JP7370042B2 - Transmission electron microscope sample support, its manufacturing method, and sample preparation method using the same - Google Patents

Description

The present invention relates to a transmission electron microscope sample support, a method for manufacturing the same, and a sample preparation method using the same.

Cryo-electron microscopy is a type of transmission electron microscopy used in fields such as structural biology and cell biology. Cryo-electron microscopy is a method in which the target sample is fixed inside thin amorphous ice by flash freezing with an aqueous solution, etc., and then observed without performing any staining or chemical fixation on the target sample.

Sample preparation for cryo-electron microscopy generally involves a structure in which a metal grid support such as copper, gold, or molybdenum is covered with an amorphous carbon thin film with a large number of holes. A grid is used. A target sample such as protein dissolved in water is dropped onto this grid, excess water is absorbed from both sides of the grid using filter paper, etc., and the sample is rapidly frozen to allow it to enter the pores of the amorphous carbon membrane. We have prepared a thin ice film that confines the target sample.

However, this method has the problem that the thickness of the water film and ice film depends on the thickness of the amorphous carbon film, and that the water film bursts and disappears during rapid freezing with liquefied ethane. It cannot be said that this is a highly accurate sample preparation method.

To address this problem, a method has also been used in which graphene oxide dispersed in an aqueous solution is dropped onto an amorphous carbon film to form a graphene oxide film by blocking the pores of the amorphous carbon film. . However, when forming a graphene oxide film by dropping, it is difficult to control the number of layers, and the electrical conductivity is inferior to metals, etc., so there are concerns that image deterioration during observation due to charge-up and resulting drift may occur. .

In order to make it hydrophilic, there is a conventional technology in which single-layer graphene is partially hydrogenated with hydrogen plasma to produce hydrogenated graphene, which is used as a transmission electron microscope sample support. The degree of corrosion is low, and since it is a single layer, its electrical conductivity and mechanical strength are low.

Special table 2016-534508 publication Special table 2016-532267 publication

Therefore, according to one aspect, an object of the present invention is to provide a transmission electron microscope sample support having excellent properties in terms of hydrophilicity and electrical conductivity, and a method for manufacturing the same.

According to one aspect, another object of the present invention is to provide a sample preparation method using a transmission electron microscope sample support having excellent properties in terms of hydrophilicity and electrical conductivity.

A transmission electron microscope sample support according to the present invention includes a support element having a plurality of holes and two layers of graphene that cover at least some of the plurality of holes. The first graphene film of the two layers of graphene is a hydrophilized graphene film in which a hydrophilic group is attached to at least a portion of the defects, and the amount of defects in the first graphene film is greater than that of the graphene film other than the first graphene film. It is characterized by having more defects than the amount of defects in the film.

Further, the method for manufacturing a transmission electron microscope sample support according to the present invention includes a method for manufacturing a transmission electron microscope sample support having a support element having a plurality of holes, and a two-layer film formed by a plasma CVD (Chemical Vapor Deposition) method. The method is characterized in that graphene is attached and the first graphene film of the two layers of graphene is subjected to ultraviolet ozone treatment.

Further, in the method for preparing a sample for a transmission electron microscope according to the present invention, an aqueous solution containing an object to be analyzed is dropped onto the above-mentioned transmission electron microscope sample support and frozen.

According to one aspect, a transmission electron microscope sample support having excellent properties in terms of hydrophilicity and electrical conductivity is obtained.

In addition, according to another aspect, by using such a transmission electron microscope sample support, it becomes possible to form a thinner ice film, making it possible to obtain better images in cryo-electron microscopy, etc. become.

FIG. 1 is a diagram showing the structure of a transmission electron microscope sample support in a state before ultraviolet ozone treatment. FIG. 2 is a diagram showing a method for manufacturing a transmission electron microscope sample support according to an example. FIG. 3 is a diagram showing the configuration of a transmission electron microscope sample support in a state after ultraviolet ozone treatment. FIG. 4 is a diagram showing changes in the Raman spectrum of bilayer graphene before and after ultraviolet ozone treatment. FIG. 5 is a diagram showing the results of Lorentzian peak fitting analysis for the 2D band of bilayer graphene. FIG. 6 is a diagram showing the relationship between the ultraviolet ozone treatment time and the D/G band intensity ratio in the Raman spectrum. FIG. 7 is a diagram showing the relationship between the ultraviolet ozone treatment time and the degree of hydrophilization for bilayer graphene on a metal substrate (copper foil catalyst layer). FIG. 8A is a diagram showing a high-resolution STEM (Scanning Transmission Electron Microscope) image of bilayer graphene before ultraviolet ozone treatment. FIG. 8B is a diagram showing a high-resolution STEM image of bilayer graphene after ultraviolet ozone treatment. FIG. 9A is a diagram showing an annular dark field image of a stack of bilayer graphene and porous amorphous carbon film after forming glassy ice. FIG. 9B is a diagram showing a typical low-loss region valence electron excitation spectrum obtained from a hole portion where bilayer graphene is bridged. FIG. 9C is a diagram showing a line profile of the ice film thickness calculated using the ratio of elastically scattered electrons to inelastically scattered electrons. FIG. 10 is a diagram showing a low-temperature TEM image of tobacco mosaic virus in glassy thin ice formed on bilayer graphene.

