JP4958313B2 - Scanning electron microscope and method of using the same - Google Patents

Description

  The present invention relates to a scanning electron microscope technique, and more specifically, scanning electron microscope capable of observing the form and structure at a high resolution without damaging even extremely fragile samples such as biological materials and organic materials. The present invention relates to a microscope and a method for using the microscope.

  Scanning electron microscopes (SEM) are often used for observation of biological samples, and surface observation of bacteria and observation of viruses and the like are performed. Conventionally, when a biological sample is observed with a scanning electron microscope, the sample is fixed with formaldehyde or the like, and gold, platinum, carbon, or the like is coated on the surface or stained with a heavy metal or the like. By such processing, the electron beam damage of the sample can be reduced, and an image with higher contrast can be obtained.

  Recently, a new SEM observation method (Indirect Secondary Electron Contrast Method: ISEC method) that enables high-contrast SEM observation of a biological sample without coating or staining has been developed by the present inventor. The obtained image is called an indirect secondary electron contrast condition (ISEC condition) and an indirect secondary electron contrast image (ISEC image) (see Non-Patent Document 1).

  In this method, an observation sample is attached to the lower surface of a thin sample support film (for example, a carbon film), and an electron beam with a low acceleration voltage is irradiated from the upper part of the sample support film. The irradiated electron beam spreads while diffusing inside the sample support film, reaches near the lower surface, and secondary electrons are emitted from the lower surface of the sample support film. Then, as a result of the secondary electrons being absorbed by the observation sample adhering to the lower surface of the sample support film, a high contrast SEM image can be obtained.

  Under ISEC conditions, it is secondary electrons generated in the sample support film that contribute to SEM image formation (contrast formation), and the incident electron beam (primary electron beam) is not directly irradiated to the observation sample. Most of the incident electron beam is absorbed by the sample support film. In addition, since the energy of secondary electrons generated under ISEC conditions is as low as several tens of eV, even if such low energy secondary electrons are absorbed by a fragile observation sample such as a biological sample, the damage can be almost ignored. .

  Moreover, in the ISEC method, an image is formed not by the secondary electrons from the observation sample but by the secondary electrons from the sample support film, so that a black contrast occurs in the observation sample. Therefore, it is possible to obtain a high-contrast observation image even from an observation sample that is difficult to have contrast.

  However, although the ISEC image obtained by the method described in Non-Patent Document 1 has a remarkably high quality compared to the SEM image obtained by a general method, it has a high quality. There is still room for improvement in terms of resolution.

  This is interpreted as secondary electrons emitted from the lower surface of the sample support film being randomly emitted in various directions. Due to such random emission of secondary electrons, the emission range of secondary electrons must be spatially widened, resulting in “blurring” in the obtained ISEC image.

Similarly, the tilted image of the observation sample obtained by tilting the sample support film is also affected by the random emission of the secondary electrons, so that the obtained tilted image is different from the original one. In addition, in the case of tilt observation, since the perpendicular direction of the lower surface of the sample support film does not coincide with the incident direction (optical axis) of the electron beam, the emitted secondary electrons are the optical axis of the incident electron beam. As a result of spreading in different directions and further expanding the secondary electron emission range, it becomes more difficult to obtain an original tilted image.
T. Ogura, “A high contrast method of unstained biological samples under a thin carbon film by scanning electron microscopy” Biochemical and Biophysical Research Communications (2008), doi: 10.1016 / j.bbrc.2008.09.097

  The present invention has been made for the purpose of developing the above-mentioned ISEC method, and can observe the form and structure of extremely fragile samples such as biological materials and organic materials with high resolution without damaging the samples. A scanning electron microscope and a method for using the same are provided.

In order to solve such a problem, a scanning electron microscope according to the present invention includes a sample holder for holding an observation sample, an electron gun disposed on the upper surface side of the sample holder, and a lower surface side of the sample holder. And a potential control unit disposed in a plane perpendicular to the optical axis of the electron beam irradiated on the observation sample, and the potential control unit is disposed on the irradiation region of the electron beam in the plane. The potential of the corresponding region can be controlled to an equipotential (V ₁ : V ₁ > V ₀ ) higher than the potential (V ₀ ) of the sample holder.

In this scanning electron microscope, a secondary electron detector is disposed below the potential control unit, and the potential (V ₂ ) of the current collector of the secondary electron detector is set to the potential (V) of the potential control unit. _It is also possible to adopt a configuration that can be controlled higher than ₁ ) (V ₂ > V ₁ ).

  The potential control unit is, for example, a conductive plate having a conductive mesh or a hole.

