JP2008027763A - Test piece carrier for transmission type electron microscope and its manufacturing method, observation and crystal structure analysis methods using the same, and test piece for transmission type electron microscope and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a test piece carrier for a transmission type electron microscope and its manufacturing method, a test piece for the transmission type electron microscope and its manufacturing method, as well as observation and crystal structure analysis methods using the test piece for the transmission type electron microscope, wherein observation using the transmission type electron microscope can obtain observation data of an object to be observed in various directions. <P>SOLUTION: A micro-grid 10 comprises a grid body 12, a catalyst layer 14 formed on the edge of the grid body 12, and a carbon nanotube 16 formed on the top surface of the catalyst layer 14. A test piece 20 comprises the micro-grid 10 and an object to be observed (a protein layer 18 for example) which is cladded at the lateral side of the carbon nanotube 16 used as a component of the micro-grid 10. The observation method is carried out in such a manner that the test piece 20 which is set up on a test piece holder is mounted on a transmission type electron microscope. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a transmission electron microscope sample holder and its manufacturing method, a transmission electron microscope sample and its manufacturing method, and an observation method and a crystal structure analysis method using a transmission electron microscope sample. .

  Generally, when analyzing a crystal structure, neutron diffraction or precise X-ray diffraction is often used, and a crystal grown to a size of several millimeters is required as a sample. However, proteins are generally difficult to crystallize, and it is very difficult to grow crystals to a size of several millimeters.

  As a method for observing and evaluating a minute and thin sample, a method using a transmission electron microscope or the like is known. In particular, the high-resolution transmission electron microscope is capable of observing a minute thin film sample at the atomic level, so that it is considered possible to analyze the structure of the minute thin film protein crystal.

  On the other hand, a technique related to a carbon nanotube structure has been proposed in which a protein dispersion is spread on a liquid surface to coat the protein, and the modified protein is coated on the side surface of the carbon nanotube (Patent Document 1).

In this carbon nanotube structure, since the protein layer coated on the side surface of the carbon nanotube is thin, it is difficult to analyze the crystal structure by neutron diffraction or X-ray diffraction. However, if a transmission electron microscope is used, the protein layer It is thought that the crystal structure analysis of
Republished Patent W02004 / 041719

  As described above, the observation object typified by protein is coated on the side surface of the supporting core typified by carbon nanotubes, and the coating layer of the supporting core coated with the side surface is observed with a transmission electron microscope. Various information including the crystal structure of the observation object can be obtained.

  However, in observation with a transmission electron microscope, the observation direction is often limited. Therefore, when the observation object is observed with orientation, predetermined observation data may not be obtained. For this reason, it is required that observation data of observation objects in various directions can be acquired in the presence of observation objects facing in various directions.

  Therefore, in view of the above problems, the present invention provides a transmission electron microscope sample holder capable of acquiring observation data of an observation object in various directions by observation using a transmission electron microscope, and manufacturing the same. Provided are a method, a sample for a transmission electron microscope and a manufacturing method thereof, and an observation method and a crystal structure analysis method using the sample for a transmission electron microscope.

The above problem is solved by the following means. That is,
A sample holder for a transmission electron microscope of the present invention is a holder body, a carrier core for carrying an observation object, and a carrier core provided on at least a part of an edge of the holder body; It is characterized by having.

  In the transmission electron microscope sample holder of the present invention, the holder body is composed of, for example, a curved surface or a plurality of surfaces depending on the shape thereof. It is suitable. For this reason, the carrying core formed in the edge part of a holding body main body will be formed facing various directions. Therefore, as a result, the observation object held on the transmission electron microscope sample holder also faces various directions, and even if the observation direction is limited due to the nature of the transmission electron microscope, It is possible to obtain observation data of the observation object in the direction of.

  In the sample holder for a transmission electron microscope of the present invention, the carrier core can be provided on the holder body via a catalyst. The catalyst is preferably at least one element selected from Co, Ni, and Fe.

  In the sample holder for a transmission electron microscope of the present invention, it is preferable that the support core includes carbon nanotubes.

  The transmission electron microscope sample of the present invention comprises the transmission electron microscope sample holder of the present invention and an observation object carried on the carrying core of the transmission electron microscope sample holder. It is a feature.

