JP2021034135A - Transmission electron microscope sample support, manufacturing method thereof, and sample preparation method using the same - Google Patents

Abstract

To provide a transmission electron microscope sample support having excellent properties in hydrophilicity and electrical conductivity.SOLUTION: A transmission electron microscope sample support includes a support element with a plurality of holes, two layers of graphene covering at least some of the holes. A first graphene film of the two graphene layers is a hydrophilized graphene film in which a hydrophilic group is attached to at least a part of the defects, and the amount of defects in the first graphene film is larger than that of a graphene other than the first graphene film. Such two graphene layers are produced, for example, by subjecting the two graphene layers formed by the plasma CVD method to ultraviolet ozone treatment.SELECTED DRAWING: Figure 3

Description

The present invention relates to a transmission electron microscope sample support, a method for producing 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. In the cryo-electron microscopy, the target sample is fixed inside amorphous thin ice and observed by instantly freezing it together with an aqueous solution or the like without performing any staining or chemical fixation on the target sample.

Sample preparation for cryo-electron microscopy generally has a structure in which a metal grid support such as copper, gold, or molybdenum is stretched with an amorphous carbon thin film or the like having a large number of holes. A grid is used. A target sample such as a protein dissolved in water is dropped onto this grid, excess water is absorbed from both sides of the grid using filter paper or the like, and the sample is rapidly frozen to enter the pores of the amorphous carbon film. A thin ice film containing the target sample has been prepared.

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

To solve this problem, a method has also been used in which graphene oxide dispersed in an aqueous solution or the like is dropped onto an amorphous carbon film to form a graphene oxide film in a form that closes the pores of the amorphous carbon film. .. However, in the formation of a graphene oxide film by dropping, it is difficult to control the number of layers and the electrical conductivity is inferior to that of metals and the like, so there is a concern that the image may be deteriorated during observation due to charge-up and drift due to the charge-up. ..

In order to make the graphene hydrophilic, there is a conventional technique for producing graphene hydride by partially hydrogenating the graphene of a single layer with hydrogen plasma and using it for a transmission electron microscope sample support, but it is hydrophilic. The degree of conversion is low, and since it is a single layer, its electrical conductivity and mechanical strength are low.

Special Table 2016-534508 Special Table 2016-532267

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

The transmission electron microscope sample support according to the present invention has a support element having a plurality of pores and two layers of graphene covering at least a part of the pores of the plurality of pores. 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 part of the defects, and the amount of defects in the first graphene film is a graphene other than the first graphene film. It is characterized by having more defects than the film.

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

Further, the sample preparation method for a transmission electron microscope according to the present invention is to drop an aqueous solution containing an analysis target onto the transmission electron microscope sample support and freeze it.

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

Further, according to another aspect, by using such a transmission electron microscope sample support, a thinner ice film can be formed, and a better image can be obtained by cryo-electron microscopy or the like. become.

FIG. 1 is a diagram showing a configuration of a transmission electron microscope sample support in a state before UV 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 UV ozone treatment. FIG. 4 is a diagram for showing changes in the Raman spectrum of bilayer graphene before and after treatment with ultraviolet ozone. FIG. 5 is a diagram showing the results of Lorentzian peak fitting analysis for the 2D band of two-layer 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 hydrophilicity for the two-layer graphene on the metal substrate (copper foil catalyst layer). FIG. 8A is a diagram showing a high-resolution STEM (Scanning Transmission Electron Microscope) image of the two-layer graphene before the ultraviolet ozone treatment. FIG. 8B is a diagram showing a high-resolution STEM image of the two-layer graphene after UV ozone treatment. FIG. 9A is a diagram showing an annular dark-field image of a laminate of two-layer graphene and a 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 the pore portion where the two-layer graphene is crosslinked. FIG. 9C is a diagram showing a line profile of ice film thickness calculated using the ratio of elastically scattered electrons and inelastically scattered electrons. FIG. 10 is a diagram showing a low-temperature TEM observation image of tobacco mosaic virus on glassy thin ice formed on two-layer graphene.

Hereinafter, a transmission electron microscope sample support according to the present invention, a method for producing the same, and a sample preparation method using the same will be described based on embodiments and examples. Duplicate explanations will be omitted as appropriate. In addition, when "~" is described between two numerical values to represent a numerical range, these two numerical values are also included in the numerical range.

