JP7304098B2 - 3D image observation method and sample grid used therefor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for tilting a sample and stereoscopically photographing it in a transmission electron microscope to observe a stereoscopic image, and a sample grid used for this method.

In the process of developing various devices and materials, microscopic observation of their fine three-dimensional structures is necessary. For example, in physical property analysis of compound derivatives composed of amphipathic molecules that self-assemble into ribbons and have a helical structure, it is required to observe the helical direction of the structure, ie, dextrorotatory or levorotatory. In order to observe such a three-dimensional structure, a plurality of samples derived from the same lot are prepared, and their surfaces are vacuum-deposited (shadowed) with platinum or the like from several angles and observed with a scanning electron microscope (SEM). method, observation with an atomic force microscope (AFM), and the like can be performed. On the other hand, when observing 3D structures using a transmission electron microscope (TEM) with a deep depth of field, it is not possible to obtain a 3D 3D image from a single observation image. A method (parallel method) is used in which two images, one in the left field of view and the other in the left field of view, are obtained, and the angle difference between them is used as the parallax angle for stereo imaging. At that time, in order to enable stereoscopic viewing, an image 1 captured by tilting the sample by rotating it by an angle θ, and an image 2 captured by tilting the sample by rotating it by an angle θ in the opposite direction, It is necessary to take two paired images of .

For example, in Patent Document 1, when a sample is set in a holder of a transmission electron microscope, the rotation axis of the holder and the height position of the set sample are matched, and the sample is observed when the holder is rotated. Disclosed is a transmission electron microscope system that includes a control processor that performs position control so that the position does not move, designates an observation area with the sample tilted, and provides stereo imaging. When the sample is set on the holder and the observation area is determined while the sample is tilted in the first direction from the reference angle by the first angle, a first image is acquired and the sample is moved from the reference angle to the first angle. A second image is obtained by tilting the same first angle in a second direction opposite to the direction, and a stereoscopic image is automatically provided from the obtained stereo pair of images.

Japanese Patent Laid-Open No. 2002-200002 discloses a method of obtaining a stereoscopic image by obtaining two images by changing the irradiation angle of an electron beam instead of tilting a sample in stereo imaging with a transmission electron microscope. Here, the first electron beam biprism was installed in the irradiation optical system, two electron beams with different angles were simultaneously irradiated to the observation area of the sample, and the transmitted electron beams were arranged in the imaging optical system. A second electron beam biprism is used to spatially separate the images, and a stereoscopic image is generated from two electron microscope images with different irradiation angles. Compared to the case where two images are obtained by alternately irradiating electron beams from two directions, the time difference between the images can be eliminated, the temporal resolution can be improved, and dynamic observation and real-time observation can be provided. It is said that it can be done.

Japanese Patent Application Laid-Open No. 2006-184202 JP 2011-40217 A

As described above, in observation with a transmission electron microscope with a deep depth of field, stereo imaging can be performed from two images, a right field image and a left field image, with different tilt angles, using this angle difference as the parallax angle. . In this case, compared to normal microscope observation, observation with a greater tilt is required to obtain an angular difference. In other words, it is required to be able to observe the specimen while tilting it to some extent, without greatly changing the main body of the transmission electron microscope. In particular, it is desirable to be able to observe a three-dimensional image by performing stereo imaging with ease and good operability even when observing minute nano-sized helical structures.

SUMMARY OF THE INVENTION The present invention has been made in view of the circumstances described above, and an object of the present invention is to provide a method for observing a three-dimensional image by tilting a sample in a transmission electron microscope and stereoscopically photographing it, and a method for observing a three-dimensional image. The object is to provide a sample grid that can be used.

The method according to the present invention is a method for observing a three-dimensional image by tilting a sample in a transmission electron microscope and stereoscopically photographing it. a step of providing a sample grid formed of a shaped plate and provided with a marker indicating the center of the long side at the periphery of the through hole, and applying the sample to the center of the long side of the through hole; placing the sample grid on the sample stage so that the stripe lines are perpendicular to an axis of rotation of the stage; rotating and tilting the sample stage about the axis of rotation to direct electrons onto the sample; and a step of obtaining a stereoscopic image by guiding the beam to obtain a stereoscopic image, wherein the electron beam is guided to the sample using the marker as a reference position.