  In the transmission electron microscope sample of the present invention, as described above, the observation core carried on the carrier core is formed because the carrier core of the transmission electron microscope sample holder is formed in various directions. Therefore, even if the observation direction is limited due to the properties of the transmission electron microscope, observation data of the observation object in various directions can be acquired.

  In the transmission electron microscope sample of the present invention, it is preferable that the observation object is a protein.

  The method of manufacturing a sample holder for a transmission electron microscope of the present invention includes a first step of preparing a holder body, and a support core for supporting an observation object on at least a part of an edge of the holder body. And a second step of forming.

  By the method for manufacturing a sample holder for a transmission electron microscope of the present invention, the sample holder for a transmission electron microscope of the present invention can be obtained.

  In the method for manufacturing a sample holder for a transmission electron microscope of the present invention, the second step includes forming a catalyst on at least a part of the edge of the holder body, and then placing the support core on the catalyst. It can be set as the process of forming.

  In the method for producing a sample holder for a transmission electron microscope of the present invention, it is preferable that the catalyst is at least one element selected from Co, Ni, and Fe.

  In the method for manufacturing a sample holder for a transmission electron microscope of the present invention, it is preferable that the support core includes carbon nanotubes.

  The method for producing a sample for a transmission electron microscope of the present invention includes a step of preparing a sample holder for a transmission electron microscope of the present invention, and the support core formed on at least a part of the edge of the holder body. And a step of supporting the observation object.

  The transmission electron microscope sample of the present invention can be obtained by the method for manufacturing a transmission electron microscope sample of the present invention.

  In the method for manufacturing a sample for a transmission electron microscope of the present invention, it is preferable that the observation object is a protein.

  The observation method of the present invention includes a step of holding the transmission electron microscope sample of the present invention in a sample holder, and mounting the sample holder on the transmission electron microscope, thereby allowing the transmission electron microscope sample to be transferred to the transmission type. And a step of observing the transmission electron microscope sample with the transmission electron microscope.

  In the observation method of the present invention, since the transmission electron microscope sample of the present invention is used, even if the observation direction is limited due to the properties of the transmission electron microscope, observation data of the observation object in various directions is acquired. It becomes possible to do.

  The crystal structure analysis method of the present invention is characterized by having an observation step of observing with the observation method of the present invention and an analysis step of analyzing the crystal structure using the observation data obtained by the observation step. .

  In the crystal structure analysis method of the present invention, since the transmission electron microscope sample of the present invention is used, observation data in various directions can be acquired even if the observation direction is limited due to the properties of the transmission electron microscope. Is possible. For this reason, it is possible to perform a more accurate crystal structure analysis than in the past.

  The protein crystal structure analysis method of the present invention is prepared by preparing the holder body of the present invention, forming a catalyst on at least a part of the edge of the holder body, and then forming a carbon nanotube on the catalyst. A sample holder manufacturing process for manufacturing a sample holder for a transmission electron microscope, and a sample manufacturing process for manufacturing a sample for a transmission electron microscope by supporting a protein on the carbon nanotubes of the sample holder for the transmission electron microscope A sample introduction step for introducing the sample for the transmission electron microscope into the transmission electron microscope by attaching the sample holder to the transmission electron microscope after holding the sample for the transmission electron microscope in the sample holder; An observation step of observing the transmission electron microscope sample with the transmission electron microscope, and using the observation data obtained by the observation step An analysis step of analyzing a crystal structure is characterized by having a.

In the protein crystal structure analysis method of the present invention, as described in the sample holder for a transmission electron microscope of the present invention, since the carbon nanotubes are formed in various directions, they are supported on the carbon nanotubes. Even if the direction of observation of proteins is limited due to the nature of the transmission electron microscope, it is possible to obtain protein observation data in various directions, and to analyze the crystal structure of the protein more accurately than before. I can do it.

  According to the present invention, it is possible to obtain observation data of an observation object in various directions by observation using a transmission electron microscope, a sample holder for a transmission electron microscope, a method for manufacturing the same, transmission electron It is possible to provide an observation method and a crystal structure analysis method using a sample for a microscope, a manufacturing method thereof, and a sample for a transmission electron microscope.