EMBODIMENT OF THE INVENTION Hereinafter, the transmission electron microscope sample support body based on this invention, its manufacturing method, and the sample preparation method using the same are demonstrated based on embodiment and an Example. Duplicate explanations will be omitted as appropriate. In addition, when a numerical range is indicated by writing "~" between two numerical values, these two numerical values are also included in the numerical range.

[Summary of embodiment of the present invention]
As a result of extensive research to achieve the above-mentioned problems, the present inventors have grown a graphene film in two layers with high controllability on a metal substrate using the plasma CVD method, bonded it to a metal grid support, and treated it with ultraviolet ozone treatment. We have obtained the knowledge that it is possible to selectively make one layer of two-layer graphene hydrophilic by applying it only to one side of the two-layer graphene. As a result, a hydrophilized graphene film with a high surface hydrophilic function is supported by a highly crystalline graphene film with high electrical conductivity inherent to graphene, which supports a transmission electron microscope sample with an ultra-thin film of two atomic layers. You can get a body.

In this way, by selectively making one layer of bilayer graphene hydrophilic, the hydrophilized graphene film functions as a film that improves the affinity with the aqueous solution containing the target sample, and the other layer becomes hydrophilic. The graphene membrane provides its inherent high electrical conductivity and also functions to strengthen the support membrane that supports the dropped aqueous solution containing the target sample. These bilayer graphenes, which have high hydrophilicity, high electrical conductivity, and high strength, overcome two conventional problems in cryo-electron microscopy: making the amorphous ice thinner and deteriorating the observed image due to charge-up. It is possible.

[Embodiment]
The transmission electron microscope sample support according to the present embodiment includes a support element having a plurality of holes (for example, the porous amorphous carbon membrane 2 or the metal grid support 1 in FIG. 3), and at least a portion of the plurality of holes. It has two layers of graphene (for example, two-layer graphene 3' in FIG. 3) that cover the holes. Of these two layers of graphene, the first graphene film (for example, the upper graphene film in FIG. 3) is a hydrophilized graphene film in which a hydrophilic group is attached to at least a portion of the defects, and the defects in the first graphene film are The amount of defects is greater than the amount of defects in graphene films other than the first graphene film (for example, the lower layer graphene film in FIG. 3).

By providing such two layers of graphene, hydrophilicity can be increased by the first graphene film, and high electrical conductivity can also be obtained by the graphene films other than the first graphene film. Furthermore, by using a two-layer graphene film instead of one layer, mechanical strength can also be increased. As a result, even when preparing a sample, instead of dropping the sample with a micropipette, etc., the sample is ejected from a nozzle using inkjet technology while controlling the amount of the droplet. It is considered to have the strength to withstand.

It should be noted that the support element described above may be a support film attached to a metal grid support having a mesh (for example, the porous amorphous carbon membrane 2 in FIG. 3) or a metal grid support (for example, the metal grid support in FIG. 3). 1). This support film may be an amorphous carbon film, or may be other commonly used materials such as a silicon nitride film or a silicon oxide film. Note that two layers of graphene may be attached to a metal grid support having a mesh without using such a support film.

The ratio of the D band intensity to the G band intensity in the Raman spectrum of the two-layer graphene is preferably 0.04 or more and 0.5 or less. Sufficient electrical conductivity can be obtained with this amount of defects. Moreover, mechanical strength is also maintained.

Further, the contact angle of the two layers of graphene measured by the θ/2 method is preferably 25° or more and 60° or less, more preferably 25° or more and 40° or less. As described above, the degree of hydrophilicity (wettability) is wide, and it can be used for various sizes of analysis targets (eg, biological samples such as proteins).

Such a transmission electron microscope sample support is made by pasting two layers of graphene formed by a plasma CVD method on a transmission electron microscope sample support having a support element having a plurality of holes (for example, S4 in FIG. 2). , is produced by subjecting the first graphene film of the two layers of graphene to ultraviolet ozone treatment (for example, S6 in FIG. 2).