[Outline of Embodiment of the present invention]
As a result of intensive research to achieve the above problems, the present inventors attach a graphene film grown in two layers with high controllability by the plasma CVD method on a metal substrate to a metal grid support, and perform ultraviolet ozone treatment. It was found that one layer of the two-layer graphene can be selectively hydrophilized by applying it only to one side of the two-layer graphene. As a result, the hydrophilized graphene film having a high surface hydrophilic function is supported by the highly crystalline graphene film having the original high electrical conductivity of graphene, and the transmission electron microscope sample support with the ultra-thin film of two atomic layers is supported. You can get a body.

By selectively hydrophilizing one layer of the two-layer graphene in this way, the hydrophilized graphene film functions as a film that improves the affinity with the aqueous solution containing the target sample, and the other The graphene film provides the original property of high electrical conductivity and also functions to enhance the strength of the support film that supports the aqueous solution containing the dropped target sample. These two-layer graphene with high hydrophilicity, high electrical conductivity, and high strength overcome the two conventional problems of further thinning of amorphous ice in cryo-electron microscopy and deterioration of 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 pores (for example, the porous amorphous carbon film 2 or the metal grid support 1 in FIG. 3) and at least a part of the plurality of pores. It has two layers of graphene covering the pores (eg, two layers of graphene 3'in FIG. 3). The first graphene film (for example, the upper graphene film in FIG. 3) of the two layers of graphene is a hydrophilized graphene film in which a hydrophilic group is attached to at least a part of the defects, and the defects of the first graphene film. The amount is larger than the defect amount of the graphene film other than the first graphene film (for example, the graphene film in the lower layer of FIG. 3).

By providing such two layers of graphene, the hydrophilicity can be enhanced by the first graphene film, and high electrical conductivity can be obtained by the graphene film other than the first graphene film. Furthermore, the mechanical strength can be enhanced by forming a graphene film having two layers instead of one layer. As a result, when preparing the sample, the impact due to the dropping of the sample is sufficient even when the sample is dropped while controlling the dropping amount from the nozzle using inkjet technology instead of dropping the sample with a micropipette or the like. It is considered to have the strength to withstand.

The support element described above is a support film (for example, the porous amorphous carbon film 2 in FIG. 3) or a metal grid support (for example, the metal grid support in FIG. 3) attached to a metal grid support having a mesh. 1). The support film may be something like an amorphous carbon film, or may be another commonly used material such as a silicon nitride film or a silicon oxide film. Instead of using such a support film, two layers of graphene may be attached to a metal grid support having a mesh.

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

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

In such a transmission electron microscope sample support, two layers of graphene formed by a plasma CVD method are attached to a transmission electron microscope sample support having a support element having a plurality of holes (for example, S4 in FIG. 2). , The first graphene film of the two layers of graphene is subjected to ultraviolet ozone treatment (for example, S6 in FIG. 2).

This ultraviolet ozone treatment is adjusted according to, for example, the target degree of hydrophilicity. For example, when the degree of hydrophilicity is increased, the intensity of ultraviolet rays is increased or the time of ultraviolet ozone treatment is lengthened. On the other hand, when the degree of hydrophilicity is lowered, the ultraviolet intensity is lowered or the time for ultraviolet ozone treatment is shortened. As a result, when the size of the analysis target contained in the sample is small, a thinner ice film can be formed by increasing the ultraviolet intensity or lengthening the treatment time to increase the degree of hydrophilicity. On the other hand, when the size of the analysis target contained in the sample is large, an ice film having a thickness including the analysis target can be formed by lowering the ultraviolet intensity or shortening the treatment time.

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

At the time of sample preparation, an aqueous solution containing an analysis target is dropped onto the transmission electron microscope sample support as described above and frozen. As a result, a thinner ice film can be produced as compared with the conventional case, so that a clearer image can be obtained by observation by cryo-electron microscopy. When the amorphous carbon film is used, the film thickness of the ice film is about 74 to 114 nm, which is suppressed to about 1/3 to 1/5 in the present embodiment.

It is preferable to use a transmission electron microscope sample support having a degree of hydrophilicity according to the size of the analysis target. This is because, as described above, an ice film having an appropriate thickness can be formed on the analysis target. More specifically, the larger the size of the analysis target, the lower the degree of hydrophilicity of the transmission electron microscope sample support is selected, and the thickness of the ice obtained by the above freezing becomes thicker. On the contrary, the smaller the size of the analysis target, the higher the degree of hydrophilicity of the transmission electron microscope sample support is selected, and the film thickness of the ice obtained by the above freezing becomes thinner.

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

<Manufacturing method of transmission electron microscope sample support>
In this embodiment, it is premised that a metal grid support with a porous amorphous carbon film to which a porous amorphous carbon film having a large number of holes is attached to the metal grid support has already been obtained. Then, for the first step of attaching the two-layer graphene before the ultraviolet ozone treatment (also referred to as UV ozone treatment) to the metal grid support with the porous amorphous carbon film, and for the upper layer of the two-layer graphene. The second step of performing the ultraviolet ozone treatment is performed.

In order to make the following description easy to understand, FIG. 1 first shows the configuration 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 further attached on the porous amorphous carbon film 2, and the transmission electron microscope sample support is these. It is a laminated body of. The two-layer graphene 3 extends over at least a part of the pores of the porous amorphous carbon film 2.

One hole of the mesh in the metal grid support 1 is, for example, a rectangle having a side of about 100 μm, and the hole of the porous amorphous carbon film 2 is, for example, a circle having a diameter of 2 μm. The film thickness of the porous amorphous carbon film 2 is about 10 to 25 nm. The film thickness of the two-layer graphene is approximately 0.67 to 0.70 nm.

Next, a method for manufacturing the transmission electron microscope sample support will be described with reference to FIG.

First, two-layer graphene 3 is generated on a copper foil catalyst layer (that is, 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. That thing.

Next, polymethylmethacrylate (PMMA), polycarbonate (PC), photoresist (PR), etc. are applied to the grown two-layer graphene 3 by a spin coater, and an organic thin film layer (protective film) having a thickness of about 100 nm to 10 μm is applied. ) Is formed (S2).

Then, after drying the organic thin film layer in the air or a vacuum environment for about 10 to 30 minutes, the metal substrate is removed by using a chemical etching method (S3). Here, as the etchant solution, _{a reactive ion etching solution such as ferric chloride (FeCl 3} ), ammonium persulfate ((NH ₄ ) ₂ S ₂ O ₈ ), hydrochloric acid (HCl), potassium hydroxide (KOH) is used. It can be used. A structure having a two-layer graphene having an organic thin film layer formed on the etchant solution is floated, and infiltration is performed until the metal substrate is completely removed. After the metal substrate is completely etched, it is transferred from the etchant solution phase to the cleaning liquid phase using _{filter paper, glass, SiO 2 or the like.} Ultrapure water, deionized water, or the like can be used as the cleaning solution.

Then, using the metal grid support 1 provided with the porous amorphous carbon film 2, the two-layer graphene 3 having the organic thin film layer formed on the cleaning liquid phase is attached so as to be scooped up, and then at room temperature to 90 ° C. It is dried under a certain temperature (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 infiltration into an organic solvent such as acetone (S5).

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

The metal grid support 1 provided with the two-layer graphene 3 obtained by vacuum drying was further subjected to heat treatment at a temperature of 200 to 600 ° C. for about 1 to 5 hours in a higher vacuum, and the organic could not be completely removed by the organic solvent. It is possible to remove the thin film layer. As a result, the effect of further cleaning the graphene surface can be expected.

After the heat treatment under high vacuum, graphene is hydrophilized with a UV ozone cleaner (S6). For UV ozone treatment, Filgen's 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 having 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 binding energies of the carbon atoms constituting graphene are 347.7 KJ / mol and 607 KJ / mol in single bond and double bond, respectively, the carbon-carbon bond in the upper graphene of the two-layer graphene is broken by irradiation with ultraviolet rays. And small defects occur. Graphene is hydrophilized by adsorbing hydrophilic groups such as hydroxyl groups (-OH) and carboxyl groups (-COOH) on the defects.

The metal grid support 1 to which the two-layer graphene 3 was transferred was placed on a slide glass, quartz glass, a Si substrate, a metal support, or the like, and installed inside the device so that ultraviolet light was irradiated only on one side. .. First, oxygen gas was first introduced into the apparatus for about 30 minutes to replace the atmospheric atmosphere in the apparatus with an oxygen atmosphere. The introduction time of oxygen gas is about 10 to 30 minutes in some cases. Then, it was irradiated with ultraviolet rays for 1 to 30 minutes. The distance between the ultraviolet lamp and the sample was 10 cm. This interval is about 5 to 20 cm in some cases.