According to this feature, the marker is used as a mark even for a sample, particularly a sample having a minute nano-sized helical structure, so that a stereoscopic image can be obtained by suppressing shadows from the thickness of the sample grid. Therefore, it is possible to arrange the sample on the sample grid and to irradiate the electron beam with ease and with good operability.

In the above-described invention, the marker may be a projecting piece projecting from the peripheral portion toward the interior of the through hole. Further, one or a plurality of the convex pieces may be provided as a pair on the opposite long sides. According to this feature, the placement of the sample on the sample grid and the irradiation of the electron beam can be performed simply and with good operability.

In the above-described invention, the sample grid may be arranged on the sample stage by aligning notches of the sample grid with projections provided on the sample stage. According to this feature, the arrangement of the sample grid on the sample stage can be performed simply and with good operability.

Further, a sample grid according to the present invention is a sample grid for observing a three-dimensional image by tilting a sample in a transmission electron microscope and stereoscopically photographing the sample, wherein the long sides of the rectangular through holes are arranged along stripe lines parallel to each other. and a marker indicating the central portion of the long side is provided on the peripheral edge of the through hole in order to provide the sample to the central portion of the long side of the through hole, and the marker is observable with the electron beam as a reference position for guiding the electron beam to the sample.

According to this feature, the marker is used as a mark even for a sample, particularly a sample having a minute nano-sized helical structure, so that a stereoscopic image can be obtained by suppressing shadows from the thickness of the sample grid. Therefore, it is possible to arrange the sample on the sample grid and to irradiate the electron beam with ease and with good operability.

In the above-described invention, the marker may be a projecting piece projecting from the peripheral portion toward the interior of the through hole. Further, one or a plurality of the convex pieces may be provided as a pair on the opposite long sides. According to this feature, the placement of the sample on the sample grid and the irradiation of the electron beam can be performed simply and with good operability.

The above-described invention may be characterized in that a notch that can be fitted to a projection provided on the sample stage is provided on the outer peripheral portion of the sample grid. Further, it may be characterized in that it is made of a metal disk, and that the notches are provided at two opposite positions on the outer peripheral portion of the disk. According to this feature, the arrangement of the sample grid on the sample stage can be performed simply and with good operability.

In the above invention, a row in which a plurality of the through-holes of the same shape are provided at predetermined intervals is provided along one of the stripe lines, and the adjacent stripes are shifted by half the length of the long side. It may be characterized by providing the columns along a line. According to this feature, the strength of the sample grid can be increased and its warpage can be suppressed, so that a good stereoscopic image can be obtained.

It is a figure which shows the method of stereo photography. FIG. 4 is a cross-sectional view showing a state in which a grid is tilted in stereo imaging; 1A and 1B are a plan view and a partially enlarged view showing a grid according to the present invention; FIG. FIG. 4 is a plan view showing how grids are set according to the present invention; It is a photograph showing an example of stereo photography using the grid according to the present invention.

FIGS. 1 to 4 show a 3D image observation method for observing a 3D image by tilting a sample in a transmission electron microscope and observing a 3D image, and a sample grid for 3D image observation used therein, according to an embodiment of the present invention. will be used to explain the details.

As shown in Fig. 1, humans can see objects three-dimensionally with their left and right eyes because there is a difference in image position and viewing direction between the left and right eyes (binocular parallax). I also use it for photography. That is, in a transmission electron microscope, an image PR obtained with the right eye and an image PL obtained with the left eye are stereoscopically photographed, and the image PR and the image PL are provided to the right and left eyes of an observer to provide a stereoscopic image. I do.