In such a scanning electron microscope, the observation sample is supported on the surface of the sample support unit fixed to the sample holder, and the surface of the sample support unit faces the potential control unit. The secondary electron which does not substantially permeate the support part and is generated in the sample support part is set to a value that can reach the observation sample support surface, and the potential control part is set to be more than the potential (V ₀ ) of the sample holder. It is used under the condition of a high potential (V ₁ : V ₁ > V ₀ ).

  Preferably, the acceleration voltage of the irradiation electron beam is set to 5 keV or less, and the thickness of the sample support portion is set to 50 nm or less. As the sample support portion, for example, a carbon film can be used.

In the scanning electron microscope of the present invention, in the plane on the lower surface side (the side opposite to the side where the electron gun is disposed) of the sample holder, and in the plane perpendicular to the optical axis of the electron beam irradiated on the observation sample A potential control unit was arranged. As a result, the observation sample is supported on the surface of the sample support portion fixed to the sample holder so as to face the potential control portion, the primary electron does not substantially pass through the sample support portion and the electron beam acceleration voltage is not transmitted through the sample support portion. Conditions are set such that secondary electrons generated in the support section reach the observation sample support surface, and the potential control section is set to a potential (V ₁ : V ₁ > V ₀ ) higher than the potential of the sample holder (V ₀ ). It was possible to carry out the observation with.

  When SEM observation is performed under such conditions, secondary electrons emitted from the lower surface of the sample support portion (the surface opposite to the side on which the electron gun is disposed) are caused by the potential control unit to be aligned with the optical axis of the electron beam. Since they are attracted in the same downward direction, random emission of secondary electrons is suppressed, and the resolution of the obtained ISEC image can be increased. For the same reason, the tilted image of the observation sample can be obtained closer to the original one.

Further, a secondary electron detector is disposed below the potential control unit, and the potential (V ₂ ) of the current collector of the secondary electron detector is higher than the potential (V ₁ ) of the potential control unit (V ₂ > V ₁ ) When set, the phenomenon that even the secondary electrons emitted from the upper surface of the sample support part (the surface on which the electron gun is disposed) is detected by the secondary electron detector and becomes a noise is suppressed. The SN ratio of the ISEC image is improved.

  Hereinafter, a scanning electron microscope and a method of using the same according to the present invention will be described with reference to the drawings. In the following, for convenience, the “vertical relationship” of other components will be described with reference to the position of the observation sample. However, it is obvious that the vertical relationship of each component may be reversed.

FIG. 1 is a conceptual diagram for explaining a configuration example of a main part of a scanning electron microscope of the present invention. An observation sample 10 is supported by a sample support portion 11 such as a carbon film, and the sample support portion 11 is a sample holder. 12 is fixed. An electron gun 13 is disposed on the upper surface side of the sample holder 12, and a potential control unit 14 (illustrated as a conductive mesh) is disposed on the lower surface side of the sample holder 12. The potential control unit 14 is arranged in a plane (indicated by B in the drawing) perpendicular to the optical axis (indicated by A in the drawing) of the electron beam irradiated to the observation sample. In B), the potential of the region corresponding to the electron beam irradiation region can be controlled to an equipotential (V ₁ : V ₁ > V ₀ ) higher than the potential (V ₀ ) of the sample holder 12, for example, Further, it may be a conductive mesh made of a conductive material such as metal or carbon, or a conductive plate having holes.

Electrons (primary electrons) irradiated from the electron gun 13 onto the upper surface of the sample support 11 diffuse inside the sample support 11, but in the present invention, when obtaining an ISEC image, the primary electrons are converted into the sample support. The acceleration voltage of the electron beam is set to a value that does not substantially pass through 11 and secondary electrons generated in the sample support 11 reach the lower surface of the sample support 11 (observation sample support surface). . Then, the potential control section 14, the potential of the sample holder 12 _{(V 0:} effective is also the potential of the sample support 11 and the observation sample 10) a potential higher than _{(V 1: V 1> V} 0) and the conditions Perform observations with. Specifically, for example, conditions are set such that the acceleration voltage of the electron beam is 5 keV or less and the thickness of the sample support 11 is 50 nm or less.

  Under such conditions, high energy primary electrons do not pass through the sample support 11 and therefore do not damage fragile biological samples and the like. On the other hand, secondary electrons generated in the sample support unit 11 are emitted from the lower surface of the sample support unit 11 to the outside, and the secondary electrons are in the same downward direction as the optical axis of the electron beam due to the presence of the potential control unit 14. Thus, random emission is suppressed, and current is collected by the secondary detector in this state.