This ultraviolet ozone treatment is adjusted depending on, for example, the target degree of hydrophilicity. For example, in order to increase the degree of hydrophilization, the intensity of ultraviolet rays may be increased or the time of ultraviolet ozone treatment may be lengthened. On the other hand, in order to lower the degree of hydrophilization, the intensity of ultraviolet rays is lowered or the time of ultraviolet ozone treatment is shortened. As a result, if the size of the analyte contained in the sample is small, a thinner ice film can be formed by increasing the intensity of the ultraviolet rays or increasing the degree of hydrophilization by increasing the treatment time. On the other hand, if the size of the analyte contained in the sample is large, by lowering the intensity of the ultraviolet rays or shortening the treatment time, it becomes possible to form an ice film thick enough to encompass the analyte.

Note that the degree of ultraviolet ozone treatment is determined by various factors, so that the ratio of D band intensity to G band intensity in the Raman spectrum of the two-layer graphene is 0.04 or more and 0.5 or less, or The contact angle of the two layers of graphene measured by the θ/2 method may be adjusted to be 25° or more and 60° or less.

During sample preparation, an aqueous solution containing the target to be analyzed is dropped onto the transmission electron microscope sample support as described above and frozen. This makes it possible to create a thinner ice film than before, making it possible to obtain clearer images when observed using cryo-electron microscopy. Note that the thickness of the ice film when an amorphous carbon film is used is about 74 to 114 nm, and in this embodiment, it can be suppressed to about 1/3 to 1/5 of that.

Preferably, a transmission electron microscope sample support having a degree of hydrophilicity depending on the size of the object to be analyzed is used. As mentioned above, this is because an ice film with an appropriate thickness can be formed on the object to be analyzed. More specifically, a transmission electron microscope sample support with a lower degree of hydrophilicity is selected as the size of the object to be analyzed becomes larger, and the thickness of the ice film obtained by the above-mentioned freezing becomes thicker. Conversely, the smaller the size of the object to be analyzed, the higher the degree of hydrophilicity selected as the transmission electron microscope sample support, and the thinner the ice film obtained by the above-mentioned freezing becomes.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to these Examples.

<Method for manufacturing a transmission electron microscope sample support>
In this example, it is assumed that a metal grid support with a porous amorphous carbon film, in which a porous amorphous carbon film having a large number of holes is attached to the metal grid support, has already been obtained. Then, a first step of pasting the two-layer graphene before ultraviolet ozone treatment (also referred to as UV ozone treatment) on the metal grid support with a porous amorphous carbon film, and and a second step of performing ultraviolet ozone treatment.

In order to make the following explanation easier to understand, FIG. 1 first shows the structure of the transmission electron microscope sample support after the first step. A porous amorphous carbon film 2 is formed on the metal grid support 1, and a two-layer graphene 3 is pasted on the porous amorphous carbon film 2, and the transmission electron microscope sample support is made of these. It is a laminate of. The two-layer graphene 3 extends over at least some of the pores of the porous amorphous carbon film 2.

Note that one hole of the mesh in the metal grid support 1 is, for example, rectangular with a side of about 100 μm, and each hole in the porous amorphous carbon film 2 is circular with a diameter of 2 μm, for example. The thickness of the porous amorphous carbon film 2 is about 10 to 25 nm. Further, the film thickness of the bilayer graphene is approximately 0.67 to 0.70 nm.

Next, a method for manufacturing a transmission electron microscope sample support will be explained using FIG. 2.

First, two-layer graphene 3 is generated on a copper foil catalyst layer (ie, a metal substrate) by a plasma CVD method using a carbon source gas (S1). For the method of producing bilayer graphene 3, see, for example, "Bilayer graphene synthesis by plasma treatment of copper foils without using a carbon-containing gas", R. Kato et al., Carbon 77 (2014) 823-828. About.

Next, polymethyl methacrylate (PMMA), polycarbonate (PC), photoresist (PR), etc. are applied to the grown bilayer graphene 3 using a spin coater, and an organic thin film layer (protective film) with a thickness of about 100 nm to 10 μm is applied. ) is formed (S2).

After drying the organic thin film layer in the air or in a vacuum environment for about 10 to 30 minutes, the metal substrate is removed using a chemical etching method (S3). Here, as the etchant solution, reactive ion etching solutions such as ferric chloride (FeCl ₃ ), ammonium persulfate ((NH ₄ ) ₂ S ₂ O ₈ ), hydrochloric acid (HCl), and potassium hydroxide (KOH) are used. Available for use. A structure including bilayer graphene with an organic thin film layer formed thereon is floated on an etchant solution, and infiltration is performed until the metal substrate is completely removed. After the metal substrate is completely etched, the etchant solution phase is transferred to the cleaning solution phase using filter paper, glass, SiO ₂ , etc. Ultrapure water, deionized exchange water, or the like can be used as the cleaning solution.