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

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

<Evaluation of hydrophilicity by contact angle (wetting property)>
The contact angle was measured by the θ / 2 method in order to evaluate the degree of hydrophilicity of the two-layer graphene 3'. A contact angle meter Dropmaster501 manufactured by Kyowa Interface Science Co., Ltd. was used for the contact angle measurement, and ultrapure water was used for the droplets. In measuring the contact angle of the two-layer graphene, the two-layer graphene 3 (UV ozone treatment time 0 minutes) on the copper foil catalyst (metal substrate) and each UV ozone treatment time (5 minutes, 10 minutes, 20 minutes) Measurements were carried out 5 times with respect to 2-layer graphene 3', and the average value was calculated.

<About crystallinity evaluation (Raman spectroscopic measurement method)>
Raman scattering spectroscopic measurements were performed on the verification examples and examples described below. An XploRa type machine manufactured by HORIBA, Ltd. was used as the Raman spectrum measuring device. 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 spectroscope 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 5 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. For monolayer graphene in use excitation wavelength 514.5 nm, 2D band, G band, the peak position of the D-band, respectively 2700 cm ^-1, 1582cm ^-1, seen in the vicinity of 1350 cm ^-1. The G band is due to a normal 6-membered ring, and the D band is a peak due to a defect in the normal 6-membered ring. The 2D band is due to the overtones of the D band. If both G-band and 2D-band are observed in Raman spectrum, the membrane 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. When the excitation wavelength used is further shortened, the 2D band shifts to the higher wavelength side.

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

<Evaluation of electrical conductivity by sheet resistance>
As a characteristic evaluation of electrical conductivity, the sheet resistance of graphene transferred onto a PET film having a thickness of 118 μm was measured using a non-contact resistance measuring device (model: EC-80 manufactured by Napson Corporation). First, a heat 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 by a chemical etching method. Ammonium persulfate ((NH ₄ ) ₂ S ₂ O ₈ ) was used as the etchant solution. The heat-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 surface side of the heat-release sheet / graphene laminate. Then, the heat release sheet was peeled from the heat release sheet / graphene / PET film laminate by heat treatment at 80 ° C. in the atmosphere to obtain a graphene / PET film laminate. The electrical conductivity according to this example was measured 5 times for a sample of a graphene / PET film laminate of 4.0 cm × 4.0 cm and evaluated by the average value.

<Evaluation of 2-layer graphene before and after UV ozone treatment by STEM>
Two-layer graphene was observed before and after UV ozone treatment with a scanning transmission electron microscope (STEM). For STEM observation, a JEM-2100F model equipped with a DELTA aberration correction device manufactured by JEOL Ltd. was used with an acceleration voltage of 60 kV. The cold cathode field emission gun had a zero loss energy resolution of 0.4 eV and was used with an electron probe diameter of approximately 0.1 nm and a current value of 30 pA.

An Annular Dark Field (ADF) image was observed using a Gatan imaging filer detector GIF Quantum for STEM image observation. The observation was carried out using a heating holder manufactured by JEOL Ltd. in order to prevent contamination of the sample due to contamination coming from the environment in the electron microscope, and controlled at about 500 ° C.
<About sample preparation by ice embedding method>
Sample preparation was performed by the ice embedding method using a metal grid support 1 (that is, a transmission electron microscope sample support) equipped with UV ozone-treated two-layer graphene 3'. In the sample preparation by the ice embedding method, a bitrobot manufactured by FEI was used. First, the environment in the device was set to a humidity of 90% or more and a temperature of 5 ° C or less. Next, the metal grid support 1 was fixed with X tweezers and installed inside the chamber of the apparatus. After that, an aqueous solution containing tobacco mosaic virus (TMV: Tabaco Mosaic Virus) was dropped from the side of the metal grid support 1 by 1.7 μmL with a micropipette, and the excess solution was sucked up with a filter paper to instantly liquefy the inside of the ethane. Instant freezing was performed by infiltrating the virus.

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

In addition, a cryo-holder-626 Gatan US manufactured by Gatan was used for transporting the frozen sample into the electron microscope and maintaining the low temperature in the electron microscope. The temperature in the electron microscope was maintained at about -175 ° C. by a Gatan Smart Controller.

<Measurement of glassy thin ice thickness by electron energy loss spectroscopy>
In order to measure the film thickness of glassy thin ice prepared by the ice-wrapping method, electron energy loss spectroscopy (STEM-EELS), which is a combination of STEM and an electron spectroscope, was performed.