  Note that “substantially does not transmit” when “primary electrons do not substantially pass through the sample support 11” means that transmission of primary electrons from the sample support 11 is completely suppressed. It doesn't mean everything. The meaning of “substantially not transmitting” here (that is, how much permeation can be tolerated) depends on the vulnerability of the observation sample to damage by the electron beam. Any amount of primary electron transmission that does not cause damage is acceptable. However, as is clear from the above description, in the ISEC method, the primary electrons transmitted through the sample support are only noise components of the ISEC image, so it is preferable to keep the amount of transmitted primary electrons as low as possible.

In a general SEM, the secondary detector is arranged on the upper side of the sample holder 12 (sample support portion 11). However, in the configuration example shown in FIG. 15 and the potential (V ₂ ) of the current collector 16 (shown as a conductive mesh) of the secondary electron detector 15 is higher than the potential (V ₁ ) of the potential controller 14 (V ₂ > V _1). ) Is set.

  In the configuration in which the secondary detector is disposed on the upper side of the sample holder 12 (sample support unit 11), not only the secondary electrons emitted from the lower surface of the sample support unit 11 but also the upper surface of the sample support unit 11 is emitted. Even the secondary electrons thus detected are detected by the secondary electron detector because the latter causes noise.

  According to the scanning electron microscope having such a configuration, secondary electrons emitted from the lower surface of the sample support 11 can be attracted in the optical axis direction of the incident electron beam without diffusing in a random direction. The resolution of the ISEC image is improved, and the “distortion” of the tilted image of the observation sample can be removed.

  As the sample support part 11, a carbon support film generally used in transmission electron microscope observation can be used, but a silicon-based material film or a metal thin film can also be used. Moreover, it is preferable that the thickness of the sample support part 11 is 50 nm or less.

  Moreover, it is necessary to select an appropriate value for the acceleration voltage of the incident electron beam depending on the material and film thickness of the sample support portion. For example, in Non-Patent Document 1, a carbon film having a thickness of 40 nm is used as the sample support portion. And a high-contrast ISEC image is obtained under the condition of an electron beam acceleration voltage of 1.5 kV.

  FIGS. 2A and 2B schematically show the difference between the case where the potential control unit provided in the scanning electron microscope of the present invention is provided (FIG. 2A) and the case where there is no potential control (FIG. 2B). FIG. 5 is a schematic diagram when the sample support is perpendicular to the optical axis of the incident electron beam (no tilt). Here, the potential control unit is illustrated as a conductive mesh.

  When there is a potential control unit (FIG. 2A), random emission of secondary electrons does not occur due to the conductive mesh 14 disposed below the observation sample 10. On the other hand, when the conductive mesh 14 is not disposed (FIG. 2B), secondary electrons emitted from the lower surface of the sample support portion are randomly emitted, which is a cause of “blurring” in the ISEC image. As a result, the resolution decreases.

  FIGS. 3A and 3B show the case where the potential control unit included in the scanning electron microscope of the present invention is provided when the sample support is tilted (FIG. 3A) and the case where there is no potential control unit (FIG. It is a figure for showing the difference of B)) typically. Also here, the potential controller is illustrated as a conductive mesh.

  Even when the sample support portion is tilted, as described with reference to FIGS. 2A and 2B, when there is a potential control portion (FIG. 3A), the sample support portion is placed below the observation sample 10. Since the arranged conductive mesh 14 attracts secondary electrons in a direction coinciding with the optical axis of the incident electron beam, random emission does not occur, so that a more accurate tilt image can be obtained. On the other hand, when the conductive mesh 14 is not disposed (FIG. 3B), secondary electrons emitted from the lower surface of the sample support portion are randomly emitted, which causes “blurring” in the ISEC image. Not only will it become an obstacle to obtaining an accurate tilt image itself.

  4 (A) to 4 (D) show one type of oral bacteria as an observation sample, which is attached to the lower part of a 40 nm thick carbon film fixed to a copper grid (mesh) and held by an aluminum sample holder. It is an ISEC image obtained by the ISEC method. In this ISEC image, as shown in FIG. 1, a copper grid mesh (metal mesh) as a potential control unit is parallel to a carbon film as a sample support part on the lower surface side of the observation sample (that is, the observation sample is irradiated). The image was taken in a plane perpendicular to the optical axis of the electron beam. The distance between the carbon film and the copper grid mesh (metal mesh) is 5 mm.