As shown in FIG. 1(a), one method of obtaining the image PR and the image PL is to make the center beam C of the electron beam incident on the specimen 1 set on the specimen stage 31 (specimen grid 10, see FIG. 4). In this method, the angle is made to correspond to the positions of the left and right eyes, and the movement is controlled by a constant angle θ to the left and right. On the other hand, if the angle of incidence of the center beam C of the electron beam on the specimen 1 is fixed and the specimen 1 is tilted corresponding to the directions of the left and right eyes, the images PR and PL can be similarly obtained. At this time, it is preferable to increase the tilt angle θ in order to obtain a large binocular parallax in stereo photography.

Here, as shown in FIG. 2( a ), in the observation of microstructures, for example, observation of submicron nano-level microstructures such as molecular spirals, a fine mesh is provided to support a sample that can be fine. A sample 1 (see FIG. 1) is applied to the sample grid 10 and observed. The electron beam C passes through the mesh holes 15 (15') of the sample grid 10 and forms an image on the imaging means (not shown) below.

As shown in FIG. 2B, when the sample stage is tilted to the left or right by an angle θ for stereo imaging, the sample grid 10 also rotates by an angle θ, but the thickness d of the sample grid 10 (usually , about 10 μm), the edge A interferes with the electron beam C and forms a shadow in the narrow mesh hole 15′. Therefore, it is preferable to make the mesh holes 15 wide at least in the direction of inclination.

Here, in a general parallel method stereo imaging, it is preferable to be able to perform imaging by tilting the sample stage at an angle θ of 10 to 25 degrees to both sides, typically about 15 degrees. Since the influence of the shadow of the edge A described above has a sine on the projected image, in the case of the sample grid 10 having a thickness d of about 10 μm, the shadow area is about 2.6 μm. can. If the part of the sample to be observed enters this area, it cannot be observed. is determined according to the desired inclination.

As shown in FIGS. 3 and 4, the mesh-like sample grid 10 is a mesh of through-holes having regular rectangular cross-sections when viewed from the top near the center of a disk made of a metal such as copper, tungsten, or molybdenum. A hole 15 is machined. The mesh holes 15 are arranged in rows so that their long sides are parallel to each other along stripe lines L (here, L1 to 3 are schematically shown), preferably to ensure the strength of the sample grid 10. , above the stripe line L2, a plurality of mesh holes 15 of the same shape are provided at predetermined intervals, and adjacent stripe lines L1 and L3 are provided with half the length of the rectangular long side of the mesh hole 15. Multiple are given in the same way with only a shift. If the strength of the sample grid 10 can be ensured, handling such as setting the sample grid 10 in the sample holding hole 33, which will be described later, becomes easier, and the thickness d of the sample grid 10 can be reduced. Also, as described above, it is possible to prevent the edge of the mesh hole 15 from interfering with the beam C of the electron beam and forming a shadow.

On the other hand, the ratio of the long sides to the short sides of the rectangles of the mesh holes 15 and the arrangement thereof may impair the flatness (warp) of the sample grid 10, and in particular, it does not affect the observation of minute nano-sized helical structures. give. Therefore, it can be determined in consideration of the material strength of the sample grid 10 as well as the non-uniformity of the plane stress due to the arrangement of the mesh holes 15 . Furthermore, from the viewpoint of providing an observation with a larger inclination angle such as a three-dimensional analysis described later, the ratio of the long side to the short side of the rectangle of the mesh hole 15 is about 2:1, or the long side is increased. It can be set longer, such as 3:1, but even in this case, it is necessary to consider the influence on the flatness of the sample grid 10 .

A sample 1 (see FIG. 1) is provided on the sample grid 10, and the sample holding hole 33 on the sample stage 31 of the holder 30 is set so that the stripe line L is perpendicular to the rotation axis P of the sample stage 31. , the sample grid 10 is arranged. Here, above the sample stage 31 of the holder 30, there are shown three sample holding holes 33, which are through holes whose inner peripheral wall is tapered or stepped so as to accommodate the sample grid 10. FIG. are appropriately provided. Then, the sample stage 31 is rotated and tilted around the rotation axis P for stereoscopic photography, and a three-dimensional image is obtained as described above. Even when the sample stage 31 is rotated by an angle .theta. Shadows from the thickness d of the sample grid 10 can be suppressed, and as a result, a good three-dimensional image can be obtained while tilting the sample 1 to some extent without significantly changing the main body of the transmission electron microscope. As described above, not only can one image (PR) be acquired by tilting the sample by rotating it by an angle θ, but also by tilting the sample by rotating it in the opposite direction by a pair of angles θ. A photographed image (PL) can also be obtained.

Here, a notch 12 is provided in the peripheral edge of the disk-shaped sample grid 10, and is fitted to a fixing projection 35 provided in the sample holding hole 33, thereby rotating the sample stage 31 of the holder 30. The sample grid 10 can be easily arranged on the sample stage 31 so that the stripe line L is perpendicular to the axis P, and the operability can be improved. For example, the cutouts 12 are preferably provided at two opposite positions on the outer periphery perpendicular to the stripe line L. It is preferably provided toward the inside of the hole 33 .

Further, by providing a marker indicating the position at the center of the long side of the mesh hole 15, which is a rectangular through-hole of the sample grid 10, the sample 1 can be easily arranged at the center of the mesh hole 15, as described above. Moreover, shadows from the thickness of the sample grid 10 can be reliably suppressed. The marker can be confirmed when the sample 1 is placed on the sample grid 10 under an optical microscope, and can be confirmed by using the marker as a reference position to reliably guide the electron beam to the sample 1 in the vicinity thereof even under observation with an electron microscope. is provided as follows. This is, for example, a recess provided on the periphery of the mesh hole 15 . In addition, the projecting piece 17 protrudes toward the inside of the mesh hole 15, and at this time, the sample 1 can be easily placed by hooking it on the projecting piece 17, and the flatness of the sample grid 10 as described above is not impaired. . Moreover, it is preferable that the convex pieces 17 are provided as a pair, one on each of the opposing long sides, toward the inside of the rectangle. Regarding the projecting piece 17, it is sufficient that the sample 1 can be placed in the central portion of the mesh hole 15, and one or a plurality of such pairs as described above may be provided near the central portion of the long side, or a plurality of projecting pieces may be provided. It may be in the form of a sawtooth consisting of a convex piece.

As described with reference to FIG. 2, since the mesh hole 15 needs to be wide in the direction of inclination during stereoscopic photography, its cross-sectional shape is not limited to a rectangle and can take various shapes.

According to the sample grid 10 described above, when the sample stage 31 is tilted clockwise and counterclockwise around the rotation axis P of the holder 30 by the same angle θ, the sample 1, which is the observation object, is It is possible to reduce the area that is hidden by the shadow from the thickness d of the sample grid 10, that is, the area where image data cannot be captured. Then, it becomes easy to acquire a pair of stereo images in which the sample stage 31 is tilted by the same angle θ in the plus angle direction and the minus angle direction (clockwise and counterclockwise), which contributes to the rapid and efficient creation of stereo images. contribute. In general, transmission electron microscopes have a deep depth of field, so in principle, it is not possible to determine how the nanoscale spiral is wound (right-handed, left-handed) from a single projected image. By using such a sample grid 10, paired images (image data) can be efficiently acquired and discriminated.

As an example, FIG. 5 shows hexabenzocoronene (hexa-peri- hexabenzocoronene) derivatives showed stereoscopic images of self-assembled helical structures. When viewing the image (a) with the left eye and the image (b) with the right eye by the parallel method, the winding of the helical structure and the overlap with the tubular structure can be seen three-dimensionally. Here, the thickness of the sample grid 10 is 0.010 mm, the long side of the mesh holes is 0.080 mm, the edge is 0.040 mm, the interval between the mesh holes arranged on the stripe line L is 0.020 mm, and the adjacent stripes The distance between the mesh holes aligned on the line L is 0.020 mm, and the angle of inclination of the photographing is θ=15 degrees. Such morphological evaluation and spiral winding method can be quickly performed on the same sample, and useful structural and morphological information can be obtained.

The short sides of the mesh holes described above are approximately the same size as the existing 400-mesh sample grid, but can be changed according to the size of the sample. For example, for the observation of the nano-sized helical structure described above, 200 mesh or less can be used. Again, the flatness of the sample grid, the flatness of the support film provided thereon, and the charge-up by the electron beam, etc. It is necessary to consider the influence of

Note that the sample grid 10 as described above can also be used when imaging using a larger tilt angle such as 60 degrees is required, such as in three-dimensional tomography. In such a case as well, if the long side:short side of the rectangular mesh hole 15=2:1, the projected image (long side) will be the cosine of the rectangular mesh hole 15, resulting in a 1:1 square.

Although representative embodiments according to the present invention and variations associated therewith have been described above, the present invention is not necessarily limited to these, and can be modified as appropriate by those skilled in the art. That is, those skilled in the art will be able to find various alternatives and modifications without departing from the scope of the appended claims.

1 sample 10 sample grid 12 notch 15, 15' mesh hole 17 sample holding convex piece 30 holder 31 sample stage 33 sample holding hole 35 fixing protrusion

Claims (9)

A method for observing a stereoscopic image by tilting a sample in a transmission electron microscope and performing stereo imaging, comprising:
preparing a sample grid consisting of a mesh plate in which the long sides of rectangular through-holes are arranged along stripe lines parallel to each other, and in which a marker indicating the central portion of the long side is provided at the peripheral edge of the through-hole, applying the sample to the central portion of the long side of the through-hole;
placing the sample grid on the sample stage so that the stripe lines are perpendicular to the axis of rotation of the sample stage;
a step of rotating and tilting the sample stage around the rotation axis to guide an electron beam to the sample and perform stereoscopic imaging to obtain a three-dimensional image;
A stereoscopic image observation method, wherein the electron beam is guided to the sample using the marker as a reference position. 2. A stereoscopic image observation method according to claim 1, wherein said marker comprises a convex piece projecting from said peripheral portion toward the interior of said through hole. 3. A stereoscopic image observation method using a transmission electron microscope according to claim 2, wherein one or a plurality of said convex pieces are provided as a pair on the opposite long sides. 2. The stereoscopic image observation method according to claim 1, wherein the sample grid is arranged on the sample stage by aligning notches of the sample grid with projections provided on the sample stage. A sample grid for observing a stereoscopic image by tilting a sample in a transmission electron microscope and stereoscopically photographing the sample,
It consists of a mesh-like plate in which the long sides of rectangular through-holes are arranged along mutually parallel stripe lines, and the long sides of the through-holes are provided at the peripheral edges of the through-holes to provide the sample to the central part of the long sides of the through-holes. given a marker to indicate the middle of the edge,
A transmission type electron beam , wherein the marker is a convex piece protruding from the peripheral portion toward the inside of the through hole, and is observable by the electron beam as a reference position for guiding the electron beam to the sample. A sample grid for stereoscopic image observation with a microscope. 6. A sample grid for three-dimensional image observation by a transmission electron microscope according to claim 5 , wherein one or a plurality of said convex pieces are provided as a pair on the opposite long sides. A sample grid for observing a stereoscopic image by tilting a sample in a transmission electron microscope and stereoscopically photographing the sample,
It consists of a mesh plate in which the long sides of rectangular through-holes are arranged along stripe lines parallel to each other, and a row in which a plurality of said through-holes of the same shape are spaced apart by a predetermined interval is provided along one of said stripe lines. and the adjacent stripe lines are provided with the columns shifted by half the length of the long side,
A marker indicating the center of the long side is provided on the peripheral edge of the through hole in order to direct the sample to the center of the long side of the through hole, and the marker is a reference position for guiding the electron beam to the sample. A sample grid for three-dimensional image observation by a transmission electron microscope, characterized in that it can be observed with the electron beam as the above-mentioned electron beam. 8. The sample grid for stereoscopic image observation according to claim 5, wherein a notch is provided on the outer peripheral portion of the sample grid so that it can be fitted to a projection provided on the sample stage. 9. A specimen grid for three-dimensional image observation according to claim 8, wherein said notch is provided at two opposite positions on the outer periphery of said disk.

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