Thereafter, using the metal grid support 1 equipped with the porous amorphous carbon film 2, the two-layer graphene 3 with the organic thin film layer formed on the cleaning liquid phase was scooped up and deposited, and then the mixture was heated at room temperature to 90°C. (S4). In this example, Quantifoil (registered trademark): R2/2 Holey Carbon film TEM grids, Mo 200 mesh was used for the metal grid support 1 with the porous amorphous carbon film 2. After complete drying, the organic thin film layer is removed by soaking it in an organic solvent such as acetone (S5).

After removing the organic thin film layer, cleaning is performed by infiltrating isopropyl alcohol or the like with a solvent for removing the organic thin film layer. By drying in a vacuum environment after cleaning is completed, a laminate including the two-layer graphene 3, the porous amorphous carbon film 2, and the metal grid support 1 as shown in FIG. 1 can be obtained.

The metal grid support 1 provided with the bilayer graphene 3 obtained by vacuum drying is further heated under high vacuum at a temperature of 200 to 600°C for about 1 to 5 hours to remove the organic material that could not be completely removed by the organic solvent. It is possible to perform thin film layer removal. This can be expected to further purify the graphene surface.

After heat treatment under high vacuum, graphene is made hydrophilic using a UV ozone cleaner (S6). For UV ozone treatment, a Filgen UV253V12 equipped with five low-pressure mercury lamps was used. The ultraviolet intensity of this device is 5.2 mW/cm ² per UV lamp, and ultraviolet rays with an extremely short wavelength of 184.9 nm (647 KJ/mol) and a short wavelength of 253.7 nm (472 KJ/mol) are irradiated. Since the bond energy between carbon atoms that make up graphene is 347.7 KJ/mol and 607 KJ/mol for single and double bonds, respectively, the carbon-carbon bonds in the upper layer of two-layer graphene are broken by ultraviolet irradiation. small defects will occur. Graphene becomes hydrophilic by adsorbing hydrophilic groups such as hydroxyl groups (-OH) and carboxyl groups (-COOH) to the defects.

The metal grid support 1 onto which the bilayer graphene 3 was transferred was placed on a slide glass, quartz glass, Si substrate, metal support, etc., and placed inside the above device so that only one side was irradiated with ultraviolet light. . First, oxygen gas was introduced into the apparatus for about 30 minutes to replace the atmospheric atmosphere inside the apparatus with oxygen atmosphere. The introduction time of oxygen gas is about 10 to 30 minutes depending on the case. Thereafter, ultraviolet rays were irradiated for 1 to 30 minutes. The experiment was conducted with a distance of 10 cm between the ultraviolet lamp and the sample. This interval is about 5 to 20 cm depending on the case.

By performing such treatment, as shown in FIG. 3, the two-layer graphene 3 before UV ozone treatment becomes a highly hydrophilic graphene film in which hydrophilic groups are attached to at least some of the defects in the upper layer, and a highly hydrophilic graphene film in which hydrophilic groups are attached to at least some of the defects in the lower layer. transforms into a bilayer graphene 3' having a graphene film that maintains its original high electrical conductivity.

That is, the transmission electron microscope sample support according to this example includes a metal grid support 1, a porous amorphous carbon film 2, and a two-layer graphene 3 including a highly hydrophilic graphene film and a highly electrically conductive graphene film. It is a laminate with '.

<About hydrophilicity evaluation by contact angle (wettability)>
In order to evaluate the degree of hydrophilization of the bilayer graphene 3', the contact angle was measured by the θ/2 method. Contact angle meter Dropmaster 501 manufactured by Kyowa Interface Science Co., Ltd. was used to measure the contact angle, and ultrapure water was used as the droplet. In measuring the contact angle of two-layer graphene, two-layer graphene 3 on a copper foil catalyst (metal substrate) (UV ozone treatment time 0 minutes) and each UV ozone treatment time (5 minutes, 10 minutes, 20 minutes) were used. Measurements were performed five times on the two-layer graphene 3', and the average value was calculated.

<About crystallinity evaluation (Raman spectroscopy measurement method)>
Regarding the verification examples and examples described below, Raman scattering spectroscopic measurements were performed. As the Raman spectrum measurement device, an XploRa model manufactured by Horiba, Ltd. was used. A semiconductor laser was used as the excitation laser, the excitation wavelength was 638 nm, the laser beam spot was 1 μm in diameter, the spectrometer had 600 gratings, the aperture was 300 μm, the slit was 100 μm, and the output of the laser source was 9.8 mW. The Raman spectrum was acquired by integrating five measurements with an exposure time of 5 seconds.

It has been shown that the peak positions of the 2D band, G band, and D band depend on the number of graphene layers and the laser wavelength at the time of Raman spectrum acquisition. In the case of single-layer graphene at an excitation wavelength of 514.5 nm, the peak positions of the 2D band, G band, and D band are found around 2700 cm ^-1 , 1582 cm ^-1 , and 1350 cm ^-1 , respectively. The G band is a peak resulting from a normal six-membered ring, and the D band is a peak resulting from a defect in the normal six-membered ring. Furthermore, the 2D band is based on overtones of the D band. If both the G band and 2D band are observed in the Raman spectrum, the film is identified as graphene. It is generally known that as the number of graphene layers increases, the peak position of the 2D band shifts to the higher wavelength side and the half-width of the peak increases. Furthermore, as the excitation wavelength used becomes shorter, the 2D band shifts to higher wavelengths.

<About 2D peak fitting of Raman spectrum>
It is known that in the 2D band of graphene, a double resonance scattering process occurs during the electron scattering process by phonons, and the half-width changes as the number of layers increases, such as single-layer graphene, bilayer graphene, and trilayer graphene. It is known that fitting can be performed using one Lorentzian peak for single-layer graphene, four for two-layer graphene, and a total of six Lorentzian peaks for three-layer graphene.

<About evaluation of electrical conductivity by sheet resistance>
As a characteristic evaluation of electrical conductivity, the sheet resistance of graphene transferred onto a 118 μm thick PET film was measured using a non-contact resistance measuring device (model: EC-80, manufactured by Napson Co., Ltd.). First, a thermal release sheet was attached to the graphene film formed on the copper foil catalyst layer (metal substrate), and then the copper foil catalyst layer (metal substrate) was removed using a chemical etching method. Ammonium persulfate ((NH ₄ ) ₂ S ₂ O ₈ ) was used as the etchant solution. The thermal release sheet/graphene laminate obtained by etching the copper foil catalyst layer (metal substrate) was washed with ultrapure water, and a PET film was attached to the graphene side of the thermal release sheet/graphene laminate. Thereafter, the thermal release sheet was peeled from the thermal release sheet/graphene/PET film laminate by performing heat treatment at 80° C. in the atmosphere to obtain a graphene/PET film laminate. The electrical conductivity in this example was measured five times on a 4.0 cm x 4.0 cm graphene/PET film laminate sample and evaluated based on the average value.

<About STEM evaluation of bilayer graphene before and after UV ozone treatment>
Bilayer graphene was observed before and after UV ozone treatment using a scanning transmission electron microscope (STEM). For STEM observation, a JEM-2100F model equipped with a DELTA aberration corrector manufactured by JEOL Ltd. was used with an accelerating voltage of 60 kV. The zero-loss energy resolution of the cold field emission gun was 0.4 eV and was used with an electron probe diameter of approximately 0.1 nm and a current value of 30 pA.

For STEM image observation, an annular dark field (ADF) image was observed using an imaging filter detector GIF Quantum manufactured by Gatan. In order to prevent the sample from being contaminated by contamination from the environment inside the electron microscope, the observation was carried out while controlling the temperature to approximately 500° C. using a heating holder manufactured by JEOL Ltd.
<About sample preparation using ice embedding method>
Sample preparation by an ice embedding method was carried out using a metal grid support 1 (ie, a transmission electron microscope sample support) provided with a UV ozone-treated bilayer graphene 3'. A bitrobot manufactured by FEI was used for sample preparation using the ice embedding method. First, the environment inside the apparatus was set such that the humidity was 90% or higher and the temperature was 5° C. or lower. Next, the metal grid support 1 was fixed using X tweezers and placed inside the chamber of the apparatus. Thereafter, 1.7 μmL of an aqueous solution containing Tobacco Mosaic Virus (TMV) was dropped onto the metal grid support 1 from the side using a micropipette, the excess solution was sucked up with a filter paper, and the mixture was instantly poured into liquefied ethane. Flash freezing was performed by infiltrating the

<About cryo-electron microscopy>
A JEOL TEM system equipped with a Schottky field emission gun, a DELTA aberration corrector, and a double Wien filter monochromator was used for low-temperature TEM observation of the frozen sample. A Gatan OneView CMOS camera was used to take the observation images.

In addition, a cryo-transfer holder manufactured by Gatan (cryo-holder-626 Gatan US) was used to transport the frozen sample into the electron microscope and maintain the low temperature within the electron microscope. The temperature inside the electron microscope was maintained at approximately -175°C using a Gatan Smart Controller.

<Measurement of the thickness of glassy thin ice using electron energy loss spectroscopy>
To measure the film thickness of glassy thin ice created by the ice embedding method, we used electron energy loss spectroscopy (STEM-EELS), which combines STEM with an electron spectrometer.

ここでＩ_tはスペクトルの全強度、Ｉ₀はゼロロス電子の強度を示している。また全非弾性散乱の平均自由行程λは以下の式により与えられる。

ここで、Ｅ₀、β、Z_effはそれぞれ電子顕微鏡の加速電圧、対物絞りの大きさ、そして膜厚測定における当該試料の有効原子番号を示す。また有効原子番号は下記の式により求められることが知られている。

ここでｆ_nおよびＺ_nはそれぞれ各元素の電子の総数、各元素の原子番号を示す。本測定における電子顕微鏡の加速電圧は６０ｋＶ、対物絞りの大きさは７６rad、また氷膜すなわち水の有効原子番号は７．４２である。このことから６０ｋＶの加速電圧使用時の氷における全非弾性散乱の平均自由行程は５２．９ｎｍと計算される。本測定では加速電圧６０ｋＶを使用し、エネルギー範囲－１０～９０ｅＶの範囲において各測定箇所０．０１秒にて測定を実施した。 With the STEM-EELS method, the film thickness of the target sample can be measured from the ratio of inelastically scattered electrons to elastically scattered electrons. It is known that when the film thickness of the target sample is t and the mean free path of total inelastic scattering is λ, the relative film thickness of the target sample is given by the following equation.

Here, I _t represents the total intensity of the spectrum, and I ₀ represents the intensity of zero-loss electrons. Further, the mean free path λ of total inelastic scattering is given by the following equation.

Here, E ₀ , β, and Z _eff respectively indicate the acceleration voltage of the electron microscope, the size of the objective aperture, and the effective atomic number of the sample in film thickness measurement. It is also known that the effective atomic number can be determined by the following formula.

Here, f _n and Z _n represent the total number of electrons of each element and the atomic number of each element, respectively. In this measurement, the acceleration voltage of the electron microscope was 60 kV, the size of the objective aperture was 76 rad, and the effective atomic number of the ice film, that is, water, was 7.42. From this, the mean free path of total inelastic scattering in ice when using an accelerating voltage of 60 kV is calculated to be 52.9 nm. In this measurement, an accelerating voltage of 60 kV was used, and the measurement was carried out for 0.01 seconds at each measurement point in the energy range of -10 to 90 eV.

<Analysis results by Raman spectroscopy>
Figure 4 shows bilayer graphene 3 on a copper foil catalyst layer (metal substrate) without UV ozone treatment (UV ozone treatment 0 minutes) (solid line), after UV ozone treatment for 10 minutes (dot-dashed line) and after 20 minutes (thin dotted line). ) shows a typical Raman spectrum for bilayer graphene 3'. In FIG. 4, the vertical axis represents intensity (arbitrary unit), and the horizontal axis represents Raman shift [cm ⁻¹ ].

As shown in FIG. 4, prominent G bands and 2D bands are confirmed in all Raman spectra. In addition, a small amount of D band originating from defects is observed in the two-layer graphene 3' after 10 and 20 minutes of UV ozone treatment.

Further, FIG. 5 shows detailed curve fitting analysis results of the 2D band. In Figure 5, the thin dot-dash line represents the Lorentzian subpeak, the thick dot-dash line represents the Raman spectrum of bilayer graphene 3' after 10 minutes of UV ozone treatment, and the solid line represents the Raman spectrum of bilayer graphene 3' after UV ozone treatment of 0 minutes. The thin dotted line represents the Raman spectrum of two-layer graphene 3′ treated with UV ozone for 20 minutes.

As a result, the 2D bands of all Raman spectra were fitted by a total of four Lorentzian subpeaks, indicating bilayer graphene, and the stacked structure of bilayer graphene obtained by implementing this Raman spectroscopy can be seen in Figure 4. The intensity ratio of the G/2D band is about 2 to 3, which indicates an AB laminated structure.

Furthermore, considering that the laser spot used in this example was 1.0 μm, a slight increase in the D band was observed after UV ozone treatment, but no defects exceeding 1.0 μm were introduced, and 2 It is thought that the layered structure is maintained.

Next, FIG. 6 shows the relationship between the intensity ratio of the D band and G band in the Raman spectrum and the UV ozone treatment time. In FIG. 6, the vertical axis represents the D/G band intensity ratio (arbitrary unit), and the horizontal axis represents the UV ozone treatment time [minutes].

FIG. 6 shows that the D/G band intensity ratio tends to increase with UV ozone treatment time. Based on the relationship between the Raman spectrum and the D/G ratio shown in Figure 4, it can be seen that even though the structure as a two-layer graphene is maintained by UV irradiation, defects begin to form at a certain site in the upper layer. It is thought that the amount of defects increases due to the expansion of defects or the start of new defect formation at different sites, and an increase in the D band originating from defects is confirmed.

<Evaluation results of electrical conductivity based on sheet resistance>
As an evaluation of electrical conductivity, sheet resistance was compared with and without UV ozone treatment. For comparison of electrical conductivity in this example, a sample subjected to UV ozone treatment for 20 minutes was used. The average sheet resistance of the bilayer graphene 3 without UV ozone treatment was 328±2.3Ω/□, and the average sheet resistance of the bilayer graphene 3' after 20 minutes of UV ozone treatment was 364±10Ω/□. From this result, the rate of increase in sheet resistance due to UV ozone treatment for 20 minutes is approximately 11%. High electrical conductivity was maintained even in samples subjected to a hydrophilic process using UV ozone treatment for about 20 minutes, which is much better than conventionally used amorphous carbon films, graphene oxide, etc. It is considered to be electrically conductive. Therefore, even in TEM observation at low temperatures, it is considered to have sufficient electrical conductivity to suppress image deterioration due to charge-up and the like.

<Wettability evaluation results>
FIG. 7 shows the relationship between the UV ozone treatment time and the contact angle between graphene and ultrapure water as a result of the degree of hydrophilicity due to UV ozone treatment on the two-layer graphene 3 on the copper foil (metal substrate). In FIG. 7, the vertical axis represents the contact angle [°], and the horizontal axis represents the UV ozone treatment time [minutes].

As shown in FIG. 7, the contact angle decreased linearly until the UV ozone treatment time was about 10 minutes, and after 10 minutes, it was about 30°. When UV ozone treatment was carried out for 10 minutes or more, no significant change was observed in the contact angle, but the average deviation tended to increase. This is probably because the amount of carbon-carbon bonds broken by ultraviolet irradiation increases, and the number of defects in the upper layer of two-layer graphene increases and the defects themselves expand, causing the surface of the lower graphene that has not been made hydrophilic to come into contact with water. This is thought to be due to the increase in area. In hydrophilizing graphene by UV ozone treatment, it is possible to freely create two-layer graphene having a wide range of hydrophilicity degrees from about 25° to about 80° by selecting the UV ozone treatment time.

<High-resolution STEM observation results>
An example of STEM observation of bilayer graphene before and after UV ozone treatment is shown in FIGS. 8A and 8B. FIG. 8A shows a STEM-ADF observation image of bilayer graphene 3 before UV ozone treatment. In FIG. 8A, a stacked layer is formed with orientation shifted by about 13 degrees, and a moiré pattern can be observed. No defects were observed in both graphene films before UV ozone treatment. This shows that highly crystalline two-layer graphene was formed by the plasma CVD method, and no defects were introduced during the transfer process to the metal grid support 1.

FIG. 8B shows a STEM-ADF image of bilayer graphene 3' subjected to UV ozone treatment for 20 minutes. Defects due to missing carbon atoms are seen after UV ozone treatment. The contrast of an ADF image in STEM observation is observed to be approximately proportional to the square of the atomic number. Therefore, the dark portions seen in FIG. 8B indicate regions corresponding to single-layer graphene. The size of the defect confirmed in this example was a defect consisting of about several hundred defects, as observed in FIG. 8B. However, not a single carbon atom is observed to be missing in the underlying single-layer graphene portion. This indicates that defect formation occurs only in the upper layer, and the dissociation of carbon-carbon bonds by UV ozone treatment for about 20 minutes and the introduction of hydrophilic groups formed by UV irradiation act only in the upper layer. it is conceivable that. This result is not different from the above Raman spectrum analysis result.

The results of these Raman spectroscopy analyses, wettability evaluations, and high-resolution STEM observations show that the UV ozone treatment performed on bilayer graphene 3 does not introduce defects into the lower layers of bilayer graphene, This shows that it is possible to make the material highly hydrophilic. This indicates that highly hydrophilic graphene can be formed on highly electrically conductive graphene by simple surface treatment.

<Results of measuring the thickness of glassy thin ice using electron energy loss spectroscopy>
Glassy ice was prepared on the bilayer graphene 3' using the transmission electron microscope sample support having the bilayer graphene 3' obtained in this example, and the ice film thickness was measured using energy loss spectroscopy (EELS). ).

FIG. 9A shows an annular dark-field image (STEM-ADF image) of the stack of bilayer graphene 3' and porous amorphous carbon film 2 after forming glassy ice. The broken line in FIG. 9A indicates the location where measurements were performed in this example.

FIG. 9B shows a typical valence electron excitation spectrum in the low-loss region obtained from the hole portion where the bilayer graphene 3' is bridged. Peaks attributed to interband transitions of ice are observed at 8.8 eV, 10.6 eV, and 14.5 eV. Furthermore, peaks attributable to surface and volume plasmons are observed at 17 eV and 21 eV, respectively. Therefore, the presence of ice is recognized on the two-layer graphene 3' that bridges the pores. Further, a line profile of the ice film thickness calculated using the ratio of elastically scattered electrons to inelastically scattered electrons is shown in FIG. 9C. The average thickness of the ice film in the crosslinked part of the bilayer graphene 3' is 24.5 nm ± 2.9 nm, and a fairly uniform ice film is formed within the crosslinked part of the pore with a diameter of 2.0 μm. is suggested.

<Demonstration using cryo-electron microscopy>
Next, the virus was observed using the transmission electron microscope sample support obtained in this example. Glassy thin ice containing the target sample, TMV having a linear structure with a diameter of 18 nm and a length of 30 nm, was formed on the bilayer graphene 3' by an ice embedding method. FIG. 10 shows a TEM image of TMV embedded in glassy ice formed on bilayer graphene 3'.

In FIG. 10, it can be seen that many linear objects are lined up, and the periodic structure of the TMV can be observed with high resolution and high contrast. This result is considered to be due to the high signal/noise ratio, high electron beam transparency, and high electrical conductivity expected from graphene.

As described above, UV ozone treatment limited to one side of bilayer graphene gives high hydrophilicity to the upper layer, which increases the affinity between the graphene surface and the aqueous solution, which leads to the thinning of glassy ice to a higher level. Furthermore, graphene's inherent high electrical conductivity suppresses image deterioration during TEM observation, making it highly adaptable to low-temperature biological structure observation.

1 Metal grid support 2 Porous amorphous carbon membrane 3 Two-layer graphene (before UV ozone treatment)
3' Bilayer graphene (after UV ozone treatment)

Claims (9)

a support element having a plurality of holes;
two layers of graphene covering at least some of the plurality of holes;
has
The first graphene film of the two layers of graphene is a hydrophilized graphene film in which hydrophilic groups are attached to at least a portion of defects caused by ultraviolet ozone treatment ,
The amount of defects in the first graphene film is greater than the amount of defects in graphene films other than the first graphene film,
A transmission electron microscope sample support characterized in that the ratio of D band intensity to G band intensity in the Raman spectrum of the two-layer graphene is 0.04 or more and 0.5 or less. a support element having a plurality of holes;
two layers of graphene covering at least some of the plurality of holes;
has
The first graphene film of the two layers of graphene is a hydrophilized graphene film in which hydrophilic groups are attached to at least a portion of defects caused by ultraviolet ozone treatment ,
The amount of defects in the first graphene film is greater than the amount of defects in graphene films other than the first graphene film,
A transmission electron microscope sample support, wherein the contact angle of the two layers of graphene measured by the θ/2 method is 25° or more and 60° or less. The transmission electron microscope sample support according to claim 1 or 2, wherein the support element is a support film attached to a metal grid support having a mesh or the metal grid support. Two layers of graphene formed by a plasma CVD (Chemical Vapor Deposition) method are attached to a transmission electron microscope sample support having a support element having multiple holes,
The first graphene film of the two graphene layers is treated with ultraviolet ozone so that the ratio of the D band intensity to the G band intensity in the Raman spectrum of the two graphene layers is 0.04 or more and 0.5 or less. I do,
A method for manufacturing a transmission electron microscope sample support, characterized in that: Two layers of graphene formed by a plasma CVD (Chemical Vapor Deposition) method are attached to a transmission electron microscope sample support having a support element having multiple holes,
A method for manufacturing a transmission electron microscope sample support, characterized in that a first graphene film of the two layers of graphene is subjected to ultraviolet ozone treatment adjusted according to a target degree of hydrophilicity. a supporting element having a plurality of pores; and two layers of graphene covering at least some of the plurality of pores, wherein a first graphene film of the two layers of graphene is free from defects caused by ultraviolet ozone treatment. a hydrophilized graphene film having a hydrophilic group attached to at least a portion of the transmission electron microscope sample support, wherein the first graphene film has a larger number of defects than the first graphene film other than the first graphene film. An aqueous solution containing the target to be analyzed is dropped onto a transmission electron microscope sample support having a degree of hydrophilicity to form an ice film with a thickness corresponding to the size of the target to be analyzed, and then frozen. Sample preparation method. 7. The sample preparation method according to claim 6, wherein a transmission electron microscope sample support having a higher degree of hydrophilicity is selected as the size of the analysis target is smaller, and the thickness of the ice obtained by the freezing becomes thinner. a support element having a plurality of holes;
two layers of graphene covering at least some of the plurality of holes;
has
The first graphene film of the two layers of graphene is a hydrophilized graphene film in which hydrophilic groups are attached to at least a portion of defects caused by ultraviolet ozone treatment ,
The amount of defects in the first graphene film is greater than the amount of defects in graphene films other than the first graphene film,
A transmission electron microscope sample support characterized in that the two layers of graphene have an average sheet resistance of 364±10Ω/□. Two layers of graphene formed by a plasma CVD (Chemical Vapor Deposition) method are attached to a transmission electron microscope sample support having a support element having multiple holes,
Performing ultraviolet ozone treatment on the first graphene film of the two layers of graphene so that the rate of increase in sheet resistance of the two layers of graphene is 11% or less.
A method for manufacturing a transmission electron microscope sample support, characterized in that:

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