4A is an ISEC image when the potential of a copper grid mesh (metal mesh) as a potential control unit is V ₁ = + 200V, and FIG. 4B is an ISEC image when V ₁ = 0V. It is. The aluminum sample holder potential is V ₀ = 0. Comparing the shape of the bacteria displayed in these ISEC images, FIG. 4 (A) is clearly thinner (that is, sharper).

  FIGS. 4C and 4D are enlarged images of the regions indicated by broken lines in FIGS. 4A and 4B, respectively. In FIG. 4C, the bent portion of the bacteria can be clearly confirmed, whereas in FIG. 4D, it is difficult to confirm the shape of the bent portion due to the “blurring” of the image.

FIGS. 5A to 5D are ISEC images taken with the same observation sample as described above tilted by 30 °, and FIG. 5A is a copper grid mesh (metal mesh) as a potential control unit. FIG. 5B is an ISEC image when the potential is V ₁ = + 100 V, and FIG. 5B is an ISEC image when V ₁ = 0V. In this case, the aluminum sample holder potential is V ₀ = 0.

Also in the case of tilt observation, bacteria are observed sharply when the potential of the potential control unit is set to an equipotential (V ₁ = + 100 V) higher than the potential of the sample holder (V ₀ ).

  FIGS. 5C and 5D are enlarged images of spherical bacteria in the regions indicated by broken lines in FIGS. 5A and 5B, respectively. In FIG. 5D, the outline of the spherical bacteria is “blurred”, whereas in FIG. 5C, a relatively clear outline can be confirmed.

  The present invention relates to a scanning electron microscope capable of observing the form and structure of extremely fragile samples such as bacteria, viruses, protein complexes, and other biological materials and organic materials with high resolution without damaging the samples, and Provide how to use it. It has high utility value not only for research equipment but also for drug development.

It is a conceptual diagram for demonstrating the structural example of the principal part of the scanning electron microscope of this invention. It is a figure for showing typically the difference with the case where there is an electric potential control part with which the scanning electron microscope of the present invention is provided (Drawing 2 (A)) and the case where there is no (Drawing 2 (B)). In order to schematically show the difference between the case where the scanning electron microscope of the present invention is equipped with the potential control portion (FIG. 3A) and the case where there is no potential (FIG. 3B) when the sample support portion is tilted. FIG. An ISEC image obtained by the ISEC method is obtained by using one type of oral bacteria as an observation sample, attaching it to the bottom of a 40 nm thick carbon film fixed on a copper grid (mesh), and holding it with an aluminum sample holder. is there. FIG. 5 is an ISEC image obtained by photographing an observation sample similar to that in FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Observation sample 11 Sample support part 12 Sample holder 13 Electron gun 14 Potential control part 15 Secondary electron detector 16 Current collection part

Claims (6)

A sample holder for holding an observation sample;
An electron gun disposed on the upper surface side of the sample holder;
A potential control unit disposed on a lower surface side of the sample holder and disposed in a plane perpendicular to an optical axis of an electron beam irradiated on the observation sample;
The observation sample is held on the lower surface side which is a surface facing the potential control unit of the sample support unit fixed to the sample holder,
The potential control unit sets the potential of the region corresponding to the electron beam irradiation region in the plane to an equipotential (V ₁ : V ₁ > V ₀ ) higher than the potential (V ₀ ) of the sample holder. A scanning electron microscope characterized by being controllable. A secondary electron detector is disposed below the potential controller, and the potential (V ₂ ) of the current collector of the secondary electron detector is higher than the potential (V ₁ ) of the potential controller (V ₁ ). The scanning electron microscope according to claim 1, wherein ₂ > V ₁ ) can be controlled.   The scanning electron microscope according to claim 1, wherein the potential control unit is a conductive plate having a conductive mesh or a hole. A method of using the scanning electron microscope according to claim 1,
The observation sample is supported on a surface of the sample support unit fixed to the sample holder, the surface facing the potential control unit,
The accelerating voltage of the irradiated electron beam is set to a value at which primary electrons do not substantially pass through the sample support and secondary electrons generated in the sample support can reach the observation sample support surface. ,
A method of using a scanning electron microscope, wherein the observation is performed under a condition in which the potential control unit is set to a potential (V ₁ : V ₁ > V ₀ ) higher than the potential (V ₀ ) of the sample holder.   The method of using a scanning electron microscope according to claim 4, wherein an acceleration voltage of the irradiation electron beam is 5 keV or less and a thickness of the sample support is 50 nm or less.   The method of using a scanning electron microscope according to claim 4 or 5, wherein a carbon film is used as the sample support portion.