A method for preparing a cryo-em organic solvent sample

Abstract

The application belongs to the technical field of cryo-EM sample preparation, and particularly relates to a preparation method of a cryo-EM organic solvent sample. The application comprises the following steps: replacing clean filter paper in a semi-automatic freezing sample preparation device, setting temperature, humidity, water absorption intensity and time of the filter paper in a cavity, adding liquid nitrogen in a freezing and heat preservation device box, precooling, directly liquefying gaseous methane between-180 DEG C and-160 DEG C which is added subsequently, fixing an electron microscope carrier net in the freezing sample preparation device, dropping a sample containing an organic solvent on the electron microscope carrier net to form an ultrathin liquid film, quickly immersing the carrier net in a refrigerant, making the organic solvent in the liquid film form a glassy organic solvent ice layer on the carrier net under the condition of-180 DEG C to-160 DEG C, and embedding the sample in the glassy organic solvent. The method is simple in operation, convenient to use, low in cost, and can be used for preparation and observation of the organic solvent sample in the cryo-EM.

Description

Technical Field

[0001] This invention belongs to the field of cryo-electron microscopy sample preparation technology, and specifically relates to a method for preparing organic solvent samples for cryo-electron microscopy. Background Technology

[0002] Cryo-electron microscopy (cryo-EM) is a technique that uses a high-energy electron beam to irradiate samples embedded in a vitrified ice layer to observe the microstructure and function of the samples. Dubochet et al. were awarded the 2017 Nobel Prize in Chemistry for their discovery of the rapid vitrification of ultrathin biological samples using liquid ethane. The advantage of this sample preparation technique is that it preserves the original state of the sample in solution, avoiding damage caused by staining, sectioning, etc. This technique has had a profound impact on the field of biological structural science due to its powerful capabilities in studying aqueous samples. However, cryo-EM is still not a mature technology, and cryo-sample preparation, as one of the key steps in cryo-EM, has always been a challenge in the field. Of particular concern is that currently, cryo-EM is almost exclusively used for aqueous biological samples; non-aqueous organic solvent samples are rarely used for cryo-EM observation. This is because organic or polymeric materials are often synthesized or stored in organic solvents, which are easily dissolved by liquid ethane, the most commonly used cryoprotectant, making vitrification difficult. Therefore, applying cryo-sample preparation techniques for aqueous samples to organic solvent systems remains highly challenging. In summary, the bottleneck effect of organic solvent sample preparation in cryo-electron microscopy is becoming increasingly prominent. To solve this problem, it is urgent to develop a cryo-preparation method suitable for organic solvent phases. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for preparing organic solvent samples for cryo-electron microscopy.

[0004] The method for preparing organic solvent samples for cryo-electron microscopy provided by this invention includes the following specific steps:

[0005] (1) Replace the filter paper with clean paper in advance in the semi-automatic cryogenic sample preparation device, and set the temperature, humidity, water absorption capacity of the filter paper, and time in the chamber.

[0006] (2) Add liquid nitrogen into the refrigeration and insulation device box and pre-cool it so that the gaseous methane added later can be directly liquefied to between -180°C and -160°C.

[0007] (3) Fix the pretreated electron microscope grid onto the automated upper and lower extraction rods of the cryo-electron microscope sample preparation device using tweezers specifically designed for cryo-electron microscope sample preparation;

[0008] (4) A sample containing an organic solvent is dropped onto an electron microscope grid and then excess liquid is absorbed by filter paper to form an ultrathin liquid film (e.g., less than 500 nanometers thick).

[0009] (5) The carrier mesh with an ultra-thin liquid film is quickly immersed in the refrigerant, so that the organic solvent in the liquid film forms a glassy organic solvent ice layer on the carrier mesh at -180℃ to -160℃, and the sample is embedded in the glassy organic solvent.

[0010] (6) Store the electron microscope grid containing the organic solvent sample in liquid nitrogen for later use.

[0011] Furthermore, different types of organic solvents have different physicochemical properties (melting point, boiling point, viscosity, and saturated vapor pressure, etc.). Therefore, the temperature inside the cryogenic sample preparation device, the strength and time of the filter paper absorbing excess solvent, and the volume of the sample should all be adjusted to achieve the requirements of ultrathin liquid film.

[0012] Furthermore, the pretreatment of the electron microscopy grid refers to whether or not the grid is subjected to glow discharge treatment. For organic solvents with low solubility in water (e.g., solubility less than 0.1 (25℃, %w / w)), the grid is not subjected to glow discharge treatment; for organic solvents with high solubility in water (e.g., solubility greater than 0.1 (25℃, %w / w)), the grid needs to be subjected to glow discharge treatment.

