Device and method for preparing attached frozen samples

Abstract

The present invention relates to a device for preparing attached frozen samples, comprising: a freezing sample unit for providing an environment for sample freezing operation; a cooling unit for storing cooling liquid and providing cooling liquid for the freezing sample unit; a sample processing unit arranged in the freezing sample unit for atomizing the sample; a sample carrying unit arranged in the freezing sample unit and corresponding to the sample processing unit, comprising a metal base for holding a metal carrier mesh, and a pipeline unit connecting the freezing sample unit and the cooling unit for guiding the cooling liquid from the cooling unit to the freezing sample unit. The device replaces the traditional immersion freezing technology with an attached rapid freezing technology after the sample solution is atomized, greatly improving the quality of the frozen sample and the efficiency of the entire freezing process.

Description

Technical Field

[0001] The present invention generally relates to the field of frozen sample preparation for cryo-transmission electron microscopy, and in particular to a device and method for preparing attached frozen samples. Background Art

[0002] 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 amount of electron microscopy data hovers around 6 angstroms. The main reason is that the current cryo-sample preparation technology still has a series of bottleneck problems.

[0003] The ultimate goal of frozen sample preparation is to provide ultra-thin liquid film and glassy water for cryo-EM imaging. Specifically, the temperature of the sample solution film on the grid is reduced to below -160°C in a very short time, so that the liquid film directly becomes a glassy film. The commonly used frozen sample preparation method is the immersion freezing method. The principle of this method is to adhere the biomacromolecule sample in solution to the grid through a pipette, use filter paper to clamp it to form a liquid film, and then quickly put it into liquid ethane to solidify it in a short time, thereby forming a glassy film with a thickness of about 100 nanometers on the surface of the grid.

[0004] There are several major problems with using immersion freezing methods and equipment for frozen sample preparation:

[0005] 1. It is easy to cause sample particles to accumulate at the gas-liquid interface. It takes more than 10 seconds for a sample in a 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 emergence of orientation advantages, or even partial or complete protein denaturation, which greatly affects the imaging resolution and the accuracy of the three-dimensional model.

[0006] 2. It is easy to cause the appearance of crystalline ice. To freeze the sample, the aqueous solution of the sample needs to be quickly lowered to 136K (-137℃) to obtain a glassy ice layer. If the glassy water is then heated to 160K (-123℃), crystal nuclei will appear, leading to the appearance of recrystallization. The temperature range of the liquid ethane used to freeze the sample is between 89.8K (-183.3℃) and 184.5K (-88.6℃). Since the experiment is carried out in a non-confined space, there is heat exchange between the liquid ethane and the outside world, which means that the temperature of the liquid ethane is prone to instability during the experiment, leading to the appearance of crystalline ice.

[0007] 3. External force affects the morphology of the sample ice layer. As can be seen from step 1 above, the sample solution is dripped sideways onto the grid. Due to gravity, it will form a thin top and thick bottom state, while step 2 is the strong clamping of the filter paper. This operation will again exert external force on the sample, exacerbating the uneven thickness of the sample ice layer. 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.

[0008] 4. Huge waste of sample solution. Sample solution is very precious and rare. There are only a few dozen microliters of sample solution in a frozen sample experiment, but the filter paper clamping in step 2 will take away most of the sample solution, resulting in a large amount of sample waste.

[0009] 5. Influence of the external environment. The entire freezing process is carried out in the air, so the external environment (temperature, humidity, dust, etc.) will have a significant impact on the freezing results.

[0010] 6. The operation is complicated and the efficiency is very low. Only one grid sample can be obtained in one freezing process (4 to 5 minutes), and the whole process requires multiple manual interventions. If you are not careful, the grid will be damaged and recrystallized.

[0011] 7. Equipment and consumables are expensive. The price of a Vitrobot Mark IV cryo-sample preparation instrument is between RMB 600,000 and RMB 1,000,000, while a pair of special tweezers costs around RMB 10,000.

[0012] 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 advantage caused by the diffusion of sample particles to the gas-liquid interface, while fully ensuring the utilization rate of the sample and the uniformity of the thickness of the glassy film, and greatly reducing the phenomenon of sample recrystallization or contamination. This will provide a new method for high-resolution imaging of a large number of low- and medium-resolution samples, especially important macromolecular complex samples. Summary of the invention

[0013] The purpose of the present application is to replace the traditional immersion freezing method with an attached rapid freezing method, thereby greatly improving the quality of frozen samples and the efficiency of the entire freezing process.

[0014] To achieve the above-mentioned purpose, the present application proposes a device for preparing attached frozen samples, including: a freezing sample unit, used to provide an environment for sample freezing operation; a cooling unit, used to store coolant and provide coolant to the freezing sample unit; a sample processing unit, arranged in the freezing sample unit, for atomizing the sample; a sample carrying unit, arranged in the freezing sample unit and corresponding to the sample processing unit, including a metal base for holding a metal carrier mesh, and a pipeline unit, connecting the freezing sample unit and the cooling unit, for guiding the coolant from the cooling unit to the freezing sample unit.

[0015] In one example, a vacuum unit is further included, which is connected to the freezing sample unit and is used to provide a vacuum environment for the freezing sample unit.

[0016] In one example, a sample preservation unit is further included, which is disposed in the freezing sample unit and is used to preserve the sample in a vitrified state.

[0017] In one 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.

[0018] In one example, the metal substrate is a high thermal conductivity metal substrate.

[0019] In one example, the metal substrate is a copper substrate.

[0020] The present application also provides an attachment freezing method, comprising: placing a sample solution in a sample processing unit, placing a metal carrier mesh on a metal substrate; performing a vacuum treatment on the frozen sample unit through a vacuum component; the cooling liquid in the cooling unit enters the frozen sample unit through a pipeline unit to perform a freezing treatment on the metal substrate, the metal carrier mesh is connected to the metal substrate, and the cooled metal substrate transfers the low temperature to the metal carrier mesh by utilizing the thermal conductivity of the metal; starting the atomizer in the sample processing unit to perform atomization treatment on the sample solution to form liquid particles; the liquid particles are attached to the metal carrier mesh to be frozen to form a glassy film.

[0021] In one example, before freezing the metal grid, a constant temperature device is started so that the sample solution in the sample processing unit is kept at a constant temperature during the freezing process of the metal grid.

[0022] In one example, the sample solution is placed in the nebulizer in the sample processing unit.

[0023] In one example, the method further includes transferring the glass film together with the metal grid to a sample holding unit.

[0024] Compared with the prior art, the device and method provided according to the embodiments of the present application have the following beneficial effects:

[0025] First, the innovative attached sample freezing technology was proposed. Compared with the current immersion sample freezing scheme, its freezing rate is 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;

[0026] 2. The design and use of sample solution atomization will further increase the freezing rate and significantly save the amount of sample solution used. In addition, sample atomization can disorder the orientation of particles in the solution, ensuring that there will be no dominant orientation of particles in the frozen sample; and the micronization of the sample solution can simultaneously ensure the uniformity of the thickness of the glassy film.

[0027] 3. The design of the closed freezing unit and the application of the vacuum operation scheme reduce the possibility of sample contamination;

[0028] 4. Design and use of frozen substrates and porous substrates to ensure the quality of glassy films while improving the efficiency of sample preparation.

[0029] 5. Simple structure and low cost. The cost of the equipment is about 1 / 10 of the high-end freezing sample instrument on the market. If precise temperature control, humidity control and vacuum detection equipment are added, the cost will not exceed 1 / 5 of the current equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] By describing the embodiments of the present application in more detail in conjunction with the accompanying drawings, the above and other purposes, features and advantages of the present application will become more apparent. The accompanying drawings are used to provide a further understanding of the embodiments of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the present application and do not constitute a limitation of the present application. In the accompanying drawings, the same reference numerals generally represent the same components or steps.

[0031] Figure 1 A schematic diagram of a device for preparing attached frozen samples according to an embodiment of the present application is shown.

[0032] Figure 2 A cross-sectional schematic diagram of a device for preparing attached frozen samples according to an embodiment of the present application is shown.

[0033] Figure 3 A flow chart of an exemplary method for preparing an attached frozen sample according to an embodiment of the present application is shown.

[0034] Figure 4A Tomography data of a frozen sample of RNA bacteriophage Qbeta are shown.

[0035] Figure 4B The locations of the markers are shown.

[0036] Figure 4C The locations of the sample particles are shown.

[0037] Figure 5 Particle distribution statistics for ten sets of data are shown.

[0038] Fig. 6A A schematic diagram of single particle imaging of a ferritin sample is shown.

[0039] Figure 6B A three-dimensional electron density diagram of a ferritin sample is shown (resolution 2.5 angstroms).

[0040] Figure 6C A schematic diagram showing the matching of the three-dimensional electron density of the ferritin sample with the model. DETAILED DESCRIPTION

[0041] Below, the exemplary embodiments according to the present application will be described in detail with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application, and it should be understood that the present application is not limited to the exemplary embodiments described here.

[0042] As described in the background technology, the common samples of cryo-electron microscopes currently adopt the immersion freezing method, which will cause problems such as sample particles to accumulate at the vapor-liquid interface, the appearance of crystallized ice, uneven ice thickness, and waste of samples. Considering the properties of liquid atomization and the extremely high freezing rate of tiny droplets on frozen metals, the sample solution can be atomized to freeze the sample. Therefore, this application utilizes the properties of liquid atomization and the rapid freezing characteristics of metals to propose a new sample freezing method. The inventive concept is to atomize the sample and attach it to the frozen metal for freezing. This can not only increase the freezing rate to ensure the formation of glassy water, but also make the particles have disordered orientation, avoiding problems such as uneven sample thickness.

[0043] The basic principle of the method used in this application is to use metal freezing instead of liquid freezing. Studies have shown that the rate at which a thin film liquid changes from a room temperature liquid to a glassy state on a frozen metal surface can reach 10 9k / s, so this process is in the microsecond or even nanosecond level. Preferably, copper can be used as the material of the metal substrate in this application, because, at -160°C, the specific heat capacity of copper is 394J / kg·℃, and the thermal conductivity is 386.4w / (mk), while the specific heat capacity of liquid ethane used in the existing design is only 1.4726J / kg·℃, and the thermal conductivity is only 0.226w / (mk). It can be seen that if ultra-low temperature copper is used to freeze the thin film liquid, its freezing rate will be an order of magnitude higher than the existing means, which will completely solve the bottleneck problem faced by the current preparation of frozen substrate samples.

[0044] Secondly, the present application uses a method of atomizing the sample solution to replace the existing filter paper clamping method to produce a liquid film. Preferably, an ultrasonic atomizer can be used as the atomizer. For example, an ultrasonic atomizer controlled by a power supply, with a vibration frequency of 170KHz and an aperture of 1mm is selected. The 0.5-2μm liquid particles generated by the atomizer are directly sprayed onto a metal carrier mesh at liquid nitrogen temperature to achieve rapid freezing. This method can effectively accelerate the formation of a glassy film and avoid the orientation advantage of the particles. It also helps to control the thickness of the film, and it can also protect the carrier mesh and avoid the phenomenon of damage to the carrier mesh due to external forces that often occurs in existing designs.

[0045] Based on the above principle, the applicant has designed a device for preparing attached frozen samples. Figure 1 The device of the present application is further explained.

[0046] like Figure 1 and Figure 2 As shown, the device 100 for preparing an attached frozen sample includes a freezing 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. The freezing sample unit 110 and the cooling unit 120 are connected through the pipeline unit 150, the vacuum unit 160 is connected to the freezing sample unit 110, and the sample processing unit 120 and the sample preservation unit 170 are arranged in the freezing sample unit 110.

[0047] Specifically, the pipeline unit 150 connecting the freezing sample 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. The pipeline inlet 152 is connected to the cooling unit 120, and the pipeline outlet 153 is connected to the freezing sample unit 110. The cooling liquid in the cooling unit 120 can flow through the pipeline inlet 152, through the pipeline 151, and through the pipeline outlet 153 into the freezing sample unit 110, so that the sample processing unit 120 in the freezing sample unit 110 can be quickly cooled.

[0048] In some examples, the pipe unit 150 may be configured as a substrate to support the sample freezing unit 110 and the cooling unit 120. Figure 2 As shown, the pipe unit 150 is in the shape of a plate with a certain thickness, and the freezing sample unit 110 and the cooling unit 120 are installed on one surface of the plate. A pipe 151 for liquid circulation is provided inside the plate. The thickness between the wall of the pipe 151 and the surface of the plate has a certain thickness, so as to slow down the heat dissipation speed when the coolant 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 plate wall, where a sealing cover 154 can be provided. When the coolant in the pipe needs to be discharged, it can be discharged through this end. Openings can be provided on the side wall and the other end of the pipe 151, so as to form a pipe inlet 152 and a pipe outlet 153, which are respectively connected to the cooling unit 120 and the freezing sample unit 110.

[0049] The cooling unit 120 connected to the pipeline inlet 152 of the pipeline unit 150 is used to store coolant, and includes: a cooling tank 121, a cooling valve 122, a cooling cover 123, and a cooling rack 124. Among them, 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, it can also be supported on the side or other positions of the pipeline unit 150 according to operational needs or spatial arrangement. The cooling tank 121 is a cavity with a certain thickness, which is convenient for isolating the temperature exchange of the coolant contained therein with the outside world. The cooling tank 121 is provided with an opening at one end and is equipped with a cooling cover 123 for adding coolant and isolating and sealing; the cooling tank 121 is also provided with another opening, which is connected to the 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 coolant from the cooling unit 120 to the pipeline unit 150. The cooling liquid contained in the cooling tank 121 may be, but is not limited to, liquid nitrogen, and any refrigeration solution used in the prior art may be used.

[0050] The freezing sample unit 110 connected to the pipeline outlet 153 of the pipeline unit 150 is used to provide an environment for sample operation, and includes: a chamber 111 , a first chamber cover 112 , a second chamber cover 113 , a switch 114 and a chamber cover 115 .

[0051] The chamber 111 is configured as a hollow cavity with an opening. The cavity may be cylindrical as shown in the figure, or may be a polygonal column. It is understood that the shape of the cavity may be changed as needed. One end of the chamber 111 is configured as an opening with a chamber cover for taking and placing the sample processing unit 130 and the sample. The chamber cover may be provided with a power switch 114 for conveniently controlling the start and stop of the atomizer 131 and the thermostat 133 in the chamber 111.

[0052] In some embodiments, multiple chamber covers may be provided according to different operation modes, for example, Figure 1 As shown, the chamber cover is divided into a first chamber cover 112 and a second chamber cover 113. The diameter of the first chamber cover 112 is smaller than the diameter of the second chamber cover 113. The first chamber cover 112 is stacked on the second chamber cover 113. When a small range of entry and exit of the chamber 111 is required, the first chamber cover 112 can be opened and closed. When a large range of entry and exit of the chamber 111 is required, the second chamber cover 113 can be opened and closed.

[0053] The other end of the chamber 111 is set as a base and placed on the pipeline unit 150. The base can be provided with a through hole for communicating with the pipeline outlet 153 of the cooling unit 120 to facilitate the circulation of the cooling liquid. The inner side of the base can be set as a step to form bosses of 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 step, and any commonly used support method can be adopted, such as a groove, a protrusion, a bracket, etc.

[0054] The chamber 111 may contain a sample processing unit 120 , a sample carrying unit 140 and a sample preservation unit 170 to provide a vacuum and freezing operating environment for the sample.

[0055] In one example, a chamber cover 115 may be connected inside the chamber cover, and further, the chamber cover 115 may be connected to the second chamber cover 113, so that the chamber cover 115 can be lifted up at the same time when the second cover is opened. The chamber cover 115 is a hollow cavity with two ends open, one end of which is connected to the chamber cover, and the other end of which is connected to the sample processing unit 120, so that the sample processing unit 120 is above the base.

[0056] The sample processing unit 130 located between the chamber sleeve 115 and the base includes a nebulizer 131 , a nebulizer cover 132 , and a constant temperature device 133 .

[0057] The nebulizer 131 is disposed in the nebulizer cover 132 , and the outside of the nebulizer cover is connected to the chamber cover 115 , so that the nebulizer 131 is located above the base, which is convenient for guiding the sample to be atomized and deposited on the substrate 141 on the base.

[0058] A thermostat 133 is provided at one end of the atomizing hood 132, and the thermostat 133 and the atomizing hood 132 half-enclose the atomizer 131, thereby providing a constant temperature environment for the sample in the atomizer 131, so as to ensure that the temperature of the sample droplets remains constant before atomization. The other end of the atomizing hood 132 is formed into a hood-shaped cavity, which covers the substrate 141, further ensuring that the atomized sample can be deposited on the substrate 141.

[0059] A sample carrying unit 140 is provided correspondingly below the atomizing hood 132 for holding a metal carrier mesh, which includes: a base 141 and a base column 142. The thickness of the base 141 is 1-20 mm, preferably 5-15 mm, and more preferably 5-10 mm. The base 141 is arranged on the base and is pressed on the base by the base column 142. In one example, the base 141 is made of metal, preferably copper, and its heat conduction characteristics are used to make the base 141 at an ultra-low temperature cool the sample liquid to a glass state at a speed of microseconds or even nanoseconds, while maintaining a low temperature state during the experiment, so that the carrier mesh will not be affected by the change of external temperature and heat up. A loading hole is provided on the base 141 for placing the carrier mesh. Furthermore, 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 carrier meshes can be placed on each loading hole according to the number of samples to be cooled, so that the efficiency of sample preparation can be improved while increasing the sample freezing rate.

[0060] The base of the chamber 111 or the inner side wall near the base may be provided with a groove to facilitate the storage of the sample preservation unit 170. It should be understood that the storage method of the sample preservation unit is not limited to the groove, and a boss or a bracket may also be used. When the sample is cooled to a glassy state, liquid nitrogen is introduced into the chamber 111 until the groove is covered, and the sample is transferred to the sample preservation unit 170 through the carrier grid, so that the entire operation process is protected by liquid nitrogen.

[0061] The chamber 111 may also be provided with an opening on a 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.

[0062] The vacuum unit 160 for providing a vacuum environment for the freezing sample unit 110 includes: a vacuum valve 161 and a power assembly 162. The vacuum valve 161 can control the air pressure in the freezing sample unit 110. When the vacuum valve 161 is opened, the freezing sample unit 110 is connected to the power assembly 162. After the power assembly 162 is turned on, the gas in the chamber 111 begins to be extracted. When the air pressure in the chamber 111 is 0, the vacuum valve 161 and the power assembly 162 are closed, so that the chamber 111 is in a vacuum state.

[0063] 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 significantly better than the existing design that uses liquid ethane to cool the sample. Combined with the nebulizer to perform atomization pretreatment on the sample, the sample can be quickly cooled into a film in a vacuum environment, which not only greatly improves the freezing rate, but also ensures the uniformity of the thickness of the sample layer after freezing. At the same time, the structure is sophisticated and convenient for large-scale production and processing.

[0064] In combination with the description of the above principles and devices, the freezing method of the present application is further described below through specific embodiments.

[0065] Figure 3 FIG. 1 shows an exemplary method flow chart of an attached frozen sample preparation according to an embodiment of the present application. Figure 3 As shown, the exemplary method may include the following steps:

[0066] S210, preparation of samples and grids.

[0067] The sample solutions that can be used for freezing in the present application include but are not limited to biomacromolecule solutions. A suitable metal grid can be selected according to the different sample solutions, such as a Quantifoli 200 mesh pure carbon support film 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.

[0068] The selected copper grid is placed on the metal substrate 141 in the sample processing unit 130 of the freezing unit, and then about 10ul of the sample solution is dripped onto the atomizer 131 in the sample processing unit 130. In some embodiments, the metal substrate 141 can be a copper substrate with a height of 6mm, a diameter of 16mm, and 8 loading holes. The high specific heat capacity and thermal conductivity of the copper substrate allow the sample solution to be frozen on the grid surface at a rate of microseconds or even nanoseconds, so that the freezing rate is increased by an order of magnitude, ensuring the formation of glassy water.

[0069] S220, performing vacuum treatment on the frozen sample unit.

[0070] After the sample solution is placed, the chamber cover of the frozen sample unit 110 is closed to keep the chamber sealed, the vacuum valve 161 is opened, the power assembly 162 is started, and the frozen sample unit 110 is vacuum-extracted. In some embodiments, the extraction time is 10-80 seconds, preferably 30-60 seconds. When the air pressure in the chamber drops to -0.08 MPa, the extraction is stopped. The above-mentioned operation scheme of sealing the frozen sample unit 110 and performing negative pressure treatment can reduce the possibility of sample contamination.

[0071] S230, performing freezing treatment on the metal grid.

[0072] After the frozen sample unit 110 is in a vacuum state, the metal substrate 141 is cooled using a cooling technique. Specifically, the thermostat 133 is turned on to keep the sample solution on the atomizer 131 in a constant temperature environment to ensure that the sample solution will not be frozen during the cooling process of the metal substrate; then, the cooling valve 122 is opened, and the liquid nitrogen in the cooling unit 120 is introduced into the frozen sample unit 110 through the pipeline unit 150, so that the liquid nitrogen passes through the through hole of the base and covers the metal substrate 141, thereby cooling the metal substrate 141. Since the metal grid is placed on the metal substrate 141, with the help of the good thermal conductivity of the metal, the cooled metal substrate 141 transfers the low temperature to the metal grid, and the metal grid is cooled at the same time. When the temperature of the metal substrate 141 and the metal grid drops to the liquid nitrogen temperature (that is, the liquid nitrogen no longer boils violently), the addition of liquid nitrogen is stopped. This process lasts for 30 to 60 seconds.

[0073] S240, atomizing the sample solution.

[0074] The atomizer 131 is started to atomize the sample solution on the atomizer 131 into liquid particles with a diameter of 0.5 to 2 μm, and the liquid particles are directly sprayed onto the metal carrier mesh whose base has been cooled to the temperature of liquid nitrogen. The design and use of the sample solution atomization helps to increase the rate of glassy film formation, avoid the generation of sample particle orientation, and is also conducive to controlling the thickness of the sample film. In addition, since the filter paper clamping step in the prior art is omitted, the utilization rate of the sample will be greatly improved, and the damage of the carrier mesh caused by external force will be reduced.

[0075] S250, rapid attachment freezing of atomized particles.

[0076] The liquid film sprayed onto the substrate grid adheres to the ultra-low temperature metal substrate 141 and is rapidly frozen at a rate of microseconds or even nanoseconds, thereby forming a glassy film (~100nm) on the surface of the grid, avoiding the crystallization of ice caused by slow cooling. In some examples, the rate of glassy film formation can theoretically reach >10 7 k / s rate, even >10 8 k / s.

[0077] This method innovatively proposes an attached sample freezing technology. Compared with the current immersion sample freezing scheme, its freezing rate is increased by an order of magnitude. This will fundamentally solve the stacking effect of sample particles at the gas-liquid interface and improve the quality of frozen samples.

[0078] S260, the sample freezing unit 110 protects the frozen sample with liquid nitrogen.

[0079] After the atomized sample is rapidly frozen on the metal grid to form a glassy film (5s to 10s), liquid nitrogen is introduced into the freezing sample unit 110 again. For example, the liquid nitrogen level can be made 3 to 5 cm higher than the grid to ensure that the liquid nitrogen covers the sample supporting unit 140. Then, under the protection of liquid nitrogen, the metal grid is transferred from the metal base 141 to the sample preservation unit 170 to complete the freezing sample operation.

[0080] Based on the above device and method, the following embodiments can be implemented:

[0081] Example 1

[0082] Step 1: Select a copper grid with a pore size according to the particle size of the sample solution, open the first chamber cover 112, take out the sample processing unit 120, place the copper grid on the metal base 141 of the sample carrying unit 140, and press the grid and the base 141 with the base column 142;

[0083] Step 2: Place the sample processing unit 120 into the chamber 111, take out the thermostat 133, expose the nebulizer 131, drip 10 μl of the light-harvesting protein sample solution onto the nebulizer 131, and then put it back into the thermostat 133;

[0084] Step 3: Close the first chamber cover 112, open the vacuum valve 161, and start the power assembly 162 to evacuate the chamber 111 for 30 seconds. When the chamber 111 reaches a vacuum state, close the vacuum valve 151 and the power assembly;

[0085] Step 4: Turn on the switch 114 on the chamber cover and start the thermostat 133 to keep the sample solution on the nebulizer 131 at a constant temperature, for example, 20°C, to ensure that the subsequent atomization is not affected; then, open the cooling valve 150 to allow the liquid nitrogen in the cooling tank 121 to flow from the pipeline inlet 152 through the pipeline 151 and the pipeline outlet 153 through the through hole of the base of the chamber 111 into the chamber 111, so that the liquid nitrogen covers the metal substrate 141, and close the cooling valve 150. Use liquid nitrogen to freeze for about 30 seconds until the metal substrate 141 and the copper grid have the liquid nitrogen temperature;

[0086] 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 covered by the atomizer cover 132, the atomized sample particles can be directly attached to the copper grid;

[0087] Step 6: After the sample solution is attached to the copper grid, wait for 10 seconds to allow the liquid particles to be rapidly frozen on the copper grid at the temperature of liquid nitrogen to form a glassy film;

[0088] Step 7: After the glassy sample film is formed, liquid nitrogen is introduced into the freezing sample unit 110 again to cover the copper grid by 4 cm, and then the second cover 113 of the chamber is opened. At this time, the chamber cover 115 connected to the second cover 113 of the chamber and the sample processing unit 120 connected thereto are removed together, exposing the sample holding unit 140 immersed in liquid nitrogen. Under the protection of liquid nitrogen, the copper grid together with the frozen light-harvesting protein sample are transferred from the metal base 141 to the sample preservation unit 170 in the chamber 111 to complete the freezing sample operation.

[0089] Example 2

[0090] 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 is RNA bacteriophage Qbeta): the frozen sample was used to collect Tomography data, as shown in Figure A4; and the three-dimensional display software IMOD was used to display the position information of the sample particles in the glass film, as shown in Figure A4. Figure 4B and Figure 4C As shown. Among them, Figure 4A is the overall picture of data collection, Figure 4B It is a contaminant on the sample and can be used to mark the surface position of the glassy film. Figure 4C It is a display diagram of sample particles in the XY plane and the XZ plane. It can be clearly seen from the figure that the frozen sample particles are distributed in the middle of the glassy film and do not appear to gather at the gas-liquid interface.

[0091] Furthermore, using the above test method, we counted the test results of ten sets of RNA phage samples and used Figure 5 The three curves in the table represent the percentage of particles distributed in contact with the upper surface of the film, the percentage of particles distributed in the middle of the glassy film, and the percentage of particles distributed 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.

[0092] In addition, refer to Figures 6A-6C The single particle imaging schematic diagram, three-dimensional electron density schematic diagram and three-dimensional electron density and model matching schematic diagram of the ferritin sample are shown. We also used the ferritin sample for freezing sample preparation and subsequent data collection and structural analysis to obtain an electron density of 2.5 angstroms. It can be seen that the use of the device and method of the present application can obtain a three-dimensional electron density with an ideal resolution and obtain a high-quality frozen sample.

[0093] In summary, it can be seen that the technical means adopted in this application have the following advantages: First, an innovative attached sample freezing technology is proposed. Compared with the current immersion sample freezing scheme, its freezing rate is improved by an order of magnitude, which will fundamentally ensure the formation of a glassy water film, solve the accumulation effect of sample particles at the gas-liquid interface and improve the quality of frozen samples; Second, 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 particles in the solution disordered, ensuring that the frozen sample will not have a dominant orientation of particles; Third, the design of the closed sample freezing unit and the application of the vacuum operation scheme reduce the possibility of sample contamination; Fourth, the design and use of the frozen metal substrate and the porous substrate greatly improve the freezing rate while improving the efficiency of sample preparation. Fifth, the structure is simple and the cost is low; the realization of basic functions, the cost of the equipment is about 1 / 10 of the current high-end freezing sample instrument on the market. If precise temperature control, humidity control, and vacuum detection equipment are added, the cost will not exceed 1 / 5 of the current equipment.

[0094] The specific implementation methods described above further illustrate the purpose, technical solutions and beneficial effects of the present application in detail. It should be understood that the above description is only the specific implementation method of the present application and is not intended to limit the scope of protection of the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A device for preparing attached frozen samples, characterized in that: include: The freezing unit is used to provide an environment for sample freezing operation, which includes: a chamber, a chamber cover, a switch and a chamber cover. The chamber is configured as a hollow cavity with an opening, one end of the chamber is configured as an opening provided with the chamber cover, the other end of the chamber is configured as a base and is placed on the pipe unit, and the base is provided with a through hole for communicating with the cooling unit; The chamber sleeve is connected to the chamber cover, and the chamber sleeve is a hollow cavity with two ends open, one end of which is connected to the chamber cover, and the other end is connected to the sample processing unit; A cooling unit is used to store cooling liquid and provide cooling liquid for the freezing sample unit, and comprises: a cooling tank, a cooling valve, a cooling cover, and a cooling rack. The cooling rack is used to support the cooling tank; one end of the cooling tank is provided with an opening and is equipped with the cooling cover; the cooling tank is also provided with another opening, which is connected to the cooling valve, and the other end of the cooling valve is connected to the pipeline unit; the cooling tank contains coolant; The sample processing unit is arranged in the freezing sample unit and is used for atomizing the sample, and comprises: an atomizer, an atomizing hood, and a constant temperature device. Wherein, the atomizer is arranged in the atomizing cover; The outside of the atomizing hood is connected to the chamber sleeve, one end of the atomizing hood is provided with the thermostat, the thermostat and the atomizing hood half-wrap the atomizer, and the other end of the atomizing hood is formed into a hood-shaped cavity, which is covered above the substrate to ensure that the atomized sample can be deposited on the substrate; A sample carrying unit is arranged in the freezing sample unit and is arranged corresponding to the sample processing unit, comprising a metal base and a base column for holding a metal carrier mesh, wherein the base is arranged on the base and is pressed and fixed to the base by the base column, and a sample carrying unit is arranged correspondingly below the atomizing hood; and A pipeline unit connects the sample freezing unit and the cooling unit, and is used to guide the cooling liquid from the cooling unit to the sample freezing unit.

2. The device according to claim 1, wherein: It also includes a vacuum unit connected to the freezing sample unit and used to provide a vacuum environment for the freezing sample unit.

3. The device according to claim 1, wherein: It also includes a sample preservation unit, which is arranged in the freezing sample unit and is used to preserve the sample in the vitrified state.

4. The device according to claim 1, wherein: The chamber cover comprises a first chamber cover and a second chamber cover, the diameter of the first chamber cover is smaller than the diameter of the second chamber cover, and the chamber sleeve is connected to the second chamber cover.

5. The device according to claim 1, wherein: The chamber cover is provided with a power switch for controlling the start and shut down of the atomizer and the thermostatic device in the chamber.

6. The device according to claim 1, wherein: The metal substrate is a high thermal conductivity metal substrate.

7. The device according to claim 6, wherein: The metal substrate is a copper substrate.

8. An attached freezing method according to claim 1, characterized in that: include: 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 vacuumed by a vacuum assembly; The cooling liquid in the cooling unit enters the freezing unit through the pipeline unit to freeze the metal substrate. The metal grid is connected to the metal substrate. The metal thermal conductivity is utilized so that the cooled metal substrate transfers the low temperature to the metal grid. Starting 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 and frozen to form a glassy film.

9. The method according to claim 8, wherein: Before the metal grid is subjected to freezing treatment, a constant temperature device is started so that the sample solution in the sample processing unit is kept at a constant temperature during the freezing treatment of the metal grid.

10. The method according to claim 8, wherein: The sample solution is placed in the nebulizer in the sample processing unit.

11. The method of claim 8, further comprising transferring the glassy film together with the metal grid to a sample holding unit.

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