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。 In the STEM-EELS method, the film thickness of the target sample can be measured from the ratio of inelastically scattered electrons and elastically scattered electrons. It is known that the relative film thickness of the target sample is given by the following equation, where t is the film thickness of the target sample and λ is the mean free path of total inelastic scattering.

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

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

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

<Analysis results by Raman spectroscopy>
FIG. 4 shows two-layer graphene 3 (solid line) untreated with UV ozone (0 minutes UV ozone treatment) on a copper foil catalyst layer (metal substrate), 10 minutes (dashed line) and 20 minutes after UV ozone treatment (dashed line). ) Is shown in a typical Raman spectrum for the two-layer graphene 3'. In FIG. 4, the vertical axis represents the intensity (arbitrary unit), and the horizontal axis represents the Raman shift [cm ^-1 ].

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

Further, FIG. 5 shows a detailed curve fitting analysis result of the 2D band. In FIG. 5, the thin single-point chain line represents the Lorentzian subpeak, the thick single-point chain line represents the Raman spectrum of the two-layer graphene 3'after 10 minutes of UV ozone treatment, and the solid line represents the two-layer graphene 3 of UV ozone treatment 0 minutes. The fine dotted line represents the Raman spectrum of UV ozone-treated two-layer graphene 3'.

As a result, the 2D bands of all Raman spectra are fitted by a total of four Lorentzian subpeaks, resulting in two-layer graphene, and the laminated structure of the two-layer graphene obtained by performing this Raman spectroscopy can be seen in FIG. As described above, the strength ratio of the G / 2D band is about 2 to 3, indicating that the structure is an AB laminated structure.

Considering that the laser spot used in this example is 1.0 μm, a slight increase in the D band is observed after the UV ozone treatment, but no defect exceeding 1.0 μm is introduced, and 2 It is thought that the structure of the layer is maintained.

Next, FIG. 6 shows the relationship between the intensity ratio of the D band and the 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 the UV ozone treatment time. Considering based on the relationship between the Raman spectrum and the D / G ratio shown in FIG. 4, defect formation started and was formed at a site having an upper layer while maintaining the structure as a two-layer graphene by ultraviolet irradiation. It is considered that the amount of defects increases and the increase of the D band derived from the defects is confirmed by the expansion of the defects or the start of the formation of new defects at different sites.

<Evaluation result of electrical conductivity by sheet resistance>
As an evaluation of electrical conductivity, sheet resistance was compared with and without UV ozone treatment. As a comparison of electrical conductivity in this example, a sample subjected to UV ozone treatment time of 20 minutes was used. The average sheet resistance of the two-layer graphene 3 untreated with UV ozone was 328 ± 2.3 Ω / □, and that of the two-layer graphene 3'20 minutes after the UV ozone treatment was 364 ± 10 Ω / □. From this result, the increase rate of the sheet resistance by the UV ozone treatment for 20 minutes is about 11%. High electrical conductivity is maintained even in the sample subjected to the hydrophilization process by UV ozone treatment for about 20 minutes, which is much better than the conventionally used amorphous carbon film, graphene oxide, etc. It is considered to be electrically conductive. Therefore, it is considered that the TEM observation at a low temperature has necessary and sufficient electrical conductivity as a function for suppressing deterioration of the image due to charge-up or the like.

<Wetting property evaluation result>
As a result of the degree of hydrophilicity of the two-layer graphene 3 on the copper foil (metal substrate) by UV ozone treatment, FIG. 7 shows the relationship between the UV ozone treatment time and the contact angle between graphene and ultrapure water. 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 °. UV ozone treatment for 10 minutes or more did not show a large change in the contact angle, but the average deviation tended to increase. This is probably because the amount of carbon-carbon bond breakage due to UV irradiation increases, and as the amount of defects in the upper graphene of the two-layer graphene increases and the defects themselves expand, the surface of the lower graphene that has not been hydrolyzed comes into contact with water. It is thought that the increase in area has an effect. In the hydrophilization of graphene by UV ozone treatment, it is possible to freely produce two-layer graphene having a wide range of hydrophilicity from about 25 ° to 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 the two-layer graphene 3 before UV ozone treatment. In FIG. 8A, a laminate with an orientation shifted by about 13 ° is formed, and a moire pattern can be confirmed. No defects are found in both graphene membranes before UV ozone treatment. This indicates that high-crystal two-layer graphene is formed by the plasma CVD method, and that no defects are introduced in the transfer process or the like on the metal grid support 1.

FIG. 8B shows a STEM-ADF image of two-layer graphene 3'treated with UV ozone for 20 minutes. After UV ozone treatment, defects due to lack of carbon atoms can be seen. The contrast of the ADF image in STEM observation is observed in proportion to the square of the atomic number. Therefore, the dark part seen in FIG. 8B shows the region corresponding to the single-layer graphene. The size of the defect portion confirmed by this example was a defect consisting of several hundred defects as observed in FIG. 8B. However, the lack of one carbon atom is not confirmed in the lower 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 ultraviolet irradiation work only in the upper layer. it is conceivable that. This is a result that is not different from the analysis result of the Raman spectrum described above.

The results of these Raman spectroscopy analyzes, wettability assessments, and high-resolution observations by STEM show that the UV ozone treatment performed on the two-layer graphene 3 does not cause the introduction of defects into the lower layer of the two-layer graphene. It is shown that can be highly hydrophilic. This indicates that highly hydrophilic graphene can be formed on highly electrically conductive graphene by a simple surface treatment.

<Measurement result of glassy thin ice thickness by electron energy loss spectroscopy>
Using the transmission electron microscope sample support having the double-layer graphene 3'obtained in this example, the glassy ice was prepared on the double-layer graphene 3', and the thickness of the ice was measured by energy loss spectroscopy (EELS). ).

FIG. 9A shows an annular dark-field image (STEM-ADF image) of the laminate of the two-layer graphene 3'and the porous amorphous carbon film 2 after forming the glassy ice. The broken line in FIG. 9A shows the place where the measurement was performed in this embodiment.

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

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

In FIG. 10, it is observed that a number of linear objects are lined up, and the periodic structure of the TMV is observed with high resolution and high contrast. This result is considered to be due to the high signal-to-noise ratio, high electron beam transmission, and high electrical conductivity that graphene originally expects.

As described above, by applying UV ozone treatment to only one side of the two-layer graphene to give high hydrophilicity to the upper layer, the high affinity between the graphene surface and the aqueous solution makes the glassy ice thin. In addition, the high electrical conductivity, which is the original characteristic of graphene, suppresses image deterioration during TEM observation, so it can be said that it is highly adaptable to low-temperature biological structure observation.

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

Claims (10)

Support elements with multiple holes and
A two-layer graphene covering at least a part of the plurality of holes,
Have,
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 part of the defect.
A transmission electron microscope sample support characterized in that the amount of defects in the first graphene film is larger than the amount of defects in graphene films other than the first graphene film. The transmission electron microscope sample support according to claim 1, wherein the support element is a support film attached to a metal grid support having a mesh or the metal grid support. The transmission electron microscope sample support according to claim 1 or 2, wherein the ratio of the D-band intensity to the G-band intensity in the Raman spectrum of the two layers of graphene is 0.04 or more and 0.5 or less. The transmission electron microscope sample support according to any one of claims 1 to 3, wherein the contact angle of the two layers of graphene by the θ / 2 method is 25 ° or more and 60 ° or less. A two-layer graphene film formed by a plasma CVD (Chemical Vapor Deposition) method is attached to a transmission electron microscope sample support having a support element having a plurality of holes.
A method for producing a transmission electron microscope sample support, which comprises subjecting the first graphene film of the two layers of graphene to ultraviolet ozone treatment. The ratio of the D-band intensity to the G-band intensity in the Raman spectrum of the two layers of graphene is 0.04 or more and 0.5 or less, or
The contact angle of the two layers of graphene by the θ / 2 method is 25 ° or more and 60 ° or less.
The production method according to claim 5, wherein the ultraviolet ozone treatment is performed. The production method according to claim 5 or 6, wherein the ultraviolet ozone treatment is adjusted according to a target degree of hydrophilicity. A sample preparation method for a transmission electron microscope, wherein an aqueous solution containing an analysis target is dropped onto the transmission electron microscope sample support according to any one of claims 1 to 3 and frozen. The sample preparation method according to claim 8, wherein a transmission electron microscope sample support having a degree of hydrophilicity according to the size of the analysis target is used. The sample preparation method according to claim 9, 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 film thickness of ice obtained by freezing becomes thinner.

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