[0013] Furthermore, for organic solvents with melting points below 0°C and low boiling points (e.g., below 100°C) or high saturated vapor pressures (e.g., above 1 kPa), such as ethanol, the chamber temperature needs to be set between 4-10°C, the sample volume between 0.1-9 μL, the force of the filter paper to absorb excess solvent between 0-6, and the filter paper absorption time between 0-6 seconds (preferably 1-6 seconds).

[0014] Furthermore, for solvents with high boiling points (boiling point above 100°C) or low saturated vapor pressures (e.g., saturated vapor pressure below 1 kPa), the chamber temperature is set to 5-10°C above the melting point of the organic solvent, the sample volume is between 0.1-9 μL, the force of the filter paper to absorb excess solvent is between 0-6, and the filter paper absorption time is between 0-6 seconds (preferably 1-6 seconds).

[0015] Furthermore, the liquid methane has a purity between 99.99% and 99.999%.

[0016] Furthermore, the temperature of the liquid methane is between -180°C and -160°C.

[0017] The method for preparing organic solvent samples for cryo-electron microscopy provided by this invention can prepare some organic solvent samples that cannot be vitrified by liquid ethane. This solves the problem that some organic solvents cannot be observed for microstructure using cryo-electron microscopy and broadens the application of organic solvent samples in cryo-electron microscopy. Attached image description:

[0018] Figure 1 Images (medium magnification and high magnification) of frozen samples prepared using liquid methane as a solvent, provided for embodiments of the present invention.

[0019] Figure 2 Image (low magnification) of a frozen sample of a mixed organic solvent (isooctane / oil) prepared using liquid methane, provided for an embodiment of the present invention.

[0020] Figure 3 Medium magnification and high magnification images of a mixed organic solvent (isooctane / oil) prepared using liquid methane, provided in an embodiment of the present invention. Detailed implementation method:

[0021] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0022] This invention provides a method for preparing organic solvent samples for cryo-electron microscopy. Based on conventional cryo-electron microscopy sample preparation procedures, it optimizes the selection of cryo-refrigerant, chamber temperature within the cryo-refrigerant apparatus, whether the grid is subjected to glow discharge treatment, the amount of organic solvent used, and the filter paper absorption time and force. This invention uses liquid methane as the cryo-refrigerant. By developing a novel cryo-refrigerant suitable for organic solvents, the dissolution of organic solvents by conventional cryo-refrigerants (liquid ethane) is avoided. Furthermore, by setting different volumes and filter paper absorption parameters (force, time) for different solvents, it is easier to prepare ultrathin organic solvent ice layers with a glassy state. The specific steps are as follows:

[0023] (1) First, select a suitable electron microscope grid. Generally, a microgrid grid or a quantifoil grid is chosen. Depending on the properties of the organic solvent to be prepared, decide whether to perform glow discharge treatment on the grid. Select a cryogenic sample preparation device, such as the ThermoFisher Vitrobot. TM Mark IV: Replace the dry filter paper in advance, set the chamber temperature, and adjust the time and force for the filter paper to absorb the solution.

[0024] (2) Then, continuously add liquid nitrogen to the cryogenic sample preparation device to pre-cool the device. Wait for the temperature of the sample preparation device to drop to the required temperature, and then introduce methane gas into the sample preparation device to rapidly liquefy it in the low-temperature environment of pre-cooling with liquid nitrogen. When the temperature of the liquid methane reaches between -180°C and -160°C, sample preparation can begin.

[0025] (3) During sample preparation, tweezers specifically designed for cryo-electron microscopy are used to grasp the (untreated) glow discharge-treated grid. The tweezers are then fixed in a fixed position within the cryo-electron microscopy apparatus. When the grid is lifted into the chamber by the tweezers, a certain amount of organic solvent or a mixture of organic solvents is drawn up using a pipette, depending on the properties of the organic solvent. In a specific embodiment of this invention, the organic solvent used here is a mixture of pure ethanol and isooctane / oil. By adjusting the pre-set chamber temperature and the time and force with which the filter paper absorbs the solution, the filter paper removes excess organic solvent, leaving only a thin liquid layer of tens to hundreds of nanometers on the grid.

[0026] (4) Research has shown that, according to the principle of like dissolves like, the closer the polarity of the organic solvent and the refrigerant, the easier it is for the organic solvent to be dissolved by the refrigerant, thus preventing the formation of a liquid layer. When the freezing rate of the refrigerant is insufficient to vitrify the organic solvent, a crystalline organic solvent ice layer will form, which is not conducive to the observation of the sample. Therefore, compared with liquid ethane, liquid methane refrigerant has the advantage of not dissolving some organic solvents, while also having a sufficient cooling rate, so that the organic solvent is neither dissolved and a glassy ultrathin ice layer can be obtained.

[0027] (5) The organic solvent film on the cryogenic carrier is rapidly frozen by liquid methane to form a glassy organic solvent ice layer. The carrier containing the organic solvent ice layer is stored in liquid nitrogen at -196°C for later use.

[0028] (6) The cryo-electron microscopy image of the pure ethanol ice layer prepared by the present invention under the refrigerant liquid methane is shown below. Figure 1 As shown, Figure 1 a is a mesoscopic image of the ethanol ice layer. Figure 1 b is Figure 1 The high-magnification image of the area within the dashed box and the corresponding Fourier transform image (top right) show that the ethanol prepared from liquid methane did not dissolve and the resulting ice layer was amorphous.

[0029] (7) A cryo-electron microscopy low-magnification image of the isooctane / oil mixed organic solvent prepared by the present invention under the condition of liquid methane as a refrigerant is shown below. Figure 2 As shown, the mixed organic solvent sample prepared by this method can form a large number of vitrified regions, proving the reliability of the invention.

[0030] (8) The cryo-electron microscopy image of the isooctane / oil mixed organic solvent prepared by the present invention under the condition of liquid methane as a refrigerant is shown below. Figure 3 As shown. Figure 3 a is a medium magnification image of the ice layer containing mixed organic solvents. Figure 3 b is Figure 3The high-magnification image of the area within the dashed box and the corresponding Fourier transform image (top right) show that the isooctane / oil mixed organic solvent prepared from liquid methane did not dissolve, and the resulting ice layer was amorphous.

Claims

1. A method for preparing organic solvent samples for cryo-electron microscopy, characterized in that, The specific steps are as follows: (1) Replace the filter paper with clean filter paper in advance in the semi-automatic cryogenic sample preparation device, and set the temperature, humidity, water absorption capacity of the filter paper and time in the chamber; (2) Add liquid nitrogen into the refrigeration and insulation device box and pre-cool it so that the gaseous methane added later can be directly liquefied to between -180°C and -160°C; (3) Fix the pretreated electron microscope grid onto the automated upper and lower extraction rods of the cryo-electron microscope sample preparation device using cryo-electron microscope sample preparation tweezers; (4) Add a sample containing organic solvent to the electron microscope grid, and then remove the excess liquid through filter paper to form an ultrathin liquid film of less than 500 nanometers. (5) The carrier mesh with an ultra-thin liquid film is quickly immersed in the refrigerant, so that the organic solvent in the liquid film forms a glassy organic solvent ice layer on the carrier mesh at -180℃ to -160℃, and the sample is embedded in the glassy organic solvent. (6) Store the electron microscope grid containing the organic solvent sample in liquid nitrogen for later use.

2. The method for preparing organic solvent samples for cryo-electron microscopy according to claim 1, characterized in that, The temperature, humidity, the force and time of filter paper absorbing excess solvent, and the volume of the sample within the cryogenic sample preparation device are adjusted according to the melting point, boiling point, viscosity, and saturated vapor pressure of different types of organic solvents to achieve the requirements of ultra-thin liquid film.

3. The method for preparing organic solvent samples for cryo-electron microscopy according to claim 1, characterized in that, Electron microscopy screen pretreatment refers to whether or not the screen is subjected to glow discharge treatment. At 25°C, for organic solvents with a solubility in water of less than 0.1% w / w, the screen does not need to undergo glow discharge treatment. At 25°C, for organic solvents with a solubility in water of greater than 0.1% w / w, the screen needs to undergo glow discharge treatment.

4. The method for preparing organic solvent samples for cryo-electron microscopy according to claim 1, characterized in that: For organic solvents with melting points below 0°C, boiling points below 100°C, or saturated vapor pressures above 1 kPa, the chamber temperature should be set between 4-10°C, the sample volume between 0.1-9 μL, the force of the filter paper to absorb excess solvent between 0-6, and the filter paper absorption time between 0-6 seconds. For solvents with boiling points above 100℃ or saturated vapor pressures below 1 kPa, the chamber temperature should be set between 5-10℃ above the melting point of the organic solvent, the sample volume between 0.1-9 μL, the force of the filter paper to absorb excess solvent between 0-6, and the filter paper absorption time between 0-6 seconds.

5. The method for preparing organic solvent samples for cryo-electron microscopy according to claim 1, characterized in that, The liquefied methane has a purity between 99.99% and 99.999%.

Images