  The present invention will be described below with reference to the drawings. It should be noted that members having substantially the same function are given the same reference numerals as those in the drawings, and redundant description may be omitted.

(First embodiment)
FIG. 1 is a schematic diagram showing a configuration of a transmission electron microscope microgrid (hereinafter, sometimes simply referred to as a microgrid) according to the first embodiment. FIG. 1A is a plan view, and FIG. (B) is an end view of 1B-1B.

  As shown in FIG. 1, the microgrid 10 (transmission electron microscope sample holder) according to the first embodiment includes a grid body 12 (holder body) and a catalyst layer 14 (catalyst) formed on the grid body 12. ) And carbon nanotubes 16 (supported core) formed on the surface of the catalyst layer 14.

Examples of the grid body 12 include those made of metal or alloy, or those obtained by forming an oxide conductor on a metal or alloy by sputtering or pulse laser. Examples of the metal include Mo, Pt, Rh, and Cu, and examples of the oxide conductor include RuO ₂ , IrO ₂ , and SrRuO ₃ . In consideration of thermal conductivity and electrical conductivity, the material of the grid body 12 is preferably Mo, Pt, Rh, or Cu. Further, considering the heat resistance, the material of the grid body 12 is more preferably Mo, Pt, or Rh. In the first embodiment, the grid body 12 is made of Mo.

  The grid body 12 is constituted by a disk having an opening at the center, for example. And the carbon nanotube 16 is formed in the edge which comprises the opening part so that it may protrude into an opening part.

Moreover, the surface which comprises the opening part side edge part of the grid main body 12 has faced various directions. Specifically, for example, the inner peripheral surface 11 of the grid body 12 is a curved surface, and the normal line of the inner peripheral surface 11 faces different directions depending on the position. In other words, the inner peripheral surface 11 of the grid body 12 is two-dimensionally oriented in various directions on a plane parallel to the disk upper surface 13.
In addition, when the inner peripheral surface 11 of the grid main body 12 is configured by a curved surface and the connecting portion between the inner peripheral surface 11 and the disk upper surface 13 is a curved surface, the normal line of the curved surface forming the connecting portion varies depending on the position. Will turn to. In other words, the curved surface is directed in various directions three-dimensionally with an angle with respect to a plane parallel to the disk upper surface 13.

  The shape of the grid main body 12 is not limited to the above shape, and various disk shapes having openings such as a disk shape in which a plurality of openings are arranged in a lattice shape can be cited. Furthermore, the shape of the grid body 12 is not limited to a disk shape having an opening, for example, a disk shape having a notch in a part thereof, a polygonal plate shape having an opening, a crescent shape, and inner centers of two crescent shapes Any shape can be applied as long as the edge of the grid body 12 is exposed in a state where the sample is set on a sample holder or the like, such as a shape in which the portions are connected to each other.

The catalyst layer 14 is a thin-film catalyst having a predetermined thickness, for example, and is arranged at equal intervals with a predetermined area on the opening side edge of the grid body 12. Since the growth of the carbon nanotubes 16 with respect to the catalyst layer 14 does not depend on the film thickness, the thickness of the catalyst layer 14 can be applied in the range of several nm to several μm.
Since the carbon nanotubes 16 are formed only on the surface of the catalyst layer 14, the carbon nanotubes 16 can be easily formed at predetermined positions by controlling the arrangement of the catalyst layer 14. Therefore, the arrangement of the catalyst layers 14 is not limited to equal intervals. If the catalyst layers 14 are formed on at least a part of the edge of the grid main body 12, the grid main bodies can be arranged even if the catalyst layers 14 are arranged at non-equal intervals. Even if it is formed on the entire surface of 12, there is no problem.
Depending on the method of forming the carbon nanotubes 16, the carbon nanotubes may be formed at the edge of the grid body 12 without using a catalyst. In that case, it is possible not to use the catalyst layer 14.

Further, since the catalyst layer 14 is a thin film having a predetermined thickness, the surface shape of the catalyst layer 14 directly reflects the surface constituting the opening side edge of the grid body 12 and faces various directions. Yes. Therefore, as described above, the carbon nanotubes 16 exist in various states.
Note that the catalyst layer 14 is not limited to a thin film having a predetermined thickness. For example, even if the catalyst layer 14 is a film having a non-constant thickness, a porous film, or a thing interspersed with granular materials, the surface shape has various results. Applicable if facing the direction. Furthermore, the catalyst layer 14 is not limited to a single layer film, and may be a multilayer film, for example, and the constituent layers may be single crystal, polycrystal, or amorphous.

  Examples of the material of the catalyst layer 14 include metals (for example, iron groups such as Ni, Fe, and Co; platinum groups such as Pd, Pt, and Rh; rare earth metals such as La and Y; transition metals such as Mo and Mn), An alloy (including two or more metals), a metal oxide (for example, titanium oxide), a mixture thereof, or the like can be given. In consideration of thermal conductivity and electrical conductivity, the material of the catalyst layer 14 is preferably a metal or an alloy. Further, when the carbon nanotubes 16 are formed on the catalyst layer 14 by chemical vapor deposition (CVD), the material of the catalyst layer 14 is at least one element selected from Co, Ni, and Fe. Something is preferable. If the catalyst layer 14 is basically a transition metal and its oxide, the catalytic action is exhibited. In the first embodiment, Fe is applied.

  The catalyst layer 14 is formed of a material suitable for each forming method and the supporting core material when the carbon nanotube 16 is formed by a method other than the CVD method, or when a material other than the carbon nanotube is used as the supporting core. can do.

  The carbon nanotubes 16 are formed only on the surface of the catalyst layer 14, and the axial direction of the nanotubes forms various angles with respect to the surface of the catalyst layer 14. FIG. 10 is a TEM image obtained by observing the carbon nanotubes 16 that actually protrude into the openings of the grid body 12 with a high-resolution transmission electron microscope (H-1500, manufactured by Hitachi, acceleration voltage: 800 kV). It can be seen from the TEM image in FIG. 10 that the carbon nanotubes are oriented in various directions. However, the carbon nanotubes shown in the TEM image of FIG. 10 are in a state of being covered with a protein layer as an observation object.

  As described above, since the surfaces constituting the edge of the grid main body 12 face in various directions, the surface of the catalyst layer 14 also faces in various directions. Accordingly, even if the axial direction of the carbon nanotubes 16 is oriented in a certain direction (for example, the vertical direction) with respect to the surface of the catalyst layer 14, the carbon nanotubes 16 having tube axes in various directions are obtained as a result. Therefore, observation data of the observation object in various directions can be acquired.

  In addition, since the carbon nanotubes 16 are very excellent in thermal conductivity and electrical conductivity, when a material having good thermal conductivity and electrical conductivity is used for the grid body 12 and the catalyst layer 14 (in the first embodiment). Is a material having relatively good thermal conductivity and electrical conductivity, and Mo is applied to the grid body 12 and Fe is applied to the catalyst layer 14.), suppressing damage to the observation object due to heat and charge-up I can do things. Further, when the observation object is a protein, by supporting it on the carbon nanotubes 16, the damage of the protein due to heat and electric charge can be reduced, and easy protein crystallization is also possible.

  The carbon nanotubes 16 may be single-walled carbon nanotubes or multi-walled carbon nanotubes. Carbon nanomaterials represented by carbon nanohorns, carbon nanofibers, fullerenes, etc. instead of carbon nanotubes; boron-based nanomaterials such as boron nitride nanotubes (BN nanotubes); composed of carbon, nitrogen, and boron such as BCN nanotubes Nanomaterials, etc., may be used as the support core. In addition, the present invention is not limited thereto, and any delocalized π electron can be used as the supporting core.

  Next, a method for manufacturing the transmission electron microscope microgrid according to the first embodiment will be described. FIG. 2 is a diagram illustrating a method for manufacturing a transmission electron microscope microgrid according to the first embodiment, according to processes.

  First, as shown in FIG. 2A, a grid body 12 is prepared. For example, a commercially available grid body (for example, a single-hole mesh 0.3 mm to a single-hole 1.5 mm mesh (manufactured by Mo, sold by Oken Shoji)) can be purchased, or a metal plate can be punched. The grid main body that can be purchased from Oken Shoji is not limited to the grid main body described above, and examples include 1 slot mesh to 4 slit mesh, 50-A mesh, 75A mesh, 100A mesh, 150A mesh, and the like, for example, 50 mesh. In this case, Pitch: 500 μm, Hole: 450 μm, Ber: 50 μm.

Next, as shown in FIG. 2B, the catalyst layers 14 are formed at equal intervals on the opening side edge of the grid body 12. Specifically, for example, after covering the grid body 12 with a metal mask having a predetermined opening, the catalyst layer 14 is formed by forming a metal film in the opening region by sputtering. Moreover, the shape and formation position of the catalyst layer 14 can be controlled by, for example, the opening shape and opening position of the metal mask.
The shape and formation position of the catalyst layer 14 can also be controlled by a photoresist method.

  As described above, the shape, arrangement, and material of the catalyst layer 14 are not particularly limited. Further, as specific sputtering conditions in forming the catalyst layer 14, generally used conditions can be applied according to the material of the catalyst. Furthermore, the method for forming the catalyst layer 14 is not limited to the sputtering method, and a known film forming method such as a vapor deposition method can be used.

Further, as shown in FIG. 2C, carbon nanotubes 16 are formed on the surface of the catalyst layer 14 by plasma CVD. Specific conditions for forming the carbon nanotubes 16 include, for example, a grid body 12 with a catalyst layer 14 installed in a CVD chamber having a pressure of 1.33 kPa (10 Torr) and a temperature of 1000 ° C., and C ₂ H as a carbon supply source. _Two gases are supplied at a flow rate of 50 sccm, and NH ₃ gas as a carrier gas is supplied at a flow rate of 150 sccm. Here, sccm is a unit of flow rate, and 1 sccm indicates that a fluid at 0 ° C. and 1 atm flows 1 cm ³ per minute.

In addition to the C ₂ H ₂ gas, for example, a gas such as methane, propylene, and ethanol can be used as the carbon supply source in the plasma CVD method. As the carrier gas, for example, Ar ₃ other than NH ₃ gas, for example, Ar , He, H ₂ or the like can also be used. Furthermore, as a method for forming the carbon nanotube 16, a CVD method other than the plasma CVD method can be used, and an arc discharge method, a laser ablation method, or the like can also be used.

  Similar support core formation methods can be used when using other nanomaterials as support cores instead of carbon nanotubes, and these support core formation methods can be selected according to the nanomaterial used as support core. Can be used.

  Furthermore, the shape, arrangement, and material of the catalyst layer 14 can be selected according to the nanomaterial used as the support core and the support core forming method, and the surface of the edge of the grid body 12 can be selected without the catalyst layer 14 being interposed. Alternatively, a method of directly growing the supporting core can be used.

  As described above, in the transmission electron microscope microgrid 10 according to the first embodiment, as described above, the surface of the grid main body 12 on which the carbon nanotubes 16 are formed faces various directions. As a result, the carbon nanotubes 16 are also formed in various directions (see FIG. 10). Therefore, when the observation target object is held on the carbon nanotube 16 and observed by the TEM, observation data of the observation target object in various directions can be acquired, so that more accurate information on the observation target object can be obtained.

(Second Embodiment)
FIG. 3 is a schematic diagram showing the configuration of a transmission electron microscope sample (hereinafter, sometimes simply abbreviated as a sample) according to the second embodiment. FIG. 3 (A) is a plan view, and FIG. ) Is an end view of 3B-3B.

  As shown in FIG. 3, the sample 20 according to the second embodiment includes the microgrid 10 according to the first embodiment and a protein layer 18 (observation target) that covers the side surfaces of the carbon nanotubes 16 of the microgrid 10. Is done. Details of the microgrid 10 are as described in the first embodiment.

  As shown in the enlarged view of FIG. 3, the protein layer 18 covers the entire side surface of the carbon nanotube 16 in a thin film shape. However, the protein layer 18 is not limited to a state in which the entire side surface of the carbon nanotube 16 is coated in a thin film shape, and the side surface of the carbon nanotube 16 is partially provided as long as the protein layer 18 is supported on the carbon nanotube 16 to the extent that observation using a TEM is possible. It may be in a state of being coated. Further, depending on the object to be observed, the carbon nanotube 16 may be partially supported on the end or inside thereof.

  As the type of the protein layer 18, bacteriorhodopsin is used in the second embodiment, but is not limited thereto, and other proteins such as halorhodopsin can be applied. The observation object is not limited to a protein, and for example, a high molecular compound other than a protein, a low molecular compound, an inorganic compound, a metal, or the like can be applied as long as it can be supported on a support core.

  Next, a method for manufacturing a transmission electron microscope sample according to the second embodiment will be described. 4 to 6 are views showing the method of manufacturing a sample according to the second embodiment for each process. Hereinafter, a form using bacteriorhodopsin as a protein will be described.

First, for example, a purple membrane extracted from a highly halophilic bacterium (Halobacterium Salinarum) by a centrifuge is dispersed in a 33% by volume dimethylformaldehyde aqueous solution to prepare a purple membrane developing solution 26 having a protein concentration of 8 μmol / l. . Next, as shown in FIG. 4, a purple membrane is formed with a syringe 27 on the liquid surface (area: 480 cm ² ) of the lower layer solution 24 (pH 3.5 hydrochloric acid) stretched on the Langmuir Blodget membrane (LB membrane) production device 22. When the developing solution 26 (volume: 180 μl) is dropped, the purple membrane developing solution 26 is developed on the liquid surface of the lower layer solution 24.

  The type of protein used in the second embodiment is bacteriorhodopsin as described above, and the bacteriorhodopsin contained in the purple membrane is a globular protein in which seven α helices are associated. As described above, when the purple membrane developing solution 26 is developed on the liquid surface of the lower layer solution 24, the association state of the α helix of the protein 28 contained in the purple membrane developing solution 26 collapses over time. Therefore, as shown in the enlarged view of FIG. 4, in order to make the α-helix association state in the protein 28 completely collapse, for example, it is allowed to stand for at least 5 hours. Thereafter, the movable barrier 30 is used to compress the surface film pressure to 15 mN / m, thereby producing a monomolecular film of the protein 28.

  Next, as shown in FIG. 5, the microgrid 10 according to the first embodiment is gently floated on the monomolecular film of the protein 28. Then, when left for 1 hour, the carbon nanotubes 16 of the microgrid 10 are covered with the protein layer 18 (FIG. 6), and the sample 20 is completed. The sample 20 is then gently lifted from the liquid level.

  At this time, as shown in FIG. 7, when the protein 28 on the liquid surface of the lower layer liquid 24 comes into contact with the carbon nanotube 16, it naturally undergoes a three-dimensional structural change, and the α helix changes to a β sheet. The protein 28 that has changed into a β sheet on the side surface of the carbon nanotube 16 covers the side surface of the carbon nanotube 16 in a layered manner to form a crystalline protein layer 18.

  Similarly, when the surface of the protein layer 18 covered with the carbon nanotubes 16 is in contact with the protein 28 in a monomolecular film state on the liquid surface of the lower layer liquid 24, the three-dimensional structure of the protein 28 is also the same. A structural change occurs and the surface of the protein layer 18 is further covered. Therefore, the thickness of the crystallized protein layer 18 can be controlled by controlling the time during which the microgrid 10 is left floating on the monomolecular film of the protein 28.

  Here, FIG. 7 is a schematic diagram showing how the protein layer 18 is formed by covering the carbon nanotubes 16 of the microgrid 10 with the protein 28 on the liquid surface of the lower layer liquid 24.

  As described above, as described in the first embodiment, the sample 20 according to the second embodiment can be observed in various directions when the observation target object is held in the carbon nanotube 16 and the observation is performed by the TEM. Since the observation data of the object can be acquired, more accurate information of the observation object can be obtained.

(Third embodiment)
A protein crystal structure analysis method according to the third embodiment will be described.

First, as shown in FIG. 8, the sample 20 of the present invention is placed on the sample holder base 32, and the sample 20 is fixed to the sample holder 30 by covering with the sample holder lid 34 (FIG. 9). As shown in FIG. 9, the carbon formed on the opening side edge of the grid body 12 and the opening side edge of the grid body 12 in the state where the sample 20 is set (held) on the sample holder 30. The nanotubes 16 and the protein layer 18 covered on the side surfaces of the carbon nanotubes 16 are all exposed.
In addition, the shape and material of the sample holder 30 can use a well-known thing according to the kind and observation purpose of TEM to be used.

  Next, the sample holder 30 on which the sample 20 is set is introduced into the TEM and observed. Observation is performed by detecting transmitted or diffracted electrons in a portion of the protein layer 18 covering the side surface of the carbon nanotube 16 that protrudes from the opening of the grid body 12, and obtained observation data. Protein crystal structure analysis is performed using (for example, a diffraction pattern).

  Specifically, for example, observation data acquisition and protein crystal structure analysis are performed as follows. Electron diffraction with information on the structure of a single crystal from a region defined by this aperture by switching the TEM image mode from the TEM observation mode to the diffraction mode by putting a limited field diffraction aperture in the observation area of a given protein. Get a pattern. Here, the aperture restricts the field of view in order to observe only the electron diffraction pattern from a predetermined single crystal. The electron diffraction pattern thus obtained is photographed on a silver salt film. In addition, an electron beam or Ise time pattern can also be directly taken into a computer using a CCD camera instead of photographing on a silver salt film.

  Here, bacteriorhodopsin (sample 20), which is a protein used in the second embodiment, is observed by the above-described method using a high-resolution transmission electron microscope (H-1500, manufactured by Hitachi, acceleration voltage: 800 kV). FIG. 12 shows an example of the diffraction pattern in the reciprocal space obtained as a result. FIG. 11 shows an ultra-high resolution TEM image obtained by performing inverse Fourier transform on the diffraction pattern of FIG.

  FIG. 13A shows a schematic diagram of one peptide chain in bacteriorhodopsin, which is a protein used in the third embodiment, and FIG. 13B shows a schematic diagram of an antiparallel β sheet structure.

  In general, the β-sheet structure is such that an extended peptide chain as shown in FIG. 13A is parallel or anti-parallel, as shown in FIG. The atom and the H atom of the N—H group of the adjacent peptide chain are formed by hydrogen bonding.

  On the other hand, in the ultra-high resolution TEM image of FIG. 11 obtained by the crystal structure analysis method, the peptide chains arranged in parallel and the hydrogen bonds between the peptide chains are shown as black shadows. From the shape of the black shadow in the ultrahigh resolution TEM image of FIG. 11, it can be seen that the crystal structure of the protein layer 18 is an antiparallel β sheet structure as shown in FIG.

  Furthermore, based on the distance between the diffraction spot and the transmission spot in the diffraction pattern of FIG. 12, the distance between peptide chains in the protein layer 18 can be determined to be 0.47 nm, and the unit length of amino acid residues can be determined to be 0.29 nm.

In this way, protein crystal structure analysis can be performed.
The crystal structure analysis means is not limited to the specific means described above, and known means can be used. In the third embodiment, the crystal structure analysis is performed using the TEM diffraction mode. However, as shown in FIG. 10, a TEM image can be obtained by simply observing the transmitted electrons.
Further, instead of the high-resolution transmission electron microscope, the observation can be performed by a known observation means using another TEM (for example, a scanning transmission electron microscope).

  As described above, when the protein crystal structure analysis method according to the third embodiment is used, since the carbon nanotubes 12 are oriented in various directions, it is possible to obtain observation data on protein crystals oriented in various directions. It is possible to perform more accurate protein crystal structure analysis than in the past.

It is the schematic which shows the structure of the microgrid for transmission electron microscopes of this invention, (A) Top view, (B) 1B-1B end view. It is process drawing which showed the manufacturing method of the microgrid for transmission electron microscopes concerning 1st Embodiment according to process. It is the schematic which shows the structure of the sample for transmission electron microscopes concerning 2nd Embodiment, (A) Top view, (B) 3B-3B end view. In the manufacturing method of the sample for transmission electron microscopes concerning 2nd Embodiment, it is the flowchart which showed the process of producing the protein monomolecular film. In the manufacturing method of the sample for transmission electron microscopes concerning 2nd Embodiment, it is process drawing which showed the process of floating a microgrid on the monomolecular film of protein. In the manufacturing method of the sample for transmission electron microscopes concerning 2nd Embodiment, it is the schematic diagram which showed the state in which the protein layer was formed on the carbon nanotube side surface of a microgrid. It is a schematic diagram which shows a mode that a protein layer is formed on the side surface of a carbon nanotube. It is a disassembled perspective view of the sample for transmission electron microscopes of this invention, and a sample holder. It is a perspective view which shows the state by which the sample for transmission electron microscopes of this invention was set to the sample holder. It is a TEM image of the sample for transmission electron microscopes of this invention. It is the super high resolution TEM image of the protein layer obtained in the process of the protein crystal structure analysis method of this invention. It is a diffraction pattern in a reciprocal space obtained in the course of the protein crystal structure analysis method of the present invention. In bacteriorhodopsin, (A) Schematic diagram of one peptide chain and (B) Chemical formula of antiparallel β sheet structure.

Explanation of symbols

10 ... micro grid (sample holder for transmission electron microscope)
12 ... Grid body (Main body)
14 ... Catalyst layer (catalyst)
16 ... carbon nanotube (supporting core)
18 ... Protein layer (observed object)
20 ... Sample (sample for transmission electron microscope)

Claims (15)

A holding body, and
A carrying core for carrying an observation object, the carrying core provided on at least a part of the edge of the holder body;
A sample holder for a transmission electron microscope. The supporting core is provided on the holding body through a catalyst,
The sample holder for a transmission electron microscope according to claim 1. The catalyst includes at least one element selected from Co, Ni, and Fe.
The sample holder for a transmission electron microscope according to claim 1 or 2. The support core includes carbon nanotubes,
The sample holder for a transmission electron microscope according to any one of claims 1 to 3. A sample holder for a transmission electron microscope according to any one of claims 1 to 4,
An observation object carried on the carrying core of the transmission electron microscope sample holder;
A transmission electron microscope sample. The observation object is a protein.
The transmission electron microscope sample according to claim 5. A first step of preparing a holder body;
A second step of forming a supporting core for supporting an observation object on at least a part of the edge of the holding body;
A method for producing a sample holder for a transmission electron microscope, comprising: The second step is a step of forming the support core on the catalyst after forming a catalyst on at least a part of the edge of the holding body.
The manufacturing method of the sample holder for transmission electron microscopes of Claim 7. The catalyst includes at least one element selected from Co, Ni, and Fe.
The manufacturing method of the sample holder for transmission electron microscopes of Claim 7 or 8. The support core includes carbon nanotubes,
The manufacturing method of the sample holder for transmission electron microscopes of any one of Claims 7 thru | or 9. Preparing a sample holder for a transmission electron microscope according to any one of claims 1 to 4,
Carrying the observation object on the carrying core formed on at least a part of the edge of the holding body; and
A method for producing a sample for a transmission electron microscope, comprising: The observation object is a protein.
The manufacturing method of the sample for transmission electron microscopes of Claim 11. Holding the transmission electron microscope sample according to claim 5 in a sample holder;
Mounting the sample holder on a transmission electron microscope to introduce the transmission electron microscope sample into the transmission electron microscope;
Observing the transmission electron microscope sample with the transmission electron microscope;
Having an observation method. An observation step of observing with the observation method according to claim 13;
An analysis step of analyzing the crystal structure using the observation data obtained by the observation step;
A crystal structure analysis method comprising: A sample holder for preparing a sample holder for a transmission electron microscope by preparing a holder body, forming a catalyst on at least a part of the edge of the holder body, and then forming carbon nanotubes on the catalyst Manufacturing process,
A sample manufacturing process for manufacturing a sample for a transmission electron microscope by supporting a protein on the carbon nanotube of the sample holder for the transmission electron microscope;
A sample introduction step of introducing the transmission electron microscope sample into the transmission electron microscope by attaching the sample holder to the transmission electron microscope after holding the transmission electron microscope sample in a sample holder;
An observation step of observing the transmission electron microscope sample with the transmission electron microscope;
An analysis step of analyzing the protein crystal structure using the observation data obtained by the observation step;
A protein crystal structure analysis method comprising: