassy water for cryo-electron microscopy imaging, specifically, to reduce the temperature of the sample solution film on the grid to below -160°C in a very short time, so that The liquid film turns directly into a glassy film. The commonly used frozen sample preparation method is the drop-in freezing method. The principle of this method is to attach the biomacromolecular sample in the solution state to the carrier net through a pipette gun, then use the filter paper to sandwich it to form a liquid film, and then quickly put it into liquid ethane to solidify it in a short time. Further, a glassy thin film with a thickness of about 100 nanometers is formed on the surface of the grid.

There are several main problems in using the drop-in freezing method and equipment for frozen sample preparation:

1. It is easy to cause sample particles to accumulate at the gas-liquid interface. It takes more than 10 seconds for the sample in the solution state to be completely frozen from being dropped into the grid. In this process, due to the large specific surface area of the liquid film, it is very easy to cause the biomacromolecules in the solution to be adsorbed on the gas-liquid interface, resulting in the accumulation of particles and the appearance of orientation advantages, and even partial or complete protein denaturation. , which greatly affects the imaging resolution and the accuracy of the 3D model.

2. It is easy to cause the appearance of crystalline ice. To freeze the sample, it is necessary to quickly lower the aqueous solution of the sample to 136k (-137°C) to obtain a glassy ice layer. If the glassy water is then heated to 160k (-123°C), crystallization nuclei will appear, resulting in recrystallization appear. The temperature range of liquid ethane used to freeze samples is between 89.8k (-183.3°C) and 184.5k (-88.6°C). Since the experiment process is carried out in a non-closed space, there is heat exchange between liquid ethane and the outside world. , which means that the temperature instability of liquid ethane is prone to occur during the experiment, resulting in the appearance of crystallized ice.

3. The external force affects the morphology of the ice layer of the sample. It can be seen from the above step 1 that the sample solution is dripped sideways on the grid, and due to the action of gravity, it will form a state of thin top and thick bottom, while step 2 is a strong pinching of filter paper. This operation will again exert an external force on the sample, aggravating the non-uniformity of the ice thickness of the sample. Since the transmission electron microscope has very strict requirements on the thickness of the sample ice layer, the morphology of the ice layer will seriously affect the quality of the electron microscope image.

4. Great waste of sample solution. The sample solution is very precious and scarce. There is only tens of microliters of sample solution in a frozen sample experiment, but the filter paper pinching in step 2 will take away most of the sample solution, resulting in a large amount of sample waste.

5. The influence of the external environment. The whole process of freezing samples is exposed to the air, so the external environment (temperature, humidity, dust, etc.) will have a significant impact on the results of freezing samples.

6. The operation is complicated and the efficiency is very low. One freezing process (4 to 5 minutes) can only get one sample on the grid, and the whole process requires manual intervention for many times, and the grid will be damaged and recrystallized if you are not careful.

7. Equipment and consumables are expensive. The price of a Vitrobot Mark IV frozen sample preparation instrument is 600,000 to 1,000,000 RMB, while the price of a pair of special tweezers is around 10,000 RMB.

In view of the above problems, the present invention proposes a device and method for preparing attached frozen samples, which can fundamentally improve the problems of particle denaturation and particle orientation advantages caused by the diffusion of sample particles to the gas-liquid interface, while fully ensuring sample utilization and The thickness of the glassy film is uniform, and the phenomenon of recrystallization or contamination of the sample is greatly reduced. This will provide a new method for high-resolution imaging of a large number of low- and medium-resolution samples, especially important samples of macromolecular complexes.

Contents of the invention

The purpose of this application is to replace the traditional drop-in freezing method with an attached fast freezing method, so as to greatly improve the quality of frozen samples and the efficiency of the entire freezing process.

In order to achieve the above purpose, the application proposes a device for preparing an attached frozen sample, including: a sample freezing unit, which is used to provide an environment for sample freezing operations; a cooling unit, which is used to store cooling liquid, and provide cooling liquid; a sample processing unit, arranged in the frozen sample unit, for atomizing the sample; a sample carrying unit, arranged in the frozen sample unit, and correspondingly arranged with the sample processing unit, including The metal base containing the metal grid, and the pipeline unit are connected to the freezing unit and the cooling unit, and are used to guide the cooling liquid from the cooling unit to the freezing unit.

In one example, a vacuum unit is also included, connected to the frozen sample unit, for providing a vacuum environment for the frozen sample unit.

In an example, it further includes a sample preservation unit, which is arranged in the freezing sample unit and is used to preserve the glassy sample.

In an example, the sample processing unit includes a constant temperature device and a nebulizer, and the constant temperature device provides a constant temperature environment for the nebulizer.

In one example, the metal substrate is a high thermal conductivity metal substrate.

In one example, the metal substrate is a copper substrate.

The application also provides an attached freezing method, comprising: the sample solution is placed in the sample processing unit, and the metal grid is placed on the metal substrate; the frozen sample unit is subjected to vacuum treatment through a vacuum component; the cooling liquid in the cooling unit Enter the frozen sample unit through the pipeline unit, and perform freezing treatment on the metal base, and the metal grid is connected with the metal base, and the cooled metal base transmits the low temperature to the metal grid by utilizing the thermal conductivity of the metal; start the The atomizer in the sample processing unit conducts atomization treatment on the sample solution to form liquid particles; the liquid particles are attached to the metal carrier and frozen to form a glassy thin film.

In an example, before the metal grid is subjected to freezing treatment, a constant temperature device is activated, so that the sample solution in the sample processing unit is in a constant temperature state during the metal grid freezing treatment.

In one example, the sample solution is placed in the nebulizer in the sample processing unit.

In an example, it also includes transferring the glassy thin film together with the metal grid to a sample storage unit.

Compared with the prior art, adopting the provided device and method according to the embodiment of the present application has the following beneficial effects:

1. Innovatively proposed the attachment-type freezing sample technology. Compared with the current input-type freezing sample solution, its freezing rate has been increased by an order of magnitude, which will fundamentally solve the accumulation effect of sample particles at the gas-liquid interface and improve the quality of frozen samples;

2. The design and use of sample solution atomization will further increase the freezing rate and greatly save the amount of sample solution used. In addition, the atomization of the sample can make the particle orientation in the solution disordered, ensuring that the frozen sample will not Predominant orientation of the particles appears; and the micronization of the sample solution can simultaneously ensure the uniformity of the thickness of the glassy film.

3. The design of the airtight freezing unit and the application of the vacuum operation scheme reduce the possibility of sample contamination;

4. The design and use of frozen substrates and porous substrates ensure the quality of glassy films and improve the efficiency of sample preparation.

5. The structure is simple and the cost is low; to realize the basic functions, the cost of the equipment is about 1/10 of the current high-end freezing instrument in the market; if precise temperature control, humidity control, and vacuum detection equipment are added, the cost will not exceed the cost of the current equipment 1/5;

Description of drawings

The above and other objects, features and advantages of the present application will become more apparent through a more detailed description of the embodiments of the present application in conjunction with the accompanying drawings. The accompanying drawings are used to provide a further understanding of the embodiments of the present application, and constitute a part of the specification, and are used together with the embodiments of the present application to explain the present application, and do not constitute limitations to the present application. In the drawings, the same reference numerals generally represent the same components or steps.

Fig. 1 shows a schematic diagram of a device for preparing an attached frozen sample according to an embodiment of the present application.

Fig. 2 shows a schematic cross-sectional view of a device for preparing an attached frozen sample according to an embodiment of the present application.

FIG. 3 shows a flowchart of an exemplary method for preparing an attached frozen sample according to an embodiment of the present application.

Figure 4A shows Tomography data for frozen samples of RNA phage Qbeta.

Figure 4B shows where the markers are located.

Figure 4C shows where the sample particles are located.

Fig. 5 shows the particle distribution statistical graph of ten sets of data.

Figure 6A shows a schematic diagram of single particle imaging of a ferritin sample.

Figure 6B shows a schematic diagram of the three-dimensional electron density of a ferritin sample (resolution 2.5 Angstroms).

Figure 6C shows a schematic diagram of the matching of the three-dimensional electron density of the ferritin sample with the model.

Detailed ways

Hereinafter, exemplary embodiments according to the present application will be described in detail with reference to the accompanying drawings. Apparently, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. It should be understood that the present application is not limited by the exemplary embodiments described here.

As mentioned in the background art, the common samples of cryo-electron microscopes currently adopt the drop-in freezing method, which will cause problems such as accumulation of sample particles to the vapor-liquid interface, crystallization of ice, uneven thickness of ice layer, and waste of samples. Considering the nature of liquid atomization and the extremely high freezing rate of tiny droplets on the frozen metal, the method of atomizing the sample solution can be used to freeze the sample. Therefore, this application utilizes the properties of liquid atomization and the fast Freezing characteristics, a new method of sample freezing is proposed. The inventive idea is to atomize the sample and attach it to the frozen metal for freezing, which can not only increase the freezing rate to ensure the formation of glassy water, but also make the particles oriented. Disordering avoids problems such as uneven sample thickness.

The rationale behind the method used in this application is to use metal freezing instead of liquid freezing. Studies have shown that the rate of thin film liquid changing from room temperature liquid to glass state on the frozen metal surface can reach 10 ⁹ k/s, so this process is on the order of microseconds or even nanoseconds. Preferably, red copper can be used as the material of the metal substrate in this application, because, also at -160°C, the specific heat capacity of red copper is 394J/kg·°C, and the thermal conductivity is 386.4w/(mk), while the existing design uses The specific heat capacity of liquid ethane is only 1.4726J/kg·℃, and the thermal conductivity is only 0.226w/(mk). It can be seen that if the ultra-low temperature copper is used to freeze the thin film liquid, the freezing rate will increase by an order of magnitude compared with the existing methods. This will completely solve the bottleneck problem faced by the current basal frozen sample preparation.

Secondly, in this application, the method of atomizing the sample solution is used to replace the existing method of sandwiching filter paper to produce a liquid film. Preferably, an ultrasonic nebulizer can be used as the nebulizer, for example, an ultrasonic nebulizer controlled by a power supply, with a vibration frequency of 170 KHz and an aperture of 1 mm. The 0.5-2μm liquid particles produced by the atomizer are directly sprayed onto the metal grid at the temperature of liquid nitrogen to realize rapid freezing. This method can effectively accelerate the formation of the glassy film, avoid the orientation advantage of particles, and also help to control the thickness of the film, and can also protect the carrier network, avoiding the damage of the carrier network due to external force that often occurs in the existing design Phenomenon.

Based on the above principles, the applicant has designed a device for preparing an attached frozen sample. The device of the present application will be further described below in conjunction with FIG. 1 .

As shown in Figures 1 and 2, the device 100 for preparing an attached frozen sample includes a frozen sample unit 110, a cooling unit 120, a sample processing unit 130, a sample carrying unit 140, a pipeline unit 150, a vacuum unit 160, and a sample preservation unit 170 . Wherein, the frozen sample unit 110 and the cooling unit 120 are connected through the pipeline unit 150 , the vacuum unit 160 is connected with the frozen sample unit 110 , and the sample processing unit 120 and the sample preservation unit 170 are arranged in the frozen sample unit 110 .

Specifically, the pipeline unit 150 connecting the freezing unit 110 and the cooling unit 120 includes: a pipeline 151 , and a pipeline inlet 152 and a pipeline outlet 153 arranged at both ends of the pipeline 151 . Wherein, the pipeline inlet 152 is connected with the cooling unit 120, and the pipeline outlet 153 is connected with the freezing unit 110, and the cooling liquid in the cooling unit 120 can flow through the pipeline inlet 152 through the pipeline 151 and enter the freezing unit 110 through the pipeline outlet 153, thereby The sample processing unit 120 in the freezing sample unit 110 is rapidly cooled.

In some examples, the piping unit 150 may be configured as a base plate to support the freezing unit 110 and the cooling unit 120 . As shown in accompanying drawing 2, the pipeline unit 150 is in the shape of a flat plate with a certain thickness. The frozen sample unit 110 and the cooling unit 120 are installed on one surface of the flat plate. The inside of the flat plate is provided with a pipeline 151 for liquid circulation. The thickness between them has a certain thickness, so as to slow down the heat dissipation rate when the cooling liquid flows through, for example, the thickness between them can be 3-20 cm, preferably, 5-15 cm. One end of the pipe 151 can pass through the flat wall, and a sealing cover 154 can be provided here, and when it is necessary to drain the coolant in the pipe, it can be discharged through this end. Openings may be provided on the side wall and the other end of the pipeline 151, thereby forming a pipeline inlet 152 and a pipeline outlet 153, which communicate with the cooling unit 120 and the freezing unit 110, respectively.

The cooling unit 120 connected to the pipeline inlet 152 of the pipeline unit 150 is used for storing cooling liquid, and includes: a cooling tank 121 , a cooling valve 122 , a cooling cover 123 , and a cooling rack 124 . Wherein, the cooling rack 124 is used to support the cooling tank 121. In one example, the cooling rack 124 supports the cooling tank 121 above the pipeline unit 150. It can be understood that the position of the cooling tank 121 is not limited to the top of the pipeline unit 150, for example , can also be supported on the side of the pipeline unit 150 or other positions according to operation requirements or space arrangement. The cooling tank 121 is a cavity with a certain thickness, which is convenient for isolating the temperature exchange between the cooling liquid contained therein and the outside. One end of the cooling tank 121 is provided with an opening and is equipped with a cooling cover 123, which is used for adding cooling liquid and isolating and sealing; the cooling tank 121 is also provided with another opening, which is connected with a cooling valve 122, and the other end of the cooling valve 122 is connected to the pipeline unit 150 , the cooling valve 122 can control the flow of cooling liquid from the cooling unit 120 to the piping unit 150 . The cooling liquid contained in the cooling tank 121 can be but not limited to liquid nitrogen, and any cooling solution used in the prior art can be used.

The frozen sample unit 110 connected to the pipeline outlet 153 of the pipeline unit 150 is used to provide an environment for sample operation, which includes: a chamber 111, a first cover 112 of the chamber, a second cover 113 of the chamber, a switch 114 and a chamber cover 115.

Wherein, the chamber 111 is set as a hollow cavity with an opening, and the cavity may be a cylinder as shown in the figure, or a polygonal cylinder. It is understood that the shape of the cavity may be changed as required. One end of the chamber 111 is provided with an opening provided with a chamber cover for taking and placing the sample processing unit 130 and samples. A power switch 114 may be provided on the chamber cover for conveniently controlling the activation and shutdown of the atomizer 131 and the thermostat 133 in the chamber 111 .

In some embodiments, a plurality of chamber covers may be provided according to different modes of operation. For example, as shown in FIG. The diameter of the first cover 112 is smaller than the diameter of the second cover 113 of the chamber. The first cover 112 of the chamber is stacked on the second cover 113 of the chamber. When it is necessary to enter and exit the chamber 111 in a small range, the chamber can be opened and closed. The first cover 112 can open and close the second cover 113 of the chamber when a wide range of access to and from the chamber 111 is required.

The other end of the chamber 111 is set as a base and placed on the pipe unit 150 , and the base may be provided with a through hole for communicating with the pipe outlet 153 of the cooling unit 120 to facilitate the circulation of cooling liquid. The inner side of the base can be set in a stepped shape, thereby forming bosses with different diameters, so as to facilitate the support of the sample processing unit 130. It can be understood that the setting of the opening here is not limited to the stepped shape, and any commonly used support method can be used, such as grooves , protrusions, brackets, etc.

The interior of the chamber 111 can contain a sample processing unit 120 , a sample carrying unit 140 and a sample storage unit 170 for providing a vacuum and a freezing operating environment for the sample.

In one example, a chamber cover 115 may be connected inside the chamber cover. Further, the chamber cover 115 may be connected with the second cover 113 of the chamber, so that the chamber cover 115 may be picked up at the same time when the second cover is opened. The chamber cover 115 is a hollow cavity with openings at both ends, one end is connected to the chamber cover, and the other end is connected to the sample processing unit 120 , so that the sample processing unit 120 is located above the base.

The sample processing unit 130 between the chamber cover 115 and the base includes: an atomizer 131 , an atomization cover 132 , and a thermostat 133 .

The atomizer 131 is arranged in the atomization cover 132, and the outside of the atomization cover is connected with the chamber cover 115, so that the atomizer 131 is located above the base, so as to guide the sample to atomize the substrate 141 deposited on the base.

One end of the atomization cover 132 is provided with a constant temperature device 133, and the constant temperature device 133 and the atomization cover 132 half-enclose the nebulizer 131, thereby providing a constant temperature environment for the sample in the nebulizer 131, so as to ensure that the sample droplets stay in the mist. The temperature was kept constant before melting. The other end of the atomization cover 132 is formed as a cover-shaped cavity, which covers the base 141 to further ensure that the atomized sample can be deposited on the base 141 .

A sample carrying unit 140 is correspondingly provided under the nebulization cover 132 for containing the metal grid, which includes: a base 141 and a base column 142 . The thickness of the substrate 141 is 1-20mm, preferably 5-15mm, more preferably 5-10mm. The base 141 is arranged on the base, and is pressed and fixed on the base by the base column 142 . In one example, the substrate 141 is made of a metal material, preferably copper. Using its heat conduction properties, the substrate 141 at an ultra-low temperature cools the liquid sample into a glass state at a speed of microseconds or even nanoseconds. The low temperature is maintained during the process, so that the carrier grid will not be affected by external temperature changes and heat up. The base 141 is provided with a loading hole for placing the loading net. Further, the base 141 can be provided with more than one loading hole, which is evenly or symmetrically arranged on the base 141, and a corresponding number of loading nets can be placed on each loading hole according to the number of samples that need to be cooled, so that it can be lifted Increase the efficiency of sample preparation while increasing the rate of sample freezing.

The base of the chamber 111 or the inner side wall close to the base may be provided with grooves to facilitate storage of the sample storage unit 170. It should be understood that the storage method of the sample storage unit is not limited to grooves, and bosses or brackets may also be used. After the sample is cooled to a glass state, liquid nitrogen is introduced into the chamber 111 to cover the groove, and the sample is transferred to the sample preservation unit 170 through the carrier grid, so that the entire operation process is under the protection of liquid nitrogen.

The chamber 111 can also have an opening on the side wall for connecting with the vacuum unit 160, so as to provide a vacuum environment in the chamber to reduce the possibility of sample contamination.

The vacuum unit 160 for providing a vacuum environment for the frozen sample unit 110 includes: a vacuum valve 161 and a power assembly 162 . The vacuum valve 161 can control the air pressure in the frozen sample unit 110. When the vacuum valve 161 is opened, the frozen sample unit 110 is connected to the power assembly 162, and the gas in the chamber 111 is started to be drawn after the power assembly 162 is turned on. When the air pressure in the chamber 111 is After 0, close the vacuum valve 161 and the power assembly 162, so that the inside of the chamber 111 is in a vacuum state.

It can be seen that the design of the freezing device 100 of the present application uses a metal base to freeze the sample, which is obviously better than the existing design that uses liquid ethane to cool the sample, and combined with the nebulizer to carry out atomization pretreatment on the sample, so that the sample can be frozen in the air. The film is rapidly cooled in a vacuum environment, which not only greatly increases the freezing rate, but also ensures the uniformity of the thickness of the sample layer after freezing. At the same time, the structure is exquisite, which is convenient for large-scale production and processing.

In combination with the above description of the principles and devices, the freezing method of the present application will be further elaborated below through specific examples.

Fig. 3 shows a flow chart of an exemplary method for the attached frozen sample preparation according to an embodiment of the present application. As shown in Figure 3, the exemplary method may include the following steps:

S210, preparation of samples and loading nets.

The sample solutions that can be used for freezing in this application include but are not limited to biomacromolecule solutions. Suitable metal grids can be selected according to different sample solutions, such as Quantifoli 200-mesh pure carbon support membrane gold grid with a pore size of 1.2 μm and a period of 1.3 μm. In some embodiments, a copper grid with good thermal conductivity can also be selected.

Place the selected copper grid on the metal substrate 141 in the sample processing unit 130 of the frozen sample unit, and then drop about 10 ul of the sample solution into the atomizer 131 in the sample processing unit 130 . In some embodiments, the metal base 141 can be a copper base with a height of 6 mm, a diameter of 16 mm, and 8 loading holes. Utilizing the high specific heat capacity and thermal conductivity of the copper substrate, the sample solution can be frozen on the surface of the grid at a rate of microseconds or even nanoseconds, which increases the freezing rate by an order of magnitude and ensures the formation of glassy water.

S220, performing vacuum treatment on the frozen sample unit.

After the sample solution is placed, close the chamber cover of the frozen sample unit 110 to keep the chamber in a sealed state, open the vacuum valve 161, start the power unit 162, and vacuum the frozen sample unit 110. In some cases, the extraction time is 10 -80s, preferably 30-60s. Stop pumping when the indoor air pressure drops to -0.08mpa. The above operation plan of sealing the freezing sample unit 110 and performing negative pressure treatment can reduce the possibility of sample contamination.

S230, performing freezing treatment on the metal carrier grid.

After the frozen sample unit 110 is in a vacuum state, the metal substrate 141 is cooled using cooling technology. Specifically, the thermostatic device 133 is turned on so that the sample solution on the atomizer 131 is in a constant temperature environment to ensure that the sample solution will not be frozen during the cooling process of the metal substrate; The liquid nitrogen in 120 is introduced into the freezing unit 110, so that the liquid nitrogen passes through the through hole of the base and passes through the metal base 141, thereby cooling the metal base 141. Since the metal grid is placed on the metal base 141, with the good thermal conductivity of the metal, the cooled metal base 141 transmits the low temperature to the metal grid, and the metal grid is cooled at the same time. When the temperature of the metal base 141 and the metal grid drops When the temperature of liquid nitrogen is reached (that is, the liquid nitrogen is no longer boiling violently), the addition of liquid nitrogen is stopped, and this process lasts for 30-60 seconds.

S240, atomizing the sample solution.

The atomizer 131 is started, the sample solution on the atomizer 131 is atomized into liquid particles with a diameter of 0.5-2 μm, and the liquid particles are directly sprayed onto the metal grid that has been lowered to the base of the liquid nitrogen temperature. The design and use of sample solution atomization help to increase the rate of glassy film formation, avoid the generation of sample particle orientation fixation, and help control the thickness of the sample film, and because the step of filter paper clamping in the prior art is omitted, It will greatly improve the utilization rate of the sample and reduce the damage of the carrier network caused by external force.

S250, Rapid attachment freezing of aerosolized particles.

The liquid film sprayed onto the base carrier net adheres to the ultra-low temperature metal substrate 141, and achieves rapid freezing at a rate of microseconds or even nanoseconds, thereby forming a glassy film (~100nm) on the surface of the carrier net, avoiding slow cooling. resulting in crystallized ice. In some examples, the formation rate of the glassy film can theoretically reach a rate of >10 ⁷ k/s, or even >10 ⁸ k/s.

This method innovatively proposes an attached freezing sample technology. Compared with the current drop-in freezing sample solution, its freezing rate is an order of magnitude higher, which will fundamentally solve the accumulation effect of sample particles at the gas-liquid interface. Improve the quality of frozen samples.

S260, the liquid nitrogen protection of the frozen sample in the sample freezing unit 110.

After the atomized sample is rapidly frozen on the metal grid to form a glassy film (5s-10s), liquid nitrogen is fed into the freezing unit 110 again, for example, the liquid level of the liquid nitrogen can be 3-5cm higher than the grid, Make sure that the liquid nitrogen is submerged in the sample holding unit 140, and then transfer the metal grid from the metal base 141 to the sample storage unit 170 under the protection of liquid nitrogen to complete the sample freezing operation.

Based on the above devices and methods, the following embodiments can be implemented:

Example 1

Step 1: Select a copper grid with an aperture according to the particle size of the sample solution, open the first cover 112 of the chamber, take out the sample processing unit 120, place the copper grid on the metal base 141 of the sample carrying unit 140, and use The base column 142 presses and fixes the grid and the base 141;

Step 2: Put the sample processing unit 120 into the chamber 111, take out the thermostatic device 133, expose the nebulizer 131, drop 10 microliters of the sample solution of light-harvesting protein on the nebulizer 131, and then put it back into the thermostatic device 133 ;

Step 3: Close the first cover 112 of the chamber, open the vacuum valve 161, and turn on the power assembly 162, vacuumize the chamber 111 for 30 seconds, and close the vacuum valve 151 and the power assembly when the chamber 111 reaches a vacuum state;

Step 4: Turn on the switch 114 on the chamber cover, start the thermostat 133, so that the sample solution on the nebulizer 131 is kept at a constant temperature, for example, 20°C, to ensure that the subsequent atomization is not affected; after that, open the cooling valve 150 , so that the liquid nitrogen in the cooling tank 121 flows into the chamber 111 from the pipeline inlet 152 through the pipeline 151 through the pipeline outlet 153 through the through hole of the base of the chamber 111, so that the liquid nitrogen does not pass through the metal base 141, and the cooling valve 150 is closed. Use liquid nitrogen to freeze for about 30 seconds until both the metal base 141 and the copper grid have the temperature of liquid nitrogen;

Step 5: Start the atomizer 131 to make the sample solution become liquid particles. Since the atomizer 131 and the copper grid are on the same axis, and the atomization cover 132 is added, the atomized sample particles can be directly attached to the Copper grid;

Step 6: Wait for 10 seconds after the sample solution is attached to the copper grid, so that the liquid particles are rapidly frozen on the copper grid with liquid nitrogen temperature to form a glassy film;

Step 7: After the glassy sample film is formed, feed liquid nitrogen into the frozen sample unit 110 again to make it submerge the copper grid by 4 cm, and then open the second cover 113 of the chamber. The chamber cover 115 connected with 113 and the sample processing unit 120 connected thereto are removed together, exposing the sample carrying unit 140 submerged in liquid nitrogen, and the copper carrier grid together with the frozen light-harvesting protein is placed under the protection of liquid nitrogen. The samples are transferred together from the metal base 141 to the sample storage unit 170 in the chamber 111 to complete the sample freezing operation.

Example 2

In order to verify the practicability of the above-mentioned device and method, the applicant used the above-mentioned device and method to conduct a sample freezing test (the test sample was RNA phage Qbeta): the frozen sample was used for the collection of Tomography data, as shown in Figure A4; The software IMOD is displayed to display the position information of the sample particles in the glassy thin film, as shown in Figure 4B and Figure 4C. Among them, Figure 4A is the overall situation of data collection, Figure 4B is the contamination on the sample, which can be used to mark the surface position of the glassy film, and Figure 4C is the display of the sample particles on the XY plane and on the XZ plane. It can be clearly seen from the figure that the frozen particles are distributed in the middle of the glassy film, and there is no phenomenon of aggregation to the gas-liquid interface.

Further, using the above test method, we counted the test results of ten sets of RNA bacteriophage samples, and used the three curves in the table of Figure 5 to represent the percentage of particles distributed in contact with the upper surface of the film, and the particles distributed in the middle of the glassy film. The percentage of the position, the percentage of the particle distribution in contact with the lower surface of the film. It can be seen that in the glassy film obtained by the device and method of the present application, most of the biomacromolecule particles are distributed in the middle of the film.

In addition, referring to the schematic diagrams of single particle imaging, 3D electron density, and matching of 3D electron density and model shown in Figure 6A-6C, we also used ferritin samples for frozen sample preparation and subsequent data collection and analysis. Structural analysis yielded an electron density of 2.5 angstroms. It can be seen that the three-dimensional electron density with ideal resolution can be obtained by using the device and method of the present application, and a high-quality frozen sample can be obtained.

To sum up, it can be seen that the technical means adopted in this application have the following advantages: 1. The attached freezing sample technology is innovatively proposed. It will fundamentally ensure the formation of glassy water film, solve the accumulation effect of sample particles at the gas-liquid interface and improve the quality of frozen samples; 2. The design and use of sample solution atomization will further increase the freezing rate and greatly save The amount of sample solution used. In addition, the atomization of the sample can make the orientation of the particles in the solution disordered, ensuring that the frozen samples will not have the dominant orientation of the particles; 3. The design of the airtight frozen sample unit and the application of the vacuum operation scheme reduce the possibility of sample contamination. Possibility; Fourth, the design and use of frozen metal substrates and porous substrates greatly improves the efficiency of sample preparation while greatly increasing the freezing rate. 5. The structure is simple and the cost is low. To realize the basic functions, the cost of the equipment is about 1/10 of the current high-end freezing instrument in the market. If precise temperature control, humidity control, and vacuum detection equipment are added, the cost will not exceed the current equipment. 1/5.

The specific implementation manners described above have further described the purpose, technical solutions and beneficial effects of the application in detail. It should be understood that the above descriptions are only specific implementation modes of the application and are not intended to limit the scope of the application. Scope of protection: All modifications, equivalent replacements, improvements, etc. made within the spirit and principles of this application shall be included within the scope of protection of this application.

Claims (10)

1. A device for preparing an attached frozen sample, comprising: The sample freezing unit is used to provide an environment for sample freezing operations; cooling unit, used to store cooling liquid, and provide cooling liquid for the frozen sample unit; A sample processing unit, set in the frozen sample unit, for atomizing the sample; The sample carrying unit is arranged in the frozen sample unit and is arranged corresponding to the sample processing unit, including a metal base for holding a metal grid, and The pipeline unit connects the sample freezing unit and the cooling unit, and is used to guide cooling liquid from the cooling unit to the sample freezing unit. 2. The device according to claim 1, further comprising a vacuum unit connected to the frozen sample unit for providing a vacuum environment for the frozen sample unit. 3. The device according to claim 1, further comprising a sample preservation unit, arranged in the freezing sample unit, for preserving the glassy sample. 4. The device according to claim 1, wherein the sample processing unit comprises a constant temperature device and a nebulizer, and the constant temperature device provides a constant temperature environment for the nebulizer. 5. The device of claim 1, wherein the metal substrate is a high thermal conductivity metal substrate. 6. The device of claim 5, wherein the metal substrate is a copper substrate. 7. An attached freezing method, comprising: The sample solution is placed in the sample processing unit, and the metal grid is placed on the metal substrate; Vacuum the frozen sample unit through the vacuum assembly; The cooling liquid in the cooling unit enters the freezing unit through the pipeline unit, and the metal substrate is subjected to freezing treatment. The metal grid is connected to the metal substrate, and the cooled metal substrate transfers the low temperature to the metal substrate by utilizing the thermal conductivity of the metal. carrying network; Start the atomizer in the sample processing unit to atomize the sample solution to form liquid particles; The liquid particles are attached to the metal grid for freezing to form a glassy thin film. 8. The method according to claim 7, wherein, before the metal grid is subjected to freezing treatment, a constant temperature device is activated so that the sample solution in the sample processing unit is at Constant temperature state. 9. The method of claim 7, wherein the sample solution is placed within the nebulizer within the sample processing unit. 10. The method of claim 7, further comprising transferring the glassy thin film together with the metal grid to a sample holding